University Microfilms, Inc., Ann Arbor, Michigan ILLUS TRATIONS— (Continued) Figure Page

This dissertation has been 64—6920 microfilmed exactly as received

JORDAN, John Maxwell, 1938- STUDIES ON METABOLISM IN PENICILLIUM CHARLESII; SOME RELATIONSHIPS BETWEEN DICARBOXYLIC ACID METABOLISM AND PRO DUCTION OF GALACTOCAROLOSE.

The Ohio State University, Ph.D., 1963 Chemistry, biological University Microfilms, Inc., Ann Arbor, Michigan ILLUS TRATIONS— (Continued) Figure Page

12 Variation with time of carbohydrate concentration of systems containing various concentrations of diammonium dihydroxymaleate ...... 88

13 Atypical behavior of system containing high level of medium n i t r o g e n ...... 99

14 Changes in hydrogen ion concentration and carbo hydrate concentration of growth medium of the RTg and RT^ series ...•••• ...... 101

15 Changes in hydrogen ion and carbohydrate concen tration of the growth medium for the DHM_ and DHM., series ...... • • 104

16 Changes in hydrogen ion and carbohydrate concen tration of the growth medium for the Funig and Fum^ series ...... 106

17 Changes in hydrogen ion and carbohydrate concen tration of the growth medium for the Mal_ and Mal^ series 110 32 18 Distribution of P -labeled components of a perchloric acid extract of P. charlesii exposed 4.5 days to orthophosphate-P^^ • • • T ..... 126 32 19 Distribution of P -labeled components of a perchloric acid extract of P. charlesii exposed 9 days to orthophosphate-P^...... 129 32 20 Distribution of P -labeled components of a . perchloric acid extract of P. charlesii exposed 13.5 days to orthophosphate-P^ . I I ~ ..... 133 32 21 Distribution of P -labeled components of a perchloric acid extract of P. charlesii exposed l8.0 days to orthophosphate-P^^T • • • • • 136

22 Distribution of P^-labeled and U.V.-light ab sorbing components of perchloric acid extract of P. charlesii exposed 4.5 days to orthophosphate- p32 ...... 146

23 Comparison of the near ultraviolet spectra.of various solutions ofJx ...... 148

24 Column chromatography ofJx...... 154

ix Fig# 9•--Variation with time of hydrogen ion concen tration of systems containing various concentrations of diam monium fumarate#

The curve defined by X represents the FUM^ system

The curve defined by closed triangles represents the FUMg system

The curve defined by the closed circles represents the FUM^ system

82 63 Fig. 10.--•Variation with time of carbohydrate concen tration of systems containing various concentrations of diam monium fumarate•

The curves are defined according to the notations of Figure 9 pH TOTAL SUGAR AS KLETT UNITS o> o ro oo o oo ro o ro ro

DAYS $8 Fig. 11.--Variation with time of hydrogen ion concen tration of systems containing various concentrations of diam monium dihydroxymaleate.

The curve defined by X represents the DHM_ system

The curve defined by closed triangles represents the DHMg system

The curve defined by the closed circles represents the DHM^ system

86 9 0

7 0

5 0 pH

3 0

1 0

2 4 6 8 DAYS Fig. 12.— Variation with time of carbohydrate concen tration of systems containing various concentrations of d±- hydroxymaleate•

The curves are defined according to the notations of Figure 11

88 TOTAL SUGAR AS KLETT UNITS

ro oo io co *< *< ro

Fig. 12 68 rate of carbohydrate release from the mycelium into the growth y.- . medium was but slightly less than the rate of carbohydrate uptake. Chromatographic evidence suggested that glucose was the major carbohydrate component of the medium during the incubation and that no unusual sugars appeared during the lag period*

Changes in pH in the DHM^ system are much in accord with previous experiences involving the "normal" Raulin-Thom medium with the exception that the date of positive inflection— the 21st

day in this case— corresponds to a much later phase in the growth

of Ft charlesii than has been noted previously* The curve re

lating the concentration as a function of time of the medium

glucose revealed that a lag occurred after 7 days growth of the

organism* However, in this case the lag is much more pronounced

than was observed in the DHM^ system*

Figure 12 summarizes changes in the carbohydrate concen

tration of the growth media for the dihydroxymaleate series* The

data entered in Table 3 demonstrate the relationship between the

initial concentration of the various ammonium carboxylic acid

salts, the amounts of oligosaccharide synthesized by P* charlesii

and the "efficiency" of oligosaccharide production in the various

experimental systems*

With but two exceptions (FUMg and RTg) the highest yields

of mycelium were obtained in those systems which contained the

highest concentration of medium nitrogen* To the extent that

comparisons of wet-weights of fungal tissue are valid, the re

lationship between initial nitrogen available and mycelium formed TABLE 3 RELATIONSHIP BETWEEN CONCENTRATION OF AMMONIUM DICAHBOXYLIC ACID SALT IN GROWTH MEDIUM AND OLIGOSACCHARIDE PRODUCTION BY P. CHARLESII

Concentration of Ammonium Total Carbo "Efficiency" of Oligosac Salt of Di- Total hydrate Iso charide Synthesis carboxylic Mycelium lated as Units of Oligosaccharide Series Acid Synthesized Hezose Units of Mycelium Designation JU Moles/Ml Grams fA Moles JiMoles/Gm

rt5 43.68 11.0 781.2 7.10 r t 2 14.56 15.0 1382.0 9.21 RTX 4.85 5.2 1302.0 25.04 RT0 14.56 9.4 882.0 9.58

dhm3 43.68 17.0 991.2 5.35 dh m2 14.56 5.6 1260.0 22.50 dhb^ 4.85 5.7 1294.0 34.90

FUM3 43.68 12.1 883 7.13 fum2 14.56 15.7 630 4.01 FUM1 4.85 6.2 772 12,45

ma l3 43.68 11.5 555 4.82 m a l 2 14.56 11.0 1108 10.07 MAI^ 4.85 6.1 1176 19.29 ILLUSTRATIONS— (Continued) Figure Page

25 A comparison of the near ultraviolet of Jx, the distillate of Jx, and authentic pyridine .... 157

26 The ultraviolet and visible absorption spectrum of J y ...... 162

27 The effect of acid on the visible and ultraviolet absorption spectrumo f J y ...... 16k-

28 Variation in carbohydrate and hydrogen ion con centration of growth-medium as a function of age of a culture of P. charlesii...... 170

29 Total carbon-1^ appearing in C0*> as a function of age of P. charlesii • ...... 173

30 Time course of^istribution of carbon-l4 from tartrate-l,*f-C in mycelium, growth medium, and C (>2 of P. c h a r l e s i i ...... 176

31 Plot of specific activity of respired CO* as a function of time of growth of P. > charlesii • . • 179

32 Plot of total activity in and specific activity of respired C"*- 0 * as a function of time of growth of P. charlesii...... 182

33 Variations in carbohydrate concentration of growth medium as function of age of P. charlesii 187

3^ Variation with age of mycelium of carbon-l4 in respired CO^ ...... 190

35 Variation with age of culture of carbon-1^ from tartrate-l,^-Cr in the mycelium and growth- medium of P. charlesii 192

36 Variation with age of mycelium of carbon-1^ distribution in mycelium, growth medium, and C ^2 of P. charlesii ...... 19^

37 'Distribution of carbon-l^f labeled components of "alcohol1! fractions of growth medium of P. charlesii ...... 200

38 Distribution of carbon-1^ labeled components of extracts of 10.5 -day mycelium of P.,charlesii . 203

x 92 probably reflects Increased capacity of Penicillium charlesii for synthesis of nitrogen-containing cellular constituents*

In confirmation of previous observations and some findings to be discussed in the next section, the absolute M amount of oligosaccharides synthesized generally approaches a maximum in that member of a series which corresponds to lowest nitrogen level in that series. Thus in the dihydroxymaleate series the highest absolute yield of oligosaccharides was ob tained in the DHM^ system which contained 4.85 micromoles per ml of diammoniumdihydroxymaleate.

Almost absolute consistency was observed when note was made of the intraseries "efficiency" of oligosaccharide formation.

In every case the ratio synthesized" reached a mn-rimum in the series member which corresponded to the lowest initial level of nitrogen in the growth medium.

The present observations are consistent with the sug gestion that maximal synthesis of oligosaccharides by P. charlesii will be obtained under conditions in which the ratio of initial organic carbon concentration to initial nitrogen concentration, of the growth medium is large. Such a view is not discordant with the concept of "shunt metabolism" which has been discussed by Foster (124). When an organism's available pathways for metabolism of a particular substrate become saturated or con trolled in some other fashion, enzyme systems "shunt" off excess substrate and deposit it in the form of a shunt product. In f this interpretation the substance defined as a shunt product 93 might be formed under conditions in which the primary metabolic pathways are not saturated but the rate of the synthesis and the absolute amount of material formed would be less than at full saturation*

In the present case, if glucose and certain dicarboxylic acids were the saturating substrates and the metabolism of these two types of organic substances were controlled, at least in part, by limiting nitrogen levels Penicillium charlesii would be expected to efficiently synthesize oligosaccharides when the organism is grown on a medium which is rich in organic precursors of galactocarolose but low in nitrogen concentration*

(3) The effect on metabolism of varying concentrations of nitrogen of the growth medium

Studies discussed in the preceding sections indicated

that the amounts of galactocarolose formed by P. charlesii varied

1) When various dicarboxylic acids were substituted

for tartaric acid

2) With the concentration of the ammonium salt of the

dicarboxylic acid when the concentration of the free-

dicarboxylic acid was held constant.

In the latter case the effect may have been a reflection

of the total amount of organic carbon compounds^present in the

growth medium and/or the total amount of nitrogen available for

synthetic processes*

If galactocarolose is produced by the organism as a by

product or as a result of saturation of primary metabolic path- 94- ways or under conditions of limited synthesis (of amino acids, lipids, nucleic acids, proteins) it might be expected that larger quantities of polygalactose would be synthesized by P.

charlesii when grown upon a low nitrogen-high carbon-source medium than one of opposite composition* A significant portion

of the range between these two extremes of growth medium con

centration is treated by experiments and data of this and the

two preceding sections*

The experiments reported in this section involved growth

of P* charlesii on the Raulin-Thom medium the final ammonium ion

concentration of which had been increased by the addition of fixed

amounts of ammonium carbonate* It is to be pointed out that am

monium carbonate does not appear in the "normal" Raulin-Thom

medium but is, in these experiments, added as the source of ad

ditional nitrogen. The carbonate rather than the sulfate or

phosphate salts of ammonia was chosen to avoid any effects which

might be due to increased or decreased sulfate or phosphate con

centrations* Additionally, the carbonate salt was more soluble

under conditions used and the organism is not known to fix sig

nificant amounts of CO^*

The growth medium was further modified to contain one-

third the normal amount of ammonium salt of the dicarboxylic acid

since under these conditions maximal synthesis of galactocarolose

obtained*

The final composition and designation of growth media

employed ere recorded in Table 4-* TABLE 4 DEFINITION OF SYSTEMS EMPLOYED IN STUDY OF OLIGOSACCHARIDE FORMATION BY P. CHARLESII

Final Mean Concentration of Ammonium Concentration Duplicate Dicarboxylic Acid Carbonate of Ammonium Flask Free Acid Nh J Salt Added Ion System Designation Moles/Ml Moles/Ml jA Moles/Ml JU Moles/Ml

0.00 4.86 0.00 ^ 0 9.23 RT1 17.72 4.86 0.00 9.23 rt2 17.72 4.86 4.86 14.09 Tartrate RT? 17.72 4.86 14.59 23.82 t RT4 17.72 4.86 32.00 41.23 RT_ 17.72 4.86 35.60 44.80 17.72 4.86 43.80 »« 53.03 MAI^ 17.72 4.86 0.00 9.23 ma l2 17.72 4.86 4.86 14.09 Malonate MAL^ 17.72 4.86 14.59 23.82 MAL^ 17.72 4.86 32.00 41.23 mal5 17.72 4.86 43.80 53.03

vo VJI TABLE 4— (Continued)

Final Mean Concentration of Ammonium Concentration Duplicate Dicarboxylic Acid Carbonate of Ammonium Flask Free Acid NHj Salt Added Ion System Designation JU Moles/Ml p. Moles/Ml JU Moles/Ml UlMoles/Ml

D H ^ 14.56 0 19.90 dhm2 14.56 4.86 24.76 Dihydroxy DHM, 14.56 14.59 33.52 maleate 3 DHM^ - 14.56 32.00 51.93 DHM_ 14.56 43.80 62.70 5

FUM^ 17.72 4.86 0 9.37 f u m 2 17.72 4.86 4.86 14.09 Fumarate FUMj 17.72 4.86 14.59 23.82 17.72 4.86 32.00 41.37 FUM- 17.72 4.86 43.80 53.03 5

vo ON 97 It was observed that during the first several days of incubation the spores, which were inoculated into the media

containing high concentrations of ammonium ion, failed to pro liferate. In each case the pH of the medium was quite high and

it was suggested that these basic conditions inhibited growth of

P. charlesii. Consequently, on the 5 th day following inoculation

of the medium the pH in duplicate flasks, which contained 53 micromoles per ml or higher concentration of ammonium ion, were

adjusted to pH values (recorded for the 5th day) by aseptic ad

dition of a standard volume of 7»5 normal sulfuric acid. The

addition of sulfuric acid .to the growth media was made with the

assumption that total-metabolism over the 26-day period would

not be adversely affected.

Observations on the metabolic patterns developed.— Con

centrations in the growth medium of hydrogen ion and residual

carbohydrate were measured as described under 'EXPERIMENTAL.'

Plots were made for at least two members of each series and these

plots show the relationship between carbohydrate concentration

and pH of the growth media as a function of time.

"Efficiency" of oligosaccharide synthesis was arbitrarily

defined as the ratio of oligosaccharide to mycelium formed in

units of micromoles of oligosaccharide per gram of mycelium.

Tartaric acid series.— Changes in the growth medium and

gross morphology of the culture are those changes which normally

obtain and these variations were used as standards with which

the fumarate, dihydroxymaleate and malonate series could be compared. With two exceptions (Table 2, lines 2 and 5* column

3 ) there appears to be a gradual increase in total mycelium formed as the concentration of nitrogen of the medium is raised.

The maximum value of the ratio formed is reached mycelium when the mean ammonia concentration is 36.81 moles/ml and this value declines when the indicated concentration is exceeded.

There was also noted a gradual, though hot continuous, increase in oligosaccharide formed at ammonium salt levels which approached the value 3 6 .81,

In the case of the tartaric acid series it was noted that when P. charlesii is cultured in the presence of high levels of nitrogen the slow decrease in pH is not succeeded by a rapid or gradual rise in the pH. This atypical behavior is represented in Figure 13.

Figure l4 affords and provides an analysis of the rates of change of pH and carbohydrate concentration of the medium which represents RTg or RT^ respectively.

Dihydroxymaleic acid series.— It has been noted that when

P. charlesii is grown in the presence of dihydroxymaleic acid, the mycelium did not form the characteristic green covering on

that area which was not in direct contact with the growth medium. ,

The culture was rigid and compact and lost little water upon

expression between layers of gauze or filter paper. The rate of

culture production approximated results obtained when tartaric

acid was present in the "normal" Raulin-Thom medium. Fig. 13*— Atypical behavior of system containing high level of medium nitrogen (ETg!).

The curve defined by closed triangles represents changes in hydrogen ion concentration of the growth medium

The curve defined by closed circles, represents changes in carbohydrate concentration

99 TOTAL SUGAR AS KLETT UNITS IOOO 200 400 500 700 900 300 800 600 100 16

24 90

X Q. 100 Fig* l*f«— Changes in pH and carbohydrate concentration of the medium for the RT,, and RT^ series*

The curve defined by the closed circles represents changes in carbohydrate concentration in the RT^ system

The curve defined by closed squares represents changes in hydrogen ion concentration of the HT^ system

The curve defined by X represents changes in carbohydrate concentration of the RT^ system

The curve defined by closed triangles represents changes in carbohydrate concentration of the RTg system

101 ILLUSTRATIONS— (Continued) Figure Page

39 Distribution of carbon-l4 labeled components of extract of 10-day mycelium of P. charlesii • • 206

40 Distribution of carbon-l4 labeled components of growth-medium concentrate...... • * • • • * 213

41 Chromatogram of 0*4 normal acid hydrolyzate of fraction • • . . . • • • • . . • • • • • • • 216

42 Chromatogram of 3«0 normal acid hydrolyzate of fraction S p ...... * ...... 219

43 Chromatography of fraction S_ in various solvent systems ...... 223

44 Chromatography of fraction S_ in various solvent systems...... 223 l4 ■ 45 Distribution of C labeled components in growth medium concentrate and acid hydrolyzates .... 232 l4 46 Distribution of C labeled components of medium concentrate after chromatography in the Butanol: Pyridine:Water (6:4:3) solvent system •••••. 240

47 Distribution of C ^ labeled components of 0.4 normal sulfuric acid hydrolyzate of fraction S^ • 242 14 48 Distribution of C labeled components of 3.0 normal sulfuric acid hydrolyzate of fraction Sp • 246 14 49 Distribution of C label on chromatograph of medium concentrate when P. charlesii incorporated glucose-u-C-^ in the presence of dihydroxymaleic a c i d ...... 253

50 Distribution of c14 label on chromatograph of 0.4 normal sulfuric, hydrolyzate of ...... 256

51 Distribution of carbon-14.labeled components after hydrolysis of fraction Sp in 3*0 normal sulfuric a c i d ...... 259

52 Chromatogram of fraction T subjected to hydrolysis in 0.2 normal and 0.4 normal sulfuric acid . . . 264

53 Chromatogram of fraction U subjected to hydrolysis in 0,2 normal sulfuric acid and 0,4 normal sulfuric acid ....•••• ...... 266 xi CD UNITS KLETT pH TOTAL SUGAR AS w*

DAYS EOT 103

Maximum "efficiency" of oligosaccharide production resulted when the mean concentration of ammonium ion in the medium was 46.3 micromoles per ml. No marked increase in total mycelial material synthesised was noted at high levels of medium nitrogen (less than 6?.6 micromoles per ml). A direct relation

ship was noted for the relationship between oligosaccharide

synthesized and medium nitrogen when the concentration of the

latter was less than or equivalent to 46.5 micromoles per ml.

In contrast to the other systems and series which were

studied, numerous monosaccharides appeared in the growth-medium

concentrate of the dihydroxymaleate systems. This observation

will be discussed in greater detail in a later section.

Changes in hydrogen ion and carbohydrate concentration

of the DHMg and DHM^ series are represented in Figure 15»

Fumarate Series.--When P. charlesii was grown in the

presence of fumaric acid only slight differences obtained (when

compared to the media containing tartaric acid) in the appear

ance of the medium and gross morphology of the mycelium. In

the presence of high levels of medium nitrogen (mean con

centration of 14 to 4l micromoles per ml of CNH^3+) growth

in the presence of fumarate resulted in a definite stimulation

of mycelium formation but not a more efficient conversion

of the organic carbon supply of the medium to oligosaccharides.

This comparison is afforded by Figures 14 and 16 and also

Table 5« Figure 16 records the observed changes in pH and

carbohydrate composition of the medium as a function of time for Fig, 15*— Changes in hydrogen ion and carbohydrate concentration of the growth medium for the DHM^ and DHM^ series*

The curves defined by closed circles represent changes in hydrogen ion and carbohydrate concentration for the DHMg system

The curves defined by X represent changes in hydrogen ion and carbohydrate concentration for the DHM^ system

Curves representing changes in hydrogen ion concentration are those which demonstrate positive slopes after 17 days of growth of the culture

10*f TOTAL SUGAR AS KLETT UNITS 900 300 500 700 100 Fig. DAYS 3 X

0 2 24 22 20 0 9 50 0 7 0 3 PO X CL VJI O. H Fig* 16.--Changes in pH and carbohydrate concentration of the medium for FUM2 and FUM^ series*

The curves defined by closed circles represent changes in hydrogen ion and carbohydrate concentration in the FUM^ system

Curves defined^by X represent changes in hydrogen ion and carbohydrate concentration of the F0M2 system

Changes in hydrogen ion concentration are reflected by the curves which have positive slopes at 12 days of growth of the culture

106 (0 o o o •>1 o o 01 o pH 04 o o TOTAL SUGAR AS KLETT UNITS o o •> ’* > O to (O to * 09 CO Ci ro O to 0 a > co -< iOT Fig. 1 6 108 two members of the fumarate series. The lag in carbohydrate removal from the medium in the case of the higher ammonium salt concentration may reflect the slow rate of metabolism of P. charlesii (in the early stages of growth) which is often observed when the pH of the medium is high. The rate of carbohydrate up take parallels but lags behind the curve representing change in hydrogen ion concentration for this system.

The key position of fumaric acid in the citric acid cycle, and as a distant precursor of the dicarboxylic alpha-amino acids, the -hydroxy alpha amino acid, and branched-chain amino

acids suggested that this acid, if efficiently transported, might

stimulate formation of cellular structural materials. Fumaric

acid could also "spare" the glucose of the growth medium for

conversion to oligosaccharides and other substances.

Maximum efficiency of oligosaccharide formation in the

presence of fumaric acid appeared to obtain at relatively low

(1^.09 micromoles/ml) levels of ammonium ion. The efficiency

ratio was not appreciably altered when the ammonium salt level t was doubled (Table 5* lines 2 and 3, column 5)» The rapid drop

off of this ratio is not easily explained but this observation

may reflect the greater conversion of the substrates to structural

elements at high ammonium ion concentrations.

Evidence to be presented in a succeeding section, and

which relates to the incorporation of carbon-1^ labeled fumarate

by P. charlesii suggests that this acid is not a good precursor

of galactocarolose. 109

Malonate series.-— Of interest was the observation that < malonic acid present in the medium at a maximum final concen tration of 52.38 micromoles per ml did not retard the growth of

Penicillium charlesii. It is a widely held experience that fungi generally do not metabolize free malonate as a sole source of organic carbon and that this symmetrical acid effects a metabolic lesion at the level of succinic dehydrogenase. That a more marked inhibition of function of P. charlesii by malonate was not observed may result from a limited capacity of the organism to transport malonate to intracellular sites at which the transported quantities would cause complete blockage of the citric acid cycle or some other function. There exists the pos sibility that the two cases in which an apparent "stimulation" in mycelium formation (MALg. and MAl^ as opposed to RTg and RT^) was observed, resulted from a slow down of the citric acid cycle with concomitant build-up of metabolites which precurse celi constituents. The key role played by malonate in fatty acid synthesis supports an hypothesis of malonate utilization in cellular syntheses by P. charlesii.

Figure 17 shows that, after an initial lag, the pH and carbohydrate concentration (in two members of the malonate series) rapidly decreased. In the case of MALg there was a retardation in rate of carbohydrate disappearance between the 8th and 13th days. In the presence of the higher concentration of diammonium malonate the pH minimum was reached only after 13 days of growth.

This was an unusual observation. In most systems studied the pH Fig. I?.— Changes in pH and carbohydrate concentration of the medium for the MALg and MAL^ series.

The curves defined by X represent changes in the pH and carbohydrate concentration of MAL^ system

The curves defined by closed circles represent changes in the pH and carbohydrate concentration of the MAL^ system

The curves representing changes in pH for the two systems are those curves which have positive slopes at l*f days of growth of the culture

110 o Ol pH OJ TOTAL SUGAR AS KLETT UNITS • in o > -< ITT Fig, 17 ILLUS TRATIONS— ( Continued ) Figure Page

54 Distribution of carbon-l4 labeled components of fraction T after chromatography in-an Ethyl Acetate:Pyridine:Water solvent system . . • • • 270

55 Distribution of carbon-l4 labeled components-of fraction TI after chromatography in the organic phase of an Ethyl Acetate:Pyridine:Water solvent system...... • ...... 273

56 Distribution of carbon-l4 labeled components of fraction T after chromatography in 80 per cent phenol...... 275 l4 57 Distribution of C labeled components of fraction U after chromatography in 80 per cent phenol 277

58 Distribution of carbon-l4 labeled components of fraction V after chromatography in 80 per cent phenol...... 282 14 59 Distribution of C labeled components of fraction W after chromatography in 80 per cent phenol ...... • . • ...... • 284 14 60 Distribution of C labeled components of fraction V after chromatography in the organic phase of an Ethyl Acetate:Pyridine:Water (36:10: 11.5) solvent system...... 286 l4 61 Distribution of C labeled components of fraction W after chromatography in the organic phase of an Ethyl Acetate:Pyridine:Water (36:10: 11.5) solvent system...... 288

62 Oxidation of NADH^ by Penicillium charlesii extracts in the presence of dihydroxymaleate (DHM) ...... - 296

63 The effect of volume of added extract on the time-course of the DHM-dependent oxidation of nadh2 ...... 2:99

64 The oxidation of NADH2 by differentially centrifuged fractions of a P. charlesii e x t r a c t ...... T~l 7~...... 302

65 The effect of concentration of dihydroxymaleate on the "initial" rate of oxidation of NADHg . • - 306 xii TABLE 5 OLIGOSACCHARIDE SYNTHESIS BY P. CHARLESII IN THE PRESENCE OF VARIOUS CONCENTRATIONS OF MEDIUM NITROGEN

Efficiency of Mean Total Total Oligosaccharide Concentration Mycelium Carbohydrate Synthesis P. charlesii of Total N Synthesized (as Hexose) JXMoles Oligosaccharide Grown on y Moles/Ml Grams j l Moles Gms Mycelium

14.1 9.21 RTo 6.5 598.9 «Ti 4.9 5.2 286.9 5.52 RT2 14.1 7.6 565.8 7.44 rt3 23 8.3 806.7 9.22 RT^ 36.8 7.0 841.3 12.00 rt5 4-2.1 8.2 577.7 7.17 rt6 53.0 9.3 524.5 ' 5.64

MAI^ 9.3 5.3 281.4 5.12 m a l 2 14.1 6.3 ,346.6 5.50 ma l 3 23.8 7.6 564.2 7.42 MAL^ 36.5 8.1 719.6 8.88 ma l5 53.0 12.4 446.2 3.60 112 TABLE 5— (Continued)

Efficiency of Mean Total Total Oli goeaccharide 0 one ent ration Mycelium Carbohydrate Synthesis P. charlesii of Total N Synthesized (as Hexose) 11 Moles Oligosaccharide Grown on pMoles/Ml Grams p Moles Gras Mycelium

FUM^ 9.32 6.5 191.0 2.94 f d m £ 14*09 7.0 815.4 11.64 FTJMj 28.8 7.2 736.5 10.23 FUM^ 36.8 10.0 868.6 8.69 FUM- 53.0 16.0 271.5 1.69 5

DHM^ 14.1 4.6 435.8 9.46 dhm£ 23.8 7.0 528.6 7.55 DM} 38.4 6.4 560.3 * 8.59 DHM^ 46.5 5.7 786.1 13.80 DBM- 67.6 7.6 443.7 5.83 5 113 114 of the medium has undergone a positive inflection between the

8th and 11th days*

Just as in other series the ratio of oligosaccharide production to mycelium produced reached a maximum when the mean- concentration of nitrogen in the medium was 36*8 ^moles/ml*

Table 6 affords a comparison of the "efficiency" of oligosaccharide formation at fixed levels of nitrogen and in the presence of various dicarboxylic acids* Of particular note from this table is the fact that essentially no differences obtained

(in efficiency of oligosaccharide formation) upon comparison of results from the defined malonate, fumarate, and dihydroxy maleate systems*

The present observations suggest that the absolute level of nitrogen available to proliferating cultures of P* charlesii may markedly affect the amount of oligosaccharides produced by

the organism* The effect directed by the nitrogen level may be

indirect and could reflect greater conversion of substrate to

structural elements of the cell* TJnder conditions of high

nitrogen concentration in the growth medium one consequence

would be the formation of large amounts of mycelium and small

amounts of oligosaccharide* This has been observed in the four

present cases* An interseries comparison of oligosaccharide

synthesis under conditions of high nitrogen concentration is

afforded by Ta&le 7*

Conversely when the nitrogen level was extremely low

there resulted lower amounts of mycelium synthesis and even TABLE 6 INTERSERIES COMPARISON OF "EFFICIENCY" OF OLIGOSACCHARIDE AT DEFINED NITROGEN LEVEL

Efficiency of Mean . Total Total Oligosaccharide P. charlesii Concentration Mycelium Carbohydrate Synthesis Grown on of Total N Synthesized as Hexose 1U Moles Oligosaccharide Medium Conte. ^Moles/Ml Grams ja Moles Gms Mycelium

Tartaric Acid 36.81 7.00 841.34 120.40

Dihydroxy 38.38 6.40 560.25 85.90 maleic acid

Fumaric acid 36.81 10.00 868.65 86.87

Malonic acid 36.81 8.10 719.55 88.80 115 TABLE 7 INTERSERIES COMPARISON OF "EFFICIENCY" OF OLIGOSACCHARIDE FORMATION AT HIGH NITROGEN CONCENTRATION

Mean Total Efficiency of Concentration Total Isolable Oligosaccharide P. charlesii of Total Mycelium Oligosaccharide Formation Grown in Nitrogen Synthesized (as Hexose) 14Moles of Oligosaccharide Presence of jiMoles/Ml Grams ^Moles Gms Mycelium

Tartrate 53.03 9.3 52 4.5 5*64

Fumarate 53.03 16.0 271.5 1.70

Malonate 53.03 12.4 446.2 3.60

Dihydroxy 62.70 7.6 443.7 5.80 maleate 911 117 medial oligosaccharide synthesis under such conditions might give rise to a relatively high ratio of oligosaccharide to mycelium synthesized. Table 8 records a comparison of the "efficiency" of oligosaccharide formation at low nitrogen concentration for four of the systems studied.

(it). The effect on metabolism of the enantiomorphs of tartaric acid

It was suggested by Maynard (7) that only one of the enantiomorphs of tartaric acid may be involved in the formation of oligosaccharide(s) by P. charlesii. Support for this hypothe sis might be gained through the observation of significant stimu lation of oligosaccharide production in the presence of one or

a specific combination of tartaric acid enantiomorphs.

Duplicate flasks were set up which contained the Baulin-

Thom medium with D(-) tartaric acid, L(+) tartaric acid or a DL mixture of tartaric acid as "secondary" carbon source. After

innoculation the cultures were developed over a period of 28

days •

At the end of the 28-day growth period the medium and

mycelium were separated by filtration. The mycelium was washed

thoroughly and the washings added to the main filtrate. The

combined volume was concentrated to 15 ml and treated with three

volumes of mixed (Dowex-l-OH and Dowex-50-H, 1:1) resin and

shaken for 1% hours at 20°C. The mixture was filtered by suction

and the filtrate concentrated to dryness. The residue was dis

solved in a minimum volume of water and stripped upon Whatman

number 3 mm chromatography paper and the dried paper developed TABLE 8 INTERSERIES COMPARISON OF "EFFICIENCY" OF OLIGOSACCHARIDE FORMATION AT LOW NITROGEN CONCENTRATION

Mean Total Efficiency of Concentration Total Oligosaccharide Oligosaccharide P. charlesii of Ammonium Mycelium Synthesized Synthesis Grown in Ion Synthesized (as Hexose) TtMoles of Oligosaccharide Presence of jUMoles/Ml Grams JU Moles Gms Mycelium

Tartrate 14.09 7.6 565.8 7.44

Fumarate 14.09 7.0 815.4 11.64

Malonate 14.09 6.3 346.6 5.51

Dihydroxy 19.90 4.6 435.8 9.46 maleate 118 119 twice in the n-Butanol:Pyridine:Water (6:4:3) solvent Bystem with a single drying between developments.

The material which remained at the starting line was eluted, the eluate concentrated, hydrolyzed with 0.3 normal or

3.0 normal sulfuric acid and the hydrolyzate neutralyzed with solid barium carbonate. The resulting barium sulfate was re moved by centrifugation and the supernatant treated with Amber- lite MB-3 resin, concentrated and chromatographed through two ascensions in the n-Butanol:Pyridine;Water (6:4:3) solvent system. The area which chromatographed as galactose was eluted and the eluate concentrated. The identification of galactose was based upon co-chromatography in two additional solvent systems and a positive response in the Fisher assay for galactose (86).

Observations on the metabolic pattern— D(-) tartaric acid.— Penicillium charlesii developed slowly during the first

5 to 6 days when grown in the presence of D(-) tartaric acid.

The growth medium retained its original pale green-yellow color throughout the 28-day growth period and there was no observable sporulation--as evidenced by formation of a green mat on the top layer of the mycelium. The mycelium did not become compact but remained a large group of segregated colonies throughout the growth period.

The white segregated groupings of mycelium were harvested and after washing pressed three times with sterile gauze and 120 thereafter pressed between four layers of filter paper according to the procedure described under 'EXPERIMENTAL.1 A total of 7*10 grams wet-weight of mycelium was isolated from 300 ml of growth me dims.

L( + ) tartaric acid.— Growth of P. charlesii and changes in morphology in the presence of L(+) tartaric acid closely * paralleled the results obtained in the presence of racemic

DL-tartrate. Observable sporulation began to occur at 6 days and at the end of the 28-day growth period the organism had developed a pronounced green layer on the mycelial felt. The medium was dark red-brown in color at this stage and the appear ances of the mycelium and medium were not different from-the appearance of the mould grown in the presence of"DL-tartrate.

At the end of the 28-day growth period the mycelium was a thick, compact and highly integrated unit. Total wet weight of mycelium obtained from 300 ml of growth medium was 9*2 grams.

DL-tartaric acid.— The mycelium developed normally and sporulation was observed after 5 days of growth. A green layer covering the top side of the mycelium was present after 8 days growth of the mould.. The medium began to assume a character istic red-brown color after 17 days. A total of 9*6 grams wet- weight of mycelium was isolated from 300 ml of growth medium.

Qualitative and quantitative analyses of the carbo hydrates of the residual growth media were performed as outlined above and under 'EXPERIMENTAL.' 121

The data recorded in Table 9 suggest that galactocarolose synthesis occurred in the presence of both optically active isomers of tartaric acid and the absolute amount of galactocarolose formed was highest when the Raulin-Thom medium contained the

L(+) dicarboxylic acid. Although differences were obtained for mycelium and galactocarolose formation when growth of the mould was allowed to occur in the presence of D or L tartrate the differences were not sufficiently large to afford the conclusion that D(-) tartrate is not involved in galactocaroloBe biogenesis*

TABLE 9 PRODUCTION OF GALACTOCAROLOSE BY P. CHARLESII IN THE PRESENCE OF ENANTIOMORPHS OF TARTARIC ACID

Analysis of Carbohydrate Fraction which was Identical with Galactose

Total Sugar Units of Galacto P. charlesii Mycelium Isolated Fraction carolose Grown in Formed (Hexose) as Unit of Mycelium Presence of Grams Moles Galactose ^Moles/Gram

DL-tartrate 9.6 693 1.01 7.22

D(-) tartrate 7.1 777 0.96 10*9^

L(+) tartrate 9.2 113^ 0.95 12.32

The absolute amount of galactocarolose synthesized in

the presence of L(+) tartaric acid is nearly twice the amount

synthesized by P. charlesii when the organism was grown in the

presence of LL tartrate or D(-) tartrate* This observation sug

gests but does not prove that L(+) tartaric acid may be the more

important of the optically active isomers of tartaric acid in the

synthesis of galactocarolose by P. charlesii. \

ILLUSTRATIONS— (Continued) Figure Page

66 The effect of EDTA on the reduction of dihydroxymaleate ...... 309

67 Specificity of the oxidant in the enzyme catalyzed oxidation of NADH^ ...... 31^

68 Oxidation of tartrate by crude extract of P. charlesii ...... 317

69 A hypothetical sequence for conversion of 5-keto- 6-deoxy-L-arabohexose to galactocarolose«... 3^7

70 A hypothetical biosynthetic sequence for 5-keto- 6-deoxy-L-arabohexose— pathway A . • • •• . . . 3^9

71 A hypothetical biosynthetic sequence for 5-keto- 6-deoxy-L-arabohexose— pathway B ...... 351

xiii 122

The question of whether or not P. charlesii preferenti ally converts one or the other of the two optically active forms of tartrate to oligosaccharides might be answered through the use of D or L tartaric acid which is labeled with carbon-1^.

Reciprocal dilution experiments which would involve the metabo- l^j. lism of DL-tartaric acid-1 ,4-C in the presence of various concentrations of L(+) tartrate or D(-) tartrate and experiments which would involve direct observation of the efficiencies of conversion of D(-) tartra.te-1,4— C"^ or L(+) tartrate-1,4-0^* into the oligosaccharides of P. charlesii would yield useful information*

The present unavailability of optically active carbon-1^ labeled tartaric acid precludes a very desirable set of experi ments that might be proposed*

(B) The Metabolism in vivo of Phosp>horylated Compounds by P* charlesii

A large number of carbon-carbon bond forming reactions

(other than carbonylation-decarbonylation processes) in bio logical systems have been shown to involve the participation of activated intermediates* Packer has categorized a number of re actions which lead to C-C bond formation as involving elimi nation of phosphate, elimination of thiol components, elimi nation of water and the action of aldolases (9*0* Carbon-carbon bond synthesis in the anabolism of carbohydrates results pri marily through reactions that are similar or equivalent to aldol condensations. 123

The three most-studied enzymes involved in reversible

C-C bond synthesis in carbohydrate metabolism are aldolase, transaldolase, and transketolase* With few exceptions these three enzymes act exclusively upon phosphorylated organic com pounds* Very few non-phosphorylated acceptor or donor substrates have been shown to participate in reactions catalyzed by transketolase. Among the exceptions are glyceraldehyde and glycol- aldehyde (95)» and formaldehyde (96) which have been Bhown to act as acceptors in the transketolase catalyzed reaction and hydroxyruvic (97) L-Erythrulose (9 8 ) and xylulose (99) have been shown to act as donors in the reaction*

Free fructose is one of the few non-phosphorylated sub strates which serves as a donor in the transaldolase reaction whereas free glyceraldehyde (100) is one of the few non-

? phosphorylated compounds known to act as an acceptor in the transketolase catalyzed process*

In the case of aldolase catalyzed reactions at least one of the substrates is always phosphorylated (101)*

Additionally, nearly all of the well-studied processes leading to the synthesis and interconversion of saccharides have been shown to involve phosphorylated substances intermediate between the initial substrates and final products (102)•

The generalizations quoted above afforded the assumption that phosphorylated substances might be involved in conversion of glucose and tartrate derived fragments to a precursor of the galactofuranoside residues of galactocarolose* . 124-

Experiments were designed to determine the nature of the glucose and tartrate derived compounds into which inorganic orthophosphate was incorporated during various stages of growth

°f Penicillium charlesii« The time intervals chosen coincided with the stages of growth at which changes in pH and total sugar of the medium or sporulation, etc., were most pronounced. At the specified time of growth of P. charlesii the medium and mycelium were examined for the presence of organic phosphate compounds. The mycelium was extracted with perchloric acid and the extract treated according to procedures described under

•EXPERIMENTAL.’ Compounds labeled with P”*2 were qualitatively examined on paper by radioautography and scanning techniques*

The data of Table 10 record the initial isotope con centrations for the various time intervals that were studied* r TABLE 10 P320. -CONTENT OF VARIOUS GROWTH MEDIA USED IN STUDY OF IN VIVO METABOLISM OF PHOSPHORYLATED COMPOUNDS BY P. CHARLESII

Total Activity Time of Incubation Present at Initiation Specific Activity of P* charlesii of Experiment of Orthophosphate Days CPM/M1 CPM/ jmmole Pi

4*5 7i,4oo 29,800 9.0 134,210 67,146 13.5 273,600 136,800 i 8 *o 482,000 241,000

Duplicate flasks were innoculated for each experiment* The

initial specific activity of the orthophosphate of the growth

medium was varied in such a fashion that phosphates isolated at 125 the termination of incubation were of essentially identical specific activity for the four growth periods.

The Raulin-Thom growth medium was modified to contain two-thirds the normal concentration of potassium biphosphate.

The medium contained magnesium chloride and potassium chloride substituted in equimolar amounts for magnesium carbonate and potassium carbonate respectively.

Exposure of P. charlesii to P3^ labeled Raulin-Thom medium for 4-.5 days

The neutralized extract of the mycelium was chromato graphed in the methanol:formic acid; water (8 0 :1 5 *5 ) solvent system. When the developed, dried chromatogram was viewed in the presence of the irradiation of a Mineralight ultraviolet light a broad area of absorption was observed. A scan of a strip of the chromatogram revealed several peaks of radiactivity and several of these areas of radioactivity coincided with regions that reduced ammoniacal silver nitrate and reacted with the periodate-benzidine reagents. Figure 18 represents the various analyses performed on a strip of the chromatogram. The areas which contained P3^ were eluted at *f°C with distilled water; the various eluted fractions were distinguished one from the other by the letters A, £, C, etc. The distribution of label among the various fractions is represented in Table 11.

The analysis of the fractions before and after hydrolysis in 1.0 normal sulfuric acid revealed several different carbo- 32 Fig. 18,— Distribution of P -labeled components of perchloric acid extract of P. charlesii exposed 4.5 days to orthophosphate-P^ .

W denotes areas that reduce ammoniacal silver nitrate.

X denotes areas that react positively in the periodate benzidine procedure.

Y denotes areas that absorb ultraviolet light.

126 w v X

Y lOOOcpm I I ( 1 (■■ 6 4 2 0 ITNE FROM DISTANCE ■ I { (1 I 0 2 4 16 14 12 10 8 3 0 0 0 cpm 0 0 0 3 RGN (INCHES) ORIGIN Fig.18 I • « T f i ' T r 18 _ _ _ _ ~ 20 ~ 128

TABLE 11 RECOVERED ACTIVITY IN ELUATES OF CHROMATOGRAM OF . 4.5-DAY OLD MYCELIUM EXTRACT

Total Volume of Eluate Total Activity in Fraction Fraction Ml CPM x 10-6

4.5 A 7.0 2.280 4.5 B 10.0 8.100 4.5 C 12.0 1.080 4.5 D 17.0 5.916 hydrate residues. Fraction A released glucose and xylose upon hydrolysis whereas fraction B released galactose. Analysis of

fractions C and D was complicated by the presence in these

fractions of numerous free sugars. Chromatographic analysis

suggested that galactose and mannose had been released from

their corresponding -1 - phosphates while ribose and xylose or

lyxose were present before and after hydrolysis aB the free

sugars. It was of interest to note the fact that the concen

tration of the pentoses in the extract was much higher than the

level of hexose.

Analysis of the mycelial extract from P. charlesii exposed to p32o^ for 9 days

Chromatography of the neutralyzed extract from the 9-day

exposed-culture revealed the distribution of labeled com

pounds that is represented in Figure 19. The areas indicated by

the letters A, B, and C were eluted and the eluates concentrated

under reduced pressure at about 15°C. The distribution of radio

activity in the various eluted fractions is indicated in Table 12. Fig. 19.--Distribution of -labeled components of perchloric acid extract of P. charlesii exposed 9*0 days to orthophosphate-p32.

129 10000 cpm A

£ OCL O o o

w rrr X ( Y r i

—| 1 1 1 1 1 • i r I i 1— -| 1 1 1 1 1 —

0 2 4 6 8 10 12 14 16 18 20

DISTANCE FROM ORIGIN . (INCHES'^

Fig. 19 130

\ 131

TABLE 12 RECOVERED ACTIVITY IN ELUATES OF CHROMATOGRAM OF 9-DAY OLD MYCELIUM EXTRACT

Total Volume Total Activity of Fraction in Fraction Fraction Ml CPM x 10“ 6

9 A 13.0 9.750 9 B 14,0 42,840 9 C 13.0 18.720

Under the conditions of the chromatography most of the radio activity present in the neutralyzed extract was immobile and attempts to completely elute the radioactivity in area A were not successful. Less than 13 per cent of the radioactivity on the chromatogram as area A could be eluted under the conditions normally employed. The possibility that fraction A contained an oligosaccharide-phosphate was considered but not proved. When eluted fraction A was' concentrated and a portion of the concen trate made 1.0 normal with respect sulfuric acid and the resulting solution sealed and heated at 95°C for 7 minutes. The cooled hydrolyzate was neutralyzed with solid barium carbonate and the resulting barium sulfate removed by centrifugation. The super natant solution was concentrated and chromatographed. Inorganic orthophosphate, galactose, and arabinose or xylose were released by hydrolysis. Hydrolysis with 0.3 normal sulfuric acid released galactose but little phosphate and no pentose. The small amount of fraction A which could be eluted intact precluded extensive % analyses other than those described. TABLES Table Page

1 Production of mycelium and oligosaccharides by P. charlesii grown in the presence of various. . dicarboxylic acids ..'*••• . 65

2 Concentration of carboxylic acids and ammonium salts present in the Raulin-Thom growth medium • 68

3 Relationship between concentration of ammonium dicarboxylic acid salt in growth medium and oligosaccharide production by P. charlesii . . . 91

k Definition of systems employed in study of oligosaccharide formation by P. charlesii . . • 95

5 Oligosaccharide synthesis by P. charlesii in the presence of various concentrations of medium nitrogen 112

6 Interseries comparison of "efficiency" of oligo saccharide formation at defined nitrogen level . 115

7 Interseries comparison of "efficiency" of oligo saccharide formation at high nitrogen concen tration ...... 116

8 Interseries comparison of "efficiency" of oligo saccharide formation at low nitrogen concen tration ...... 118

9 Production of galactocarolose by P. charlesii in the presence of enantiomorphs of tartaric a c i d ...... 121 32 10 P 0^-content of various growth media used in study of in vivo metabolism of phosphorylated compounds by P. charlesii...... 12*f

11 Recorded activity in eluates of chromatogram of 4.5-day old mycelium extract 128

12 Recovered activity in eluates of chromatogram of 9-day old mycelium extract ...... 131

xiv 132

It should be pointed out that the chromatographic results obtained upon hydrolysis of fraction A could have resulted from the cleavage of an oligosaccharide-phosphate or a combination of a.non-phosphorylated oligosaccharide and a polyphosphate com pound*

Fraction B released glucose upon 1*0 normal acid hydroly sis and no component other than glucose phosphate was present*

Fraction C released galactose upon hydrolysis in 1*0 normal sulfuric acid. Free galactose and ribose were present in this fraction before the hydrolysis.

Both fractions B and C contained relatively large amounts of a component which reduced ammoniacal silver nitrate, reacted only feebly in the periodic acid, benzidine procedure and migrated twice as rapidly as galactose in three solvent systems. This material did not contain phosphate and was not a pentose.

Analysis of mycelial extract from P. charlesii exposed to P* 0^ for 13*5 days

The distribution of radioactivity on a chromatogram of

the neutralyzed extract of 13«5-day o ld mycelium is represented in Figure 20. The indicated areas were eluted from the chromato

gram and concentrated. The distribution of radioactivity in the

various fractions is represented in Table 13*

Fraction A contained two reducing sugars prior to hy

drolysis and yielded six reducing components after treatment

with 1.0 normal sulfuric acid. Mannose, glucose, galactose,

ribose, and xylose were identified by chromatographic procedures. 32 Fig. 20.— Distribution of P -labeled components. of perchloric acid extract of P. charlesii exposed 13*5 days to orthophosphate.

Denotes areas that reduce ammoniacal silver nitrate.

Denotes areas that react with benzidine-periodate reagents.

Denotes areas that absorb ultraviolet light. 1000 CP?4 W Y X ) ( ) ( ( ) ( ITNE RM ORIGIN FROM (INCHES) DISTANCE )( )( Fig. 20 VjJ H •P- 135

TABLE 13 RECOVERED ACTIVITY IN ELUATES OF CHROMATOGRAM OF 13.5-DAY OLD MYCELIUM EXTRACT

Total Volume Total Activity

4 of Eluate in Fraction Fraction Ml CPM x 10"®

15.5 A 11.0 3.168 13.5 B 13.0 12*168 13.5 C lA.O 9.602 13.5 D 12.0 7.1^2

Fraction B contained only one component which contained phosphate and reduced ammoniacal silver nitrate before hydrolysis but seven reducing components were separated from the hydrolyzate as a result of multiple chromatography in the n-Butanol:Pyridine:

Water (6:A:3) solvent system* Glucose, galactose, mannose, xylose, and ribose were identified on the basis of chromatography*

A component which moved twice the distance of a galactose standard was also demonstrated to be present in the hydrolyzate

of fraction B*

Fraction C released glucose and inorganic orthophosphate

-‘Upon hydrolysis* Very significant quantities of the material

with R _ value 2*0 were released* gal Glucose was present as the free sugar in fraction D*

Additional glucose was released upon hydrolysis of fraction D

with 1*0 normal sulfuric acid*

Analysis of mycelial extract from P. charlesii exposed to P* 0^ for 18 days Chromatography of the neutral extract revealed the dis tribution of radioactivity that is represented in figure 21* Fig. 21.— Distribution of P-' -labeled components of perchloric acid extract of P. charlesii exposed 18 days to orthophosphate-P32.

W Denotes areas that reduce ammoniacal silver nitrate.

X Denotes areas that react with benzidine-periodate reagents.

Y Denotes areas that absorb ultraviolet light.

136 V * lOOOcpm I 0 2 ) ) ( ) ( ( 4 ITNE RM TRIG IE (INCHES) LINE STARTING FROMDISTANCE 6 1 012 10 8 Fig. 21 Fig. ) ( ) ( 14 ) ( 16 18 20

USX 138

When the Indicated axeas were eluted the distribution of isotope in the various fractions was as indicated in Table l4.

TABLE 14 RECOVERED ACTIVITY IN ELUATES OF CHROMATOGRAM OF 18-DAY OLD MYCELIUM EXTRACT

Total Volume Total Activity of Eluate in Fraction Fraction Ml CPM x 10-6

18 A 11.0 2.058 18 B 11.0 4.752 18 C 10.0 11.952

When fraction A was chromatographed in the n-Butanol:

Pyridine:Water (6:4:3) solvent system there resulted one very broad -(streaked) area which reacted with ammoniacal silver nitrate. Mannose, galactose, and xylose were present after hydrolysis•

Fraction B appeared to contain galactose before and after hydrolysis; mannose, ribose, and xylose were present after hydrolysis.

Fraction C was a mixture of numerous free sugars. The radioactivity of this fraction was not present as inorganic orthophosphate. Chromatography of fraction C in two other sol- 32 vent systems suggested that the P was associated with a small

or relatively neutral organic compound. The observation of

such a fast moving phosphorylated substance in extracts of P.

charlesii has been made on several occasions but the identity of

of the compound remains unknown. 159

The results of the present experiments afford support for the hypothesis that qualitative changes occur in the dis tribution of phosphorylated compounds present in the mycelium of P. charlesii and that the variations in distribution may be a function of age of the organism. Two unusual phosphorylated substances were extracted from the mycelium of P. charlesii; one of these was immobile during chromatography which has been shown to result in significant migration of aldose and ketose phosphates while the second substance migrates much more rapidly than free galactose.

The particular patterns of distribution of phosphorylated compounds represented in Figures 18 through 21 have essentially duplicated in three independent sets of experiments. The patterns of isotope distribution in extracts seem to suggest that the mycelium of P. charlesii contains significant quantities of low- molecular weight saccharide phosphates (hexose and pentose phos phates) during the first two weeks of growth of the mould and 32 that P added initially as orthophosphate gradually becomes as sociated with more highly polymerized cell constituents*

These experimental results suggested that the question of the nature of phosphorylated conversion products of tartrate which are incorporated into galactocarolose might be more fruit fully approached through modifications of the methods described in the.preceding paragraphs. Experiments involving the addition of P -labeled orthophosphate and carbon-l^f labeled tartaric acid to the growth medium of P. charlesii might afford the production 140 32 l4 and possible isolation of doubly labeled (with P"^ and C ) tartrate derivatives. Such doubly labeled tartrate derivatives might afford a direct means for following tartrate interconver sions that involve phosphorylated species as intermediates. The absolute desirability of such studies cannot be overemphasized.

Experiments of the type described might be made to yield ad ditional useful information related to tartrate metabolism if these experiments were conducted with specifically labeled tar- 14 14 trate (tartrate-1,4-C and tartrate-2,3-C ).

Additionally, experiences with paper chromatography have suggested that other methods are desired for the resolution of

extracts'of P. charlesii. Column chromatography of such extracts would perhaps meet the demands for increased capacity of reso

lution and greater reproducibility.

The isolation and partial characterization of Jx

Penicillium charlesii was grown for 4.5 days on a modi

fied Raulin-Thom medium which contained magnesium chloride and

potassium chloride substituted in equimolar quantities for mag

nesium carbonate and potassium carbonate respectively. The

growth medium was further modified to contain 66 per cent of

the amount of diammonium-hydrogen phosphate reported in early

studies which involved use of the Raulin-Thom medium (105).

Orthophosphate containing phosphorous-32 to a final isotope 141 concentration of 29,800 counts per minute per micromole was added to the growth medium as a means of facilitating the iso lation of phosphorylated compounds of the mycelium of P. charlesii*

At the end of the 4.5-day growth period the mycelium and

growth medium were separated by filtration and decantation. The mycelium was washed with six 30-ml volumes of double distilled water. The washed mycelium was extracted with 10 per cent per

chloric acid and the resulting extract neutralyzed with barium

carbonate. After removal of salts the extract was concentrated

according to procedures (defined for acid extraction of Penicil

lium charlesii) as defined under ’EXPERIMENTAL.* The concen

trated extract was stripped upon Whatman number 5 MM chroma

tography paper and the paper dried overnight at 22°C. The

chromatogram was then developed in the Methanol:Formic Acid:

Water (80:15:5) solvent system. At 13 hours the solvent front

had moved 19 inches and examination of the dried chromatogram

under ultraviolet light revealed the presence of an ultraviolet

light absorbing band which was bounded by the solvent front and

a point approximately 9 inches removed from the starting line.

The location of the U.V. absorbing area, with respect to the

phosphorylated components of the extract, is represented in

Figure 23.

The ultraviolet light absorbing band was cut out and

eluted exhaustively with cold distilled water. The eluate was

concentrated under reduced pressure at 20°C. Crystals began to

form when the volume of the eluate in the flask, used for concen- TABLES— ( C on tinue d ) Table Page

15 Recovered activity in eluates of chromatogram of 13.5-day old mycelium extract ...... 135

l^f Recovered activity in eluates of chromatogram of 18-day old mycelium extract ...... 138

15 Microanalytical elemental analysis of Jx • . . • 1^5

16 Infrared absorption of compound Jx ...... 152

17 Infrared absorption ofJy ...... 166

18 Distribution of carbon-l4 label in various "alcohol fractions" of Raulin-Thom growth medium ...... 196

19 Distribution of carbon-l4 in perchloric acid extracts of P. charlesii ...... 199

20 Distribution of carbon-14 in chromatographically resolved components of "alcohol" fractions of the growth m e d i u m ...... 199

21 Distribution of carbon-l4 in chromatographically resolved components of cold PCA extract of mycelium of P. charlesii-...... 202

22 Incorporation of various carbon-l^f labeled carboxylic acids by P. charlesii • ...... 210

23 Distribution of carbon-l*+ in various fractions derived from study of incorporation of tartrate- 1, it-c1^ in the presence of tartronate...... 215

2k Carbon-l4 and carbohydrate content of concen trated eluates of fractions and A ...... 221

25 Radioactivity-recovered in mycelium, residual ‘ growth medium, and sodium carbonate when P. charlesii metabolized glucose-u-Cr-^ in the presence of tartronate ••••...... 231

26 Carbon-l4 content and carbohydrate analyses of fractions A and S ^ ...... 23^

27 Specific activity of initial glucose and galactose isolated from galactocarolose .... 235

xv 1^2 tration, had been reduced by two-thirds. The precipitate here after referred to as Jx, was removed and washed with cold dis tilled water. The water washings were concentrated under reduced pressure and the concentrate kept at 4°C for 12 hours. The crys.tals which formed in the refrigerated concentrate were re moved by suction filtration and these crystals washed with

chilled (*f°C) 90 per cent aqueous ethanol. The washed crystals were added to the initial fraction of Jx. Two recrystallizations

from distilled water yielded white.needles which melted at 272-

277°C with decomposition. Two recrystallizations from absolute

ethanol yielded granular crystals that melted at 272-27^-°C with

decomposition.

(a) Solubility properties.--The white crystals were in

soluble in benzene, collidine, ethylacetate, diethylether,

petroleum ether, dimethylcellosolve, chloroform, and carbon

tetrachloride. Jx wan slightly soluble in 50 per cent methanol,

absolute ethanol, and water at 2*f°C. Jx was very soluble in

water at 50°C and higher temperatures.

(b) Crystalline form and melting point.™When repeatedly

crystallized from water, Jx yielded white needles which melted

at 27^— 277°C. Crystallization from absolute ethanol or 80 per * cent ethanol yielded granular ■plates. When recrystallization

was effected from 50 per cent methanol, Jx appeared white and

granular in appearance and melted at 273-277°C. Considerable

darkening preceded melting in all cases examined.

group tests suggested the absence of amino, amido hydroxyl, 143 aldehydic, oxo, thiol, ester, methoxy, methyl, and ether func tions. Isolated unsaturated functions were absent and the near ultraviolet absorption spectrum suggested that Jx contained a system of conjugated double bonds. Solubility and alkali titra tion data suggested that Jx contained a carboxyl group* A positive reaction with cyanogen bromide in a modification of the original Konig test (106) suggested the presence of a heterocyclic ring system. Under conditions of the Konig reaction Jx, in the presence of thiobarbituric acid, formed a rose-pink compound which was not isolated. Several pyridine carboxylic acids also formed pink products under the conditions of the test.

Jx rapidly absorbed bromine from bromine water but did not react with bromine in carbon tetrachloride. The brick red precipitate, which formed when Jx was treated with bromine water, was not isolated. Pyridine has been reported to undergp reaction in the presence of bromine water to form an isolable pyridine bromide (107). It has been suggested (108) that the compound

isolated by Grimaux (107) was identical with the hydrated per- bromide salt, C,_H,-N»Br *HBr*2H_0, which was described more 5 5 2 2 thoroughly by Trowbridge and Diehl (109).

Present experiences have shown that a large number of

N-heterocyclic aromatic compounds undergo bromine addition.

Pyridine and many of its derivatives react in bromine water to

form products that vary in color from a deep yellow-orange for

pyridine to a deep red for isonicotinylazide.

Microanalytical elemental analyses were performed by the

Schwarzkopf Microanalytical Laboratories of Woodside, New York. Ikk

These analyses confirmed the group analysis for the carboxylate function and also suggested the presence of halogen, as chloride, in the compound, Jx. The analytical data did not distinguish the chloro function as covalently bound to carbon or as a com ponent of the anionic portion of an acid-base conjugate. This latter possibility might be reflected in or represented by a perchlorate salt of an acid. The microanalytical data obtained from Jx are recorded in Table 15*

These data supported the previously made hypothesis that

Jx was unsaturated. Additionally, it was observed that Jx under went decomposition in the presence of concentrated base and this decomposition was accompanied by the release of a vapor which contained an odor similar to that of pyridine. Decomposition of

Jx by fusion with anhydrous barium hydroxide at elevated tem peratures afforded a gas which could be trapped in water or dilute acid. The trapped gas demonstrated a near ultraviolet absorption spectrum which was similar to the spectrum obtained for authentic pyridine. A comparison of these spectra is af forded by Figure 23.

Spectrophotometric studies

(1) Light absorption in the near-ultraviolet region of

the spectrum.— The absorption spectrum of Jx demonstrates a maxi mum at 258 millimicrons with secondary peaks of high extinction

at 250 and 262 millimicrons. The wavelengths of maximum ab

sorption of these three peaks were not significantly altered

upon addition of acid or base. Acidification of a solution of 145

TABLE 15 MICROANALYTICAL ELEMENTAL ANALYSIS OF Jx

Analysis Found in Test Performed (Percentage)

C 32.930 H 3.145 N 7.680 0 36.065 P 0.000 s Trace Br 0.00 I 0.00 Cl 19.710

Total 99.53 C00H 24.73 -OH 0.00

Jx effected a broadening of the peaks in the 250 and 262 milli micron region and this result was reversed by base. Solutions of Jx in aqueous methanol or ethanol demonstrated increased sharpness of the peaks at 250, 2581 and 262 millimicrons. Ad dition of mineral acids to the alcoholic solutions of Jx caused a broadening of the peaks and an increase in extinction.

Figure 23 records the spectrum of Jx in a solution of

50 mg per liter concentration which had been treated with hydro

chloric acid. The ultraviolet absorption spectrum in this

figure and all others appearing in this section were obtained with a Beckman model DB recording spectrophotometer. Figure 23

also demonstrates the spectrum of Jx in 50 per cent aqueous 52 Figure 22.— Distribution of P -labeled and UV-light absorbing components of perchloric acid extract of P. charlesii exposed 4.5 days to orthophosphate-p22#

Sn denotes areas that reduce ammoniacal silver nitrate.

Bz denotes areas that react with benzidine-periodate reagents.

UV denotes areas that absorb ultraviolet light.

146 E oo. O O O

2 4 6 8 10 12 14 1816 22 24 DISTANCE FROM ORIGIN (INCHES)

UV ( )

SN BZ Y') k W '/ / / / '/ / V / / / )

F ig .. 22

H •P- Fig. 23.— Comparison of the near ultraviolet spectra of various solutions of Jx.

•Curve A Jx at pH 4.5 or pH 1.8.

Curve B Jx in 50 per cent methanol.

Curve C Jx at pH 9*5*

148 OPTICAL DENSITY 0.2 0.8 0.4 0.6 2.0 270 260 AEEGH (m;j)WAVELENGTH 250 fig. 23 fig. 3 220 230 210 1$0 methanol. Curve C of Figure 25 shows that the presence of base

effects a sharpening of the three distinguishable peaks and a concomitant lowering of the extinctions of these.

Although dilute sodium hydroxide or potassium hydroxide

effected only minor shifts in the wavelengths of maximum ab

sorption of Jx both bases were found to cause a pronounced

lowering of the extinction of the 250, 258, and 262 peaks. Ad

ditionally the presence of dilute base appeared to effect a

sharpening of the three distinguishable peaks of Jx. Acidifi

cation of a solution of Jx, on the other hand, caused a broad

ening of these three peaks and an increase in the extinction.

The ultraviolet absorption spectrum of pyridine was

studied by Kasha (111) who noted a marked solvent effect on the

resolution of peaks observed at 250, 258, and 262 millimicrons.

The author suggested that the three peaks in the 2500 to 2900&

region resulted from low energy non-bonding to pi-star (n -- >TT*)

transitions for the non-bonded electrons of nitrogen of the

pyridine nucleus.

The experimental data, in the present case, argue for

the presence of a pyridine nucleus in Jx. The loss of resolution

of the 250, 258, and 262 peaks of Jx when an aqueous solution of

the latter was acidified may have reflected a shift out-of-range

of the n >TT * bands or alternatively a shift of U --

bands which then obscured the observation of the n — M r *

transitions. These explanations for the effect.;of acids on the

near ultraviolet absorption spectra of certain n-heterocyclia 151 aromatic compounds were advanced by Kasha (ill). The addition of base to a slightly acidic solution of Jx probably results in de-protonation of the aromatic nitrogen hypothesized to be present in Jx. The deprotonation might also be effected by certain hydroxylic solvents such as methanol or ethanol. De protonation of the heterocyclic nitrogen atom might give rise to an increase in the population of species with non-bonding electrons available for excitation.

(2) Spectrophotometric studies of Jx in the visible region of the spectrum.— When analyzed with the Beckman model

DB recording spectrophotometer Jx appeared to be transparent to light of wavelengths between 280 and 550 millimicrons.

(5) Infrared spectrophotometric studies on Jx.— Infrared spectra were obtained with potassium bromide pellets or with chloroform or carbon tetrachloride and sodium chloride optics.

The instrument used was a Beckman IR-*fA.

Examination of the infrared spectrum of Jx revealed numerous well defined and severe!, broad peaks of absorption.

Table 16 records the relative intensities and locations of bands observed in the infrared spectrum of Jx.

The physical and chemical properties of a large number of

H-heterocyclic aromatic compounds were compared to the properties of Jx but the results have not contributed significantly to a

definition of the molecular structure of Jx. Certain similari

ties in the ultraviolet and infrared absorption spectra of Jx

and a wide variety of compounds suggests that Jx contains a pyridine nucleus. TABLES— (Continued) Table Page

28 Effect of the nature of carboxylic acid "carrier" on isotope distribution from various carbon-14 precursors ...... 237

29 Eadioactivity recovered in mycelium, residual growth medium, and sodium carbonate when P. charlesii metabolized glucose-u-C in the presence of tartrate ...... 238

30 Carbon-l4 content and results of carbohydrate analyses of fractions A and ...... 244

31 Carbohydrate analyses and carbon-l4 content of fractions S, and A „ ...... 245 5 d 14 32 Distribution of carbon-l4 of glucose-u-C in the mycelium, growth medium, and CO. produced by P. c h a r l e B i i ...... 249

33 Distribution of carbon-l4 recovered when P. charlesii was allowed to incorporate glucose-u- G&- in the presence of dihydroxymaleic acid . • 252

34 Preliminary analyses of fractions A and B derived from hydrolysis of ...... 258

35 Distribution of carbon-l4 label and results of carbohydrate analyses of fractions T and U • . . 26l

36 Distribution of carbon-l4 label and results of carbohydrate analyses of fractions A^ and • • 262

37 Distribution of carbon-14 label and results of carbohydrate analyses of fractions resulting from hydrolysis of fractions T and U ...... 268

38 Distribution of carbon-l4 label in chromatographically resolved components of fraction T • 269

39 Distribution of carbon-l4 label in chromatographically resolved components of fraction U . 272 v * 40 Distribution of carbon-l4 label and results of carbohydrate analyses of fractions V and W . . • 280

41 Carbohydrate analyses of fractions V and W . . . 28l

42 Distribution of carbon-l4 label in- chromato- graphically resolved components of fractions V and W ...... 290 xvi 152

TABLE 16 INFRARED ABSORPTION OF COMPOUND Jx

Wavelength Nature of Inflection (microns) (relative intensity) o -p o . k .o very strong, broad ^•9 weak 6.1 to 6.2 (doublet) very strong, narrow 6.5 very strong, narrow 6.7 very strong, narrow 7.25 weak 7.^5 weak

8.00 f weak 8.2 to, 10.5 very strong, very broad 13.3 very strong, broad l*t.7 very strong, broad

Chromatography of Jx

Jx was subjected to column chromatography and paper chromatography. The solvent systems used in paper chromatography included an Ethanol:n-Butanol:2 per cent aqueous Ammonia (2:^:1) system and the two systems described under 'EXPERIMENTAL* for the paper chromatography of heterocyclic basis and acids. The R^ values obtained in the acidic solvent systems reflected the presence in Jx of an acidic function. Jx migrated with con siderable streaking in the solvent systems which were employed.

Column chromatography of Jx on Dowex-l-formate was con ducted according to Hurlbert and co-workers (110). A 1 x 15 cm column containing thoroughly washed Dowex-l-formate was employed.

A solution of 80 milligrams of Jx in 2.0 ml of distilled water 153 was adjusted to pH 8.2 before being applied to the top of the

column was developed by gradient elution using 1.0 molar am monium formate and 2.0 molar ammonium formate successively added

to a mixing flask maintained at 500 ml total volume.

An automatic fraction collector, provided for the accurate

collection of 10 ml fractions from the column. A total of 150

tubes containing 10 ml fractions were collected. The first ^5

tubes resulted from development of the column with distilled

'' r water. Tubes 45 through 90 were collected with increasing

gradient to 1.0 molar ammonium formate. Tubes 90 through 150

were collected with increasing gradient to 2.0 molar ammonium

formate.

Jx was found to be very weakly bound to the column of

Dowex-l-formate. The pattern for elution of Jx is represented

in Figure 2 k , No ultraviolet light absorbing material emerged

from the column beyond tube 17. Essentially all of the Jx ap-

plied to the column was collected in tubes 1 to 17*

A relatively large number of derivatives of pyridine

were synthesized or obtained commercially and some of the pro

perties of these compounds .were compared to certain properties

of Jx. These and other experimental efforts have failed to

fully define the structure of Jx.

Microanalytical data suggest that Jx may be described by

the empirical formula (C^HgN0^Cl)n . The neutralization equivalent

of Jx was shown to be 183 and if this neutralization equivalent

approximates the molecular weight of Jx the value of n in the Fig* 2k,— Column chromatography of Jx.

15^ OPTICAL DENSITY at 260m;i 40 30 20 UE NUMBERTUBE Fig. 2* Fig. 22 26 34 38 150 156 empirical formula must be unity. The empirical formula

Cj-HgNO^Cl corresponds to a molecular weight of 191. The molecu lar formula C^HgNO^Cl might represent the hydroperchlorate of pyridine but that Jx is not pyridine hydroperchlorate was sug gested by the fact that 2k per cent of the molecular weight of

Jx is accounted for by the carboxyl group (see Table 16). The perchlorates of the pyridine monocarboxylic acids were prepared and their physical and chemical properties compared to Jx. The anhydrous monoperchlorates of pyridine-monocarboxylic acids can be assigned the empirical formula C^HgNOgCl which corresponds to a molecular weight of 223. This latter fact and other properties of the pyridine monocarboxylic acid hydroperchlorates eliminated

the perchlorates as possible structural equivalents of Jx.

Additionally, all of the pyridine mono and di carboxylic acids were obtained and their properties compared to Jx. These mono and di carboxy derivatives of pyridine were converted to

the corresponding methyl and/or ethyl esters and amides but

neither the parent free-acids nor the derivatives was chemically

equivalent to Jx. The 2 and the 6-Pyridones of trigonolline

were synthesised and shown to be chemically different from Jx.

When placed in a platinum crucible and the crucible

heated in the flame of a Bunsen burner, Jx underwent combustion

in a mildly explosive manner and no detectable ash or residue was

observed. This observation and the microanalytical data sug

gested that Jx cannot contain significant amounts of non-volatile

material. Fig. 25.--A comparison of the near ultraviolet spectra of Jx, the distillate of Jx and authentic pyridine.

Curve A Jx distillate acidified.

Curve B Jx distillate.

Curve C Jx.

Durve D Pyridine.

157 OPTICAL DENSITY 0.2 0.3 0.4 0.5 0.7 0.6 0.8 2.0 0.9 280 0 7 2 6 5.230 250. 260 WAVELENGTH(mjj) 8 £ 8 . g M 240 220 159

The complete assignment of a molecular structure and metabolic function for Jx must await future research efforts#

Studies on other isolable compounds found in extracts of Penicillium charlesii

(1) Isolation and partial characterization of Jy.--When

P. charlesii was grown for 10 days on the modified Raulin-Thom medium described at the beginning of this section and extracted with 5 Per cent perchloric acid as outlined under ’EXPERIMENTAL' the neutralyzed extract was observed to be yellow-green in color.

The concentrated extract was chromatographed in the Methanol:

Formic Acid:Water (80:15:5» V/V/V) solvent system at 23°C. The developed, dried chromatogram was observed under ultraviolet light to contain a fluorescent band whose midpoint corresponded

to an Rj. of O.bj and a dark yellow-red ultraviolet-light ab

sorbing band the midpoint of which corresponded to an R^ value of 0*78* Both the R^, O.A-3 and the R^ 0.78 band were eluted and

the eluates concentrated under reduced pressure. Negligible

residue was obtained upon concentration to dryness of the R^

0.^3 fraction and this material was not further investigated.

The R^ 0.78 band yielded a yellow granular powder upon concen

tration to dryness. The yellow material was redissolved in water

and the solution concentrated to dryness. The residue was

crystallized from 60 per cent ethanol at *f°C and the amorphous

powder which resulted was washed with benzene and diethyl ether.

The washed powder was stored in vacuo over calcium sulfate. The

dry powder melted at 198-202°C. 160

The yellow material is hereafter referred to as Jy. Jy was not soluble in benzene, chloroform, absolute ethanol, abso lute methanol, diethyl ether, petroleum ether, chloroform, nitro benzene, carbon tetrachloride, dimethyl cellosolve, or toluene#

Jy was readily soluble in dilute hydrochloric, sulfuric, or acetic acid#

That a total of only O.385 grams of Jy was available precluded many of the tests that were performed in partial

characterization of Jx.

Functional group analyses suggested the presence of a

carboxyl group, a quinoid or nitro function, at least one isolated

double bond which was reactive to neutral permanganate and a

carbonyl function which underwent rapid reaction with 2,^-Dinitro-

phenylhydrazine to yield an orange-red precipitate. When treated

with 10 to 12 drops of 6 normal potassium hydroxide a dilute

solution of 1.0 ml volume of Jy becomes green in color and this

base-induced change in color was not completely reversed upon

acidification with concentrated hydrochloric acid.

Jy migrates slightly more rapidly than does Jx in the

Methanol:Formic Acid:Water (80:15:5» V/V/V) solvent system and

significantly more rapidly than Jx in the solvent systems de

scribed for heterocyclic compounds under ’EXPERIMENTAL.' Jy was

not subjected to column chromatography.

Jy was found to be strongly absorbing at and below 25^

millimicrons and the brilliant yellow color of the compound sug

gested that it should absorb light in the visible region of the l6l spectrum. Figure 26 represents the spectrum of Jy in the *f60 to 300 millimicron region and the effect on this spectrum of traces of mineral acid. Figure 27 demonstrates the 460 to 200 millimicron spectrum of Jy and the effect of increasing acid concentration. The maximum in the visible portion of the spectrum shifts from 358 millimicrons to 3 ^ millimicrons for the highest concentration of acid present (curve 7) while the minimum between 260 and 320 millimicrons experienced a blue shift and an increased extinction.

The infrared spectrum of Jy was essentially identical to that for Jx. The peaks obtained for Jy are recorded in

Table 17.

The similarities in their infrared spectra and other physical properties suggested that Jx and Jy might be inter related metabolites of P. charlesii. The conditions and pro cedures used in isolation of the two compounds were essentially identical. That so little data is available which might be used to establish molecular structures of these two compounds precludes the advancement of dogma which relate their in vivo function and significance.

(2) Isolation and partial characterization of Jw.~When

10-day old Penicillium was subjected to direct extraction with

95 Per cent ethanol as described under 'EXPERIMENTAL' the re sulting extract was brown in nature. Concentration to dryness of the alcoholic extract yielded a syrup-like residue which was dissolved in a minimum amount of water and precipitated by the TABLES— (Continued ) Table Page

43 Comparison of carbohydrate composition of fractions T, U, V, and W from chromatography of dihydroxymaleate-supplemented Raulin-Thom medium...... • ...... 291 /j 44 Comparison of specific activity of glucose-u-C at initiation of experiment and galactose isolated from galactocarolose. 292

^5 Demonstration of the dihydroxymaleat® dependent oxidation of NADH_~definition of the system employed ...... 295

46 Absorption properties and protein-content of fractions of extract of P. charlesii ...... 3^4

xvii Fig. 26.— The ultraviolet and visible absorption spectrum of Jy.

A Solution of Jy in distilled water,

B Aqueous solution of Jy made 0.10 molar with respect to sodium hydroxide.

C Aqueous solution of Jy made 0.20 molar with respect to hydrochloric acid.

162 OPTICAL DENSITY 20 0 0-4 0-3' * 2 • 400 440 420 4 0 0 300 300 *340 320 *340 320 300 300 0 0 4 420 440 400 - « . i . — . « i AEEGH N MILLIMICRONS IN WAVELENGTH . i# 26Fig# _ ..i . ... - ; _»i . i ■ « ; ■ i , i vO d 0 20 4 2*20200 240 2^0 200 Fig. 27•--The effect of acid on the visible and ultra violet absorption spectrum of Jy.

Final Concentration of HC1 Curve Designation Normality

A 0.00 B 0.05 C 0.10 D 0.27 E 0.4l F 0.54 G 0.67

164 OPTICAL DENSITY -9 0 0-8 2 -7 0 0-2 0-6 0-4 0-5

0 0 6 4 0 2 4 WAVELENGTH ( ( WAVELENGTH 0 8 3 . f f t f 0 4 3 27 li cr ) s n ro ic illim m 0 0 3

260 220 200 166

TABLE 17 INFRARED ABSORPTION OF Jy

Wavelength Microns Absorption Characteristics

2.9 to 3.2 strong, broad 3.45 medium, narrow 6.35 strong, narrow 6.4 medium, medium 6.55 weak, narrow 6.72 strong, narrow 7.20 weak, broad 7.50 weak 7.95 strong, medium 8.10 weak, medium 8.6 to 8.8 medium, broad 9.0 to 10.0 very strong, very broad, split at 9*4 microns 10.5 weak, medium 12.3 to 12.5 medium, broad 13.45 strong, medium 14.60 strong, medium 15.05 medium, broad addition of absolute ethanol to 90 per cent. One to 2 grams of the sticky precipitate, were obtained from a total of about 25 grams pressed (wet) weight of P. charlesii. The material was designated Jw. Jw was insoluble in anhydrous organic solvents and slightly soluble in cold water. Jw was readily solubilized by water above 40°C.

Functional group tests indicated the absence of isolated unsaturation, methyl groups, amido, amino, sulfhydryl, carboxylate, nitroso, quinoid, and phenolic hydroly groups. Jw reacted 167

rapidly with 2,4 dinitrophenylhydrazine in the cold to yield an

orange-red precipitate. The sticky Jw slowly decolorizes bromine water in the cold (4°C).

Paper chromatographic studies revealed that Jw was not

mobile in the n-Butanol:Pyridine:Water (6:4:3) solvent system

or the Methanol:Formic AcidiWater (80:15:5) solvent system, Jw

reduced ammoniacal silver nitrate after chromatography of the

former and Jw responded positively to the Nelson procedure for

reducing sugar.

An aqueous solution containing 2 mg/ml of Jw was trans

parent in the near ultraviolet and visible regions of the 4 ' spectrum and the infrared spectrum suggested that Jw was highly

hydrated. Attempts to dehydrate Jw by desiccation over

were not successful,

(D) Studies On the Time-Course of Utilization of Tartaric Acid-1,4-C^ by P, charlesii

The observation that tartaric acid is incorporated into

galactocarolose by P. charlesii (5) and almost negligibly into

the tetronic acids elaborated by this mould (23) suggested that

tartaric acid utilization by P, charlesii might provide a con

venient vehicle through which the course of production of

galactocarolose might be studied. Additionally, such a study

might provide some insight into the role of this dicarboxylic

acid in general metabolism of P. charlesii. If tartaric acid

functions as a "secondary" carbon source it might be suggested 168 that tartrate is actively metabolized only after the "primary" organic carbon source, glucose, has been utilized.

An experiment was designed to determine the rate of up take of tartrate from the growth medium and the rate at which carbons 1 and 4- of tartrate are metabolized to carbon dioxide by Penicillium charlesii.

A 'faetabolic train" was set up according to Figure 2 under 'EXPERIMENTAL.' The design of the apparatus was such that the following obtain:

(1) Growing mycelium in the "closed" system was supplied

with a continuous source of "CO^-free" air.

(2) Respired carbon dioxide could be trapped and

measured quantitatively.

(3) Changes in the relative concentrations of medium

metabolites could be measured sensitively.

C^f) Conditions differed little if any from normal

circumstances of surface growth of P. charlesii.

The incubation flask contained 100 ml of the Raulin-Thom l^f growth medium to which was added tartaric acid-1,k-C to a

final isotope concentration of about 700,000 cgn/ml. Carbon-14

labeled carbon dioxide was trapped in sodium hydroxide as de

scribed under 'EXPERIMENTAL.' Aliquots removed at regular inter

vals from the sodium hydroxide trap were counted as suspensions

in a tricarb liquid scintillation counter. The solvent-phosphor

system and counting techniques for solutions of sodium carbonate

were described under 'EXPERIMENTAL.' Standard volumes of 169 l*f Na_C 0_ were converted to BaC Ik 0, for specific activity

determinations.

Aliquots removed from the growth medium were subjected

to the following analyses:’

(1) Total sugar.

(2) Total hexose.

(3) Residual glucose.

(A-) Residual radioactivity.

(5) Hydrogen ion concentration.

Additionally, chromatographic analyses were performed

which employed the solvent systems Ethylacetate:Pyridine:Water

(36:10:11.5)» n-Butyric acid:n-Butanol:Water (2:2:1), n-Amyl-

alcohol:5 Molar aqueous Formic Acid (1:1), 70 per cent aqueous

phenol, and Methanol:Formic Acid:Water (80:15:5).

Variation of total sugar, total hexose, glucose, and hydrogen ion concentrations as a function of age of P. charlesii

When a plot was obtained for the variation with time of

the total sugar, hexose, and glucose in the growth medium the

regularly observed pattern shown in Figure 28 was observed.

This statement also applies to changes in pH of the medium data

corresponding to which are also plotted in the figure.

The curves representing the various carbohydrate analyses

axe superimposable for the first 17 days of growth of the mould.

This suggested that glucose was the major carbohydrate present

during this period. Material which was immobile in the acid and

basic solvent systems used appeared after 5 days of incubation. Fig. 2.8.— Variation of carbohydrate and hydrogen ion concentration of the growth medium as a function of age of P. charlesii.

Curve Designation

The open circles represent total sugar.

The triangles represent glucose concentration.

The designation X represents changes in reducing sugar.

Closed circles represent changes in pH.

170 001 o co MEDIUM 03 m g r o w t h

X in "P p r e s e n t

(PER CENT OF IN IT IA L ) carbohydrate c> ■ ro ro -I 01 Ol CD CJI ro

TIME OF GROWTJH (DAYS) u x Fie. 28 INTRODUCTION

Penicillium charlesii Q. Smith, isolated from mouldy

Italian maize, was shown to produce several heterocyclic carboxylic acids and two unique oligosaccharides when the organism was grown on the Raulin-Thom medium or the Czapek-Dox medium Cl).

The molecular structures of the carboxylic acids (2) and .the oligosaccharides C3* *0 were subsequently reported. One of the oligosaccharides, mannocarolose, was shown to be a homogeneous polymannoside which contained 8 to 9 mannopyranose units -linked alpha-1,6. The second oligosaccharide was given the name galacto-

carolose and shown to consist exclusively of galactofuranose units linked beta-1,1?.

Interest in galactocarolose is a reflection of the unusual

structure and mutual linkages of the galactofuranose units. Five

additional, well-documented reports of the occurrence in nature

of galactofuranose in glycosidic linkage have appeared and in all,

cases the particular glycoside was non-homogeneous. These natu

rally occurring galactofuranosides include the galactomannan of

Trichophyton granulosum (126), the acidic extracellular polysac

charide of Gibberella fu.likuroi (127), the complex polysaccharide

of Cladophora rupestris (l8), and umbilicin the unusual galacto-

furanoside found in certain lichens (17)*

1 172

This immobile material reduced ammoniacal silver nitrate and reacted faintly with the periodate-benzidine reagentB. The concentration of this material increased with age of the mycelium however, its absolute amount appeared to be much lower than glucose over the first 15 to 17 days. The chromatographic procedures and subsequent visualization were not sufficiently sensitive to afford quantitation of the relationship between the glucose concentration and the concentration of the component of the growth medium which was immobile under the conditions of chromatography.

Variation of the carbon-1^ content of the medium, mycelium, and respired carbon dioxide as a function of age of P. charlesii

Figure 29 represents a plot of total activity in respired carbon dioxide as a function of days of growth of P. charlesii.

Attention is directed to the polyphasic nature of the curve.

Although carbon-1^ began to appear in respired carbon dioxide after the organism had grown for only 2 days, a lag period of l^t about 6 days preceded a rapid rate of C 0^ release by P. l^f charlesii. The rate of C 02 release between the 15th and l6th days approximated the rate for the 2nd through 5th days of growth. 1 it At 17 days the rate of C 0^ production increased and the ac

celeration continued through the 28th day of the experiment. The lit C (>2 curve of Figure 29 does not lend itself to facile expla nation. It does, however, afford the suggestion that tartrate lit oxidation, based on C 0^ release, approached its maximum at a Fig. 29.— Total carbon-1^ appearing in CO., as function of &S® P • charl6sii•

173 TOTAL ACTIVITY to/ C'*Oa CPM )C IQ"5 tr\ V

H***1 oq

*1&x 175 stage in which the glucose content of the medium had already been drastically reduced, as shown in Figures 28 and 29«

This data is not incompatible with the previously made suggestion that one of tartaric acid's functions is that of a

"secondary" carbon source in the Raulin-Thom medium and that the mould will most actively metabolize tartrate when the

"primary" organic carbon supply has been exhausted or reduced to low levels. That this argument is limited by additional un known or poorly understood factors is suggested by the plots in

Figure 30. This figure summarizes data obtained on'the time course of carbon-14 distribution in the growth medium, mycelium, and respired carbon dioxide of P. charlesii. Attention is directed to the differences in magnitude of the left-ordinate t which relates carbon-14 in the medium and mycelium to the right- ordinate which relates carbon-l4 in respired carbon dioxide.

In this experiment removal of carbon-l4 from the growth medium began after 24 hours of growth of P. charlesii. The rate for this process was quite rapid, particularly over the first 5 14 days and although the rate of C 0^ evolution was small the up- 14 take of tartaric acid-1,4-C was rapid during this period.

A Comparison of this figure with Figure 28 revealed that when ap proximately 40 per cent of the initial tartrate had been removed from the medium the pH had changed only 0.4 of a unit; these changes occurred during the first 4 days of growth. Tartrate disappearance from the growth medium did not appear to be ac companied by rapid changes in hydrogen ion concentration. This suggested that tartrate uptake and ammonium ion uptake from the Fig. 30.— Time course of distribution of carbon-l4 from tartrate-1,in mycelium, growth-medium, and C^O, of P. charlesii. c

176 o£ *sT,i .ZZ,T JO S^yQ 1 < . — 3 O O<3 ■ w *■> o/- ciiy n eim h Mycelium rhd Medium in Activity Tor/M- o o oh ACTIVITY TorhL *i ©o **-i o- o O counts O a xio~b in C!H0 i o -Cr c ° x _ C O C> O C7O

V — — CV 178 medium occurred concomitantly, such that the hydrogen ion con

centration of the medium was essentially constant during the

period in reference.

Figure 30 further shows that, at aborit 6 days, when the

glucose content of the medium had been reduced by 50 per cent

of the initial value the tartaric acid concentration had suf

fered an identical dimunition. This, observation provides basis

for the suggestion that complete removal of glucose from the

growth medium of P. charlesii was not absolutely required for

tartaric acid uptake to occur.

Between the 12th and 25th days of incubation the rates

of evolution and carbon-l4 uptake by the mycelium were

approximately equal although the absolute amounts of carbon-l4

involved in these two cases differed by a factor of 10, 14 An analysis of changes in specific activity of C 0^

released by P. charlesii might provide information which relates

to the question of the time at which conversion of tartrate to

carbon dioxide proceeds most rapidly. Figure 31 represents the lif specific activity of respired C 0^ ae a function of time. Except

for values recorded on the 13th and l^fth days the data approximate

a smooth curve which increases in slope through the 15th day,

levels off and rapidly increases from the 19th through 25th days.

The specific activity of carbon dioxide decreased beyond the 25th

day of growth and the phint at which this decrease began cor

responded to the time at which activity concentrated in the

mycelium began to re-appear in the medium. These latter obser- Fig. 31«— Plot of specific activity of respired CO^ as a function of time of growth of P. charlesii.

179 21 20 20 ID 10

17 16 i d 14 13 12 to II

O 0 ? G 5‘ 4 G- 3 O 2 I

3 II 12 13 14 15 1-3 1? 13 IS2Q2.I2? 23 24 25 2G 2? DAYS OF GROWTH 091

Fig. 31 l8l vations are recorded in Figure 32. The latter figure also shows

that the total amount of radioactivity in respired COg continued

to increase during the 25th through 28th days, a period in which

the specific activity of the carbon dioxide decreased and the

total amount of carbon-1^ in the mycelium decreased. These three

observations can be made harmonious if it is assumed that

release of some intracellulary concentrated metabolite (as, for

example, through autolysis), which derives primarily from glucose

or carbons 2 and 3 of the original tartrate.significantly.dilute lif the C Og derived from tartaric acid.

Inspection of Figure 32 reveals the relationship between

the total activity appearing in carbon dioxide release by P.

charlesii and the specific activity of that carbon dioxide. Al

though the left and right ordinates differ by a magnitude of 10,

this figure affords a direct comparison between the two variables

represented. One salient feature of the two curves is that the

specific activity of carbon dioxide decreased while the total

C 0^ increased after 25 days of growth. With the exclusion of

the points corresponding to the 13th and l*fth days on the specific

activity curve, the data collected over the first 25 days of

growth can be interpreted to suggest that carbons 1 and 4 of

tartaric acid were metabolized at a gradually increasing rate.

An alternative situation in which glucose of the system were

metabolized to carbon dioxide prior to the conversion tartaric l^f lA- acid-1,4—C to carbon dioxide would have yielded C 0^ of very

high specific activity after the onset of active tartaric acid STUDIES ON METABOLISM IN PENICILLIPM CHARLESII:

SOME RELATIONSHIPS BETWEEN DICARBOXYLIC

ACID METABOLISM AND PRODUCTION OF

GALACTOCAROLOSE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

John Maxwell Jordan, B.S.

The Ohio State University 1963

Approved bjrea bv

Adviser Department of Agricultural Biochemistry Until quite recently no information had been published which related to the biosynthesis of any naturally occurring galactofuranoside. The results of studies related to galactocarolose were reported by Gander who observed that carbon-l4 from a number of substrates was incorporated into the polyga- l4 l^f 14- lactose (5); glucose-l-C , glucose-2-C , glucose-u-C , l^f 1^ acetate-l-C , and tartaric acid-l,Jf-C were found to supply carbon-1 for the individual galactose units that were isolated.

When the specific activity of the starting carbon-l^f labeled substrates was compared with the specific activity of the galac tose isolated in the particular cases a broad pattern of in corporation was suggested. The data were interpreted in terms of a condensation of a unit, derived from tartrate, with a glucose- derived unit to yield a Cg precursor of the hexofuranose units*

The eventual conversion of this condensation product into galactocarolose would be expected to occur through activated forms of the Cg precursor*

The experiences of a large number of investigators has led to the concept-that oligosaccharide formation usually occurs

through the agency of activated glycosyl compounds and that three important types of processes are operative in the polymerization*

These processes include (a) action of phosphorylases, (b) action of transglycosylases, and (c) the action of nucleoside diphospho-

glycoside transglycosylases*

The phosphorylases are generally regarded as being in

volved in degradative reactions and a similar function is assigned Fig. 32.— Plot of total activity in and specific activity of respired C1 0_ as function of time of growth of P. charlesii.

Curve Designation

The. curve defined by closed triangles reflects changes in total carbon-l4 in CO^.

The curve defined by X represents specific activity of "respired" CPM MILLIMOLE IN Ba(A>3 * | 0~q

«<>. CO

-a

^ CPM X IO‘r TOTAL ACTIVITY

£8T l8Jf oxidation. With the exception of the two points mentioned previously, the C 0^ specific activity curve shows no sharp and dramatic inflection prior to the 20th day of growth of P. charlesii. The gradual increase of the specific activity of the lit respired C 0^ over three-fourths of the growth period is in conflict with the view that oxidation of tartaric acid occurs only after most of the glucose has disappeared from the growth medium. lA- The unusual specific activities of C 0^ recorded for the 13th and l^th days of incubation do not appear to be pro cedural artifacts. The significance of these values (at 13 and l*t days) is not known.

Qualitative definition of the form in which tartaric acid-1,^-C is present in the mycelium of P. charlesii

Experiments of the previous section yielded data which demonstrated a significant rate of tartaric acid uptake by P. charlesii during the early stages of growth of the organism.

The mould proliferated rapidly; the entire surface of the growth medium was covered by the mycelium after k days and that surface of the mycelium not in direct contact with the growth medium was covered with a green matting of spores at 6 days. At the time of initiation of visible sporulation the tartaric acid concen tration of the medium was approximately 50 per cent of the •14 original value and little C 0^ had been produced. Beyond the period at which sporulation was initiated the concentration of 185 tartaric acid "by the mycelium increased in rate and this increased rate of uptake was maintained throughout the duration of the experiment. It may be extremely fortuitous that initiation of sporulation and rapid uptake of tartrate occurred at approxi mately the same stage of growth of Penicillium charlesii and the arbitrary and indirect comparison of these two phenomena is not intended to imply that they are or are not related through the metabolic machinery of the mould* lif That a relatively small amount of C 0^ was being produced during the first 10 days of growth suggested that the concen- l^f trated tartaric acid-1,4— C was being stored as the acid or that

the isotopic compound was being converted to other substances which were restricted to the intracellular regions of the mould mycelium. In the absence of experimental evidence to the con

trary it could be assumed that intracellular tartaric acid or

its conversion-product was related to a precursor of the galacto-

furanosyl residues of galactocarolose. A number of other as

sumptions might be made but all of these have in common the re

sulting desirability of knowing the molecular form(s) in which

l i j . concentrated carbon-1^ from tartaric acid-1,4— C is held within

the cells of P. charlesii.

An experiment, similar to that described in the previous

section, was designed with the purpose of permitting P. charlesii

to concentrate approximately one-half of the tartaric acid-1,4— 14- C which had been added to the growth medium. The mycelium and

medium which corresponded to this 50 per cent concentration were 186 fractionated by standard techniques in an attempt to determine 14 if and to what extent tartaric acidi-l,4-C had been converted to other substances.

Because of technical difficulties the "metabolic train" was operated at £6°C in this experiment. In the previous ex periment the incubation was carried out at 23°C. The absolute lij. level of DL-tartaric acid-1,4-C employed in this experiment was one-third the level used in the experiment of the previous section. With these two exceptions all conditions of the two experiments were identical.

The growth of the mycelium was quite slow during the first 6 days of incubation. The mycelium covered the available surface of the growth medium after about 7 days and the presence of green areas on the upper mycelium could be detected only after

9 to 9.5 days. Figure 33 shows that the removal of carbohydrate from the growth medium was not slow under the conditions of this experiment. At 6 days two-thirds of the carbohydrate, initially present, had been removed from the medium. 14 On the other hand, the uptake of tartaric acid-1,4-C was relatively slow. After 6 days of growth approximately 65 per cent of the carbon-l4 originally present was still in the growth medium and most of the remaining 35 per cent was in the mycelium. The rate of concentration of tartaric acid was ap proximately constant over the 4th to 8th day period. After 8,5 days the rate of disappearance of carbon-14 from the medium in creased quite markedly and this was accompanied by a similarly Fig. 33.--Variations in carbohydrate concentration of growth medium as function of age of P. charlesii.

Curve Designation

The open circles represent changes in the concentration of total sugar.

The closed triangles represent changes in the con centration of reducing sugar.

The curve defined by X represents changes in the glucose concentration.

187 188 O (jtjjnoles ml.) CARDOHYDRATE IN MEDIUM CONCENTRATTO'N OF ‘ - tp - tp c r\

i5ays of g r o w t h 189 rapid concentration of isotope within the mycelium. Evolution l^f of C Og, slow over the first 8 days of growth of the mould, obtained its maximal rate during the 8.5 to 10.5 day interval.

These observations are recorded in Figure Figure 35 lustrates the rate of disappearance of carbon-l^f from the growth medium and the rate of concentration of carbon-14 of DL-Tartrate- 14 1,4-C in the mycelium.

Figure 36 summarizes the data recorded in Figures 34 and

35* It is observed that the maximal rates for decrease in medium and increase in mycelial carbon-14- concentrations correspond l4 quite well to the maximal rate of production of C 0^. It may be of some interest to emphasize that, in this experiment, the 14 maximum rate of tartaric acid-C uptake was reached after most

(93 per cent) of the medium glucose had been concentrated and/or metabolized by the mycelium. This latter observation was afforded by a comparison of Figure 35 and Figure 36. With respect to this point attention should be directed to Figures 28 and 30 of the previous experiment. It was observed that maximal rates of disappearance of glucose and tartaric acid from the medium were coincident with respect to the age of the culture.

The differences observed in these two cases may reflect the differences in conditions of growth employed in the two experiments.

After 10.5 days the medium and the mycelium were sepa rated and treated according to the procedures outlined below and under 'EXPERIMENTAL.* Fig. J>b• Variation with age of mycelium of carbon-lA- in respired CC^.

190 a 1” IN RESPIRED C^Oa CPM X. IQ'6

c/i o o

O > to

H- D oq ■n

-p-

«c 191 3 to numerous of the transglycosylases. During the past five years the view has gained support among investigators in the field that the nucleoside diphosphoglycoside transglycosylases compose the most important class of enzymes which function in glycoside and oligosaccharide formation.

The importance of this latter process has been discussed by Bobbins and Lipmann (6). The authors considered the question of glycogen synthesis in muscle and compared the activity of phosphorylase-a with the TJDPG-Glycogen-transglycosylase pathway.

The suggestion was advanced that "phosphorylase-a may not be concerned physiologically with glycogen synthesis but rather glycogen utilization."

This latter concept has been extended to a number of other systems in which an oligosaccharide can be formed by a nucleoside diphosphoglycoside transglycoeylase catalyzed-reaction or some alternative process. Upon this basis the possibility of nucleoside-diphosphoglycoside compounds as participants in reactions which form galactocarolose has been investigated by

Maynard (7)»

The product of the condensation of a glucose-derived carbon fragment with a fragment derived from tartrate might be

formed prior to nucleoside involvement in the overall scheme of

galactocarolose biogenesis and the delineation of the mechanism of such a condensation could further the understanding of sub

sequent processes involved.

ft Fig* 35.— Variation with age of culture of carbon-l*f from tartrate-1,in mycelium and growth medium of P. charlesii. "" C1* IN MYCELIUM and GROWJH MEDIU M

- £ r l/x O o

o 5 in H* Oq O -T1 VH VJ1 G) Q

o o ° MYCELIUM GROW M E D fU M 193

CPM X IQ' 6 Fig. 56.— Variation with age of mycelium of carbon-l^t distribution in. mycelium, growth medium, and of P. charlesii.

19^ co C14 IN MYCELIUM .AND MEDIUM OF RCNARLESK cpm XIO” 40 60 50 20 30 AS F GROWTH OF DAYS i. 36 Fig. MYCELIUM MEDIUM CO 6.0 4.0 2.0 5.0 to 195 .o o to V) K iLl a o g u C' o E OJ : Initial separation of medium.and mycelium was effected by filtration. The mycelium was washed exhaustively with 20 ml volumes of cold, double distilled water and the washings added to the main filtrate.

The filtrate plus mycelial washings was concentrated to 18.7 ml* The components of the concentrated growth medium were fractionated with absolute ethanol according to methods described under 'EXPERIMENTAL.1 The distribution of carbon-l1* in the various "alcohol” fractions of the medium is recorded in Table 18.

TABLE 18 DISTRIBUTION OF CARB0N-l*f LABEL IN VARIOUS "ALCOHOL FRACTIONS" OF RAULIN-THOM GROWTH MEDIUM

Total Volume Total Radioactivity Present Fraction Ml CPM x 10-6

Growth medium plus 18.7 25.601 mycelial washings (concentrated) 80 per cent ethanol 9.0 2.2 ,8 lk soluble Absolute ethanol wash of 65.0 0.160 80 per cent ethanol insoluble 80 per cent ethanol 11.0 2.356 insoluble

The thoroughly washed mycelium was cut into small pieces and the resulting mince suspended in cold (0°C) 0.12 molar per chloric acid in a Waring Blendor cup which was pre-cooled in an ice-water bath. The mixture was homogenized for 5 minutes over 197 one minute intervals and the homogtnate placed in a sonic oscil lator cup and subjected to sonication for 30 minutes. The soni-

cation was performed with a Raytheon 10 kc ultramagnetostrictive

sonic oscillator which was operated at 1.25 amperes. During the

sonication the cup which contained the sample was cooled by a

continuous stream of water at 0 to 4°C. The sonicate was stirred

for 24 hours at 2 to 3°C by a magneto-mixer in a cold room which

was maintained at the stated temperature of 2 to J°C. At the end

of this time period the residue was removed by centrifugation

and the supernatant stored at 3°C. The residue was washed with

cold double-distilled water and the washings added to the main

supernatant. The separation of supernatant from residue was

accomplished by centrifugation in a Servall refrigerated centri

fuge. The combined supernatants were designated "cold PCA

extract."

The washed residue was suspended in approximately 175 ml

of 1.2 per cent perchloric acid and the mixture placed in a

500 ml round bottom flask. The flask and contents were brought

to and held at 60° to 70°C for 24 hours. The flask was fitted

with a reflux condenser to prevent concentration of the acid

under the conditions of extraction. After 24 hours the flask and

contents were cooled to room temperature and allowed to stand for

30 minutes at 0 to 3°C. The mixture was centrifuged at 15*000 x g

in a refrigerated centrifuge which was operated at 0°C. The

residue was washed twice with 50 ml volumes of distilled water, 198

the washings were added to the main supernatant and this com

bination was designated "hot PCA extract."

The washed residue from the latter treatment was dried

in a thermostatically-controlled oven at 80°C over a ^8-hour

period.

The two perchloric acid extracts were neutralized to

pH 6.5 *>y slow addition of 3*6 normal potassium hydroxide. After

standing for two hours at -10°C the precipitated potassium per

chlorate was removed by centrifugation.

Relative distributions of radioactivity among the various

fractions are represented in Table 19* The data of this table

suggest that treatment of the mycelial residue with hot perchloric

acid resulted in the release of little radioactivity.

The various soluble fractions were subjected to paper

chromatography in several solvent systems. The formic acid-

containing solvent system, which has been used to advantage in

the chromatographic resolution of sugar phosphates was found to

be suitable for resolution of acidic and neutral-polymeric com

pounds. This latter solvent system (methanol, formic acid,

water; 80:15:5) (6l) resolved the PCA extracts of P. charlesii

into several components. In the present case descending chro-

* matography was performed on Whatman number 3 nun paper at ^ C .

The dried chromatograms were scanned with the strip-scanner

described under 'EXPERIMENTAL.' The scans which resulted are

represented in Figure 37 • The patterns obtained suggested that

. l4 P. charlesii had converted DL-tartaric acid-1,4-C to a number 199 TABLE 19 DISTRIBUTION OF CARBON-l4 IN PERCHLORIC ACID EXTRACTS OF P. CHARLESII

Total Quantity Total Activity Recorded of Fraction ■6 Fraction Ml Mg CPM x 10'

Cold PCA extract 300 3.160 Hot PCA extract 150 0.571 Residue from hot 882 2.492 PCA extract of radioactive substances at the time of termination of the present experiment.

The areas indicated by the letters A, B, and C were eluted from the chromatograms and the eluates concentrated to small volumes. The total carbon-l4 content of each concentrated eluate was determined by liquid scintillation counting. The data thus obtained are recorded in Tables 20 and 21.

TABLE 20 DISTRIBUTION OF CARBON-14 IN CHROMATOGRAPHICALLY RESOLVED COMPONENTS OF "ALCOHOL" FRACTIONS OF THE GROWTH MEDIUM

Total Radioactivity Total Volume in Fraction Fraction Ml CPM x 10“6

Alcohol soluble A 8.0 0.586 B 6.0 2.472 C 6.0 0.353 Alcohol insoluble A 6.0 0.019 B 5.5 0.201 Fig. 37•— Distribution of carbon-l4 labeled com ponents of "alcohol" fractions of growth medium of P. charlesii.

200 201

MEDIUM CONCENTRATE

E OQ. o O O

I ALCOHOL INSOLUBLE FRACTION E oa. O O O

ALCOHOL SOLUBLE FRACTION t £ uC l O O 0 1 yr BC

TARTRATE STANDARD

E. OCL O O O

2 4 6 810 12 14 16 18 20 DISTANCE FROM ORIGIN (INCHES)

Fig. 37 4

A necessary consequence of a plus C,, condensation Cor whatever type condensation is involved in linking glucose and

tartrate-derived fragments) is that the compound which results

or one of its conversion products must be derivatized at carbon-5

(of the eventual hexofuranosyl precursor) in such a fashion that

this compound exists preferentially in the furanose form. The presence at C^, for example, of an oxo, an -o-phosphoryl, or

deoxy function would meet the stipulated requirement. It is

conceivable that a 5-ketogalactose, 5-o-phosphoryl-galactoee, or

5-keto-6-deoxygalactose could function in this process and any

one of these three substances would satisfy conformational

requirements•

It would be instructive, therefore, to define the nature

of the fragments of the condensation which derive from glucose

and other such factors that influence galactocarolose synthesis

as are experimentally approachable and relevant. This dissertation

summarizes experimental efforts directed toward these ends.

The problem of the nature and function of intermediates

which lie between glucose, tartrate, and galactocarolose (in the

anabolic pathway of the latter) was attacked through the use of 14 isotopic tracer compounds. Glucose-u-C , various carbon-14

labeled dicarboxylic acids, and 1 and/or -2-C^* labeled acetate

have been employed to establish precursor-product relationships.

Additionally, orthophosphate containing was employed in an

attempt to establish the nature of changes with age (of Penicil-

lium charlesii) of organic phosphate compounds. 202

TABLE 21 DISTRIBUTION OF CARBON-14 IN CHROMATOGRAPHICALLY RESOLVED COMPONENTS OF COLD PCA EXTRACT OF MYCELIUM OF P. CHARLESII

Total Radioactivity Total Volume in Fraction Fraction m i : CPM x 10-^

Cold PCA extract A 7.5 6.125 B 6.0 1.549 C 6.2 11.549

That the pattern of carbon-l4 distribution of Figure ?8 represents mycelial metabolism of tartaric acid and not artifacts

of procedure was supported by an experiment in which 10-day old

P. charlesii, which had been allowed to concentrate and metabolize 14 tartaric acid-1,4-C was extracted with perchloric acid and the

neutralyzed extract resolved into a number of components by

column chromatography.

Penicillium charlesii was grown for 10 days on the Raulin-

Thom medium which was supplemented with tartaric acid-1, 4-c14.

At 10 days the mycelium was collected, washed thoroughly with

distilled water and extracted with 0,6 M perchloric acid in the

cold. The extract was neutralyzed and the solution which resulted

was placed on a 1 x 15 cm column of Dowex-1 Cl (200 to 400 mesh).

The pH of the solution applied to the column was 8.0. Develop

ment of the column was effected through elution with distilled

water which was followed by 0*01 normal hydrochloric acid.

Samples of 10 ml each were collected from the column using an

automatic fraction collector. Radioactivity in the emergent Fig. 3 8 . — Distribution of carbon-1^ labeled com ponents of extracts of 10.5-day mycelium of P. charlesii.

203 20

14 16 13 12 Fig. 33 10 DISTANCE DISTANCE FROM (INCHES) ORIGIN 6 8 4 2 0 Hot ExtractHot

uidoQOOl — -*■ —— ■ m doQQQ j------*—«------uidoQOOl V 205 fractions was quantitated through the use of a liquid scintil lation counter. Figure 39 records a plot of total activity against the tube number of the 10 ml samples collected.

The radioactive fraction emergent from the column in tubes 62 to 72 represents the position at which standard DL- l4 tartaric acid-1,4-C is eluted from the column. This point l4 was established by applying a solution of tartaric acid-1,4-C to a column of Dowex-l-Cl in a control experiment.

The data of the present and the two preceding experiments l^j. signify that tartaric acid-1,4-C was removed from the Raulin-

Thom growth medium at a rate which may not depend on the absolute level of glucose in the. medium; concentrated tartaric acid-1,4- 14 C was converted to numerous metabolites as early as 10 days after innoculation of the culture medium. Significantly small 14 amounts of concentrated tartrate-C were converted to carbon

dioxide during the period in which glucose disappearance from the

medium occurred most rapidly. The absolute quantity of carbon-l4 14 14 from tartrate-C which appeared in C 0^ was quite low in com

parison to the total. carbon-l4 which was removed from the growth

medium and significant quantities of tartrate carbon were con

verted to cellular structural, materials after 10 days growth of

the mould, numerous carbon-l4 labeled orgamic compound were

extracted from the 10 and 10.5-day old cultures and it is sug

gested that the amount of free teu*taric acid in the mycelium

(at 10 days of growth) is small compared to other forms into 14 which tartrate-C has been distributed. Fig. 39.— Distribution of carbon-14 labeled components of extract of 10-day mycelium of P. charlesii.

206 T

COUNTS PER MINUTE X I0 3

Loz 208

It is tempting to suggest that some of the carbon-1^ labeled substances isolated from Penicillium charlesii (by acid extraction) are involved in the biogenesis of the tartrate- derived precursor of galactocarolose. The formal offering of such a suggestion must await further characterization of the carbon-1^ labeled isolates in reference,

. (E) Incorporation of Various Carbon-l4 Labeled Carboxylic Acids by Penicillium charlesii

Penicillium charlesii growing upon the Raulin-Thom medium has been shown to produce galactocarolose in the presence of several dicarboxylic acids substituted for tartaric acid in the growth medium. Existing knowledge related to the role of these dicarboxylic acids in galactocarolose production would be augmented by data which establish that the dicarboxylic acids, in reference, do or do not contribute portions of their carbon chains to the carbon chain of the hexose units of galactocaro lose. Studies related to a search for this definitive infor mation represent inquiries as to whether or not dicarboxylic s acids other than tartrate serve as precursors of galactocarolose and if said dicarboxylic acids are precursors with what efficien cies are they incorporated into galactocarolose.

Studies were undertaken which involved the addition to the Raulin-Thom growth medium of several carbon-1^ labeled di carboxylic acids and carbon-14 labeled acetate. The additions of carbon-l4 labeled acids were made at the time of innoculation of the growth medium or alternatively the additions were made at

16 days after initiation of growth. 209 iZf Respired C 0^ was trapped in sodium hydroxide and the mycelium and residual medium were prepared for isotope content analysis according to the procedures defined under 'EXPERIMENTAL.'

The cultures were grown upon 25 ml of growth medium and the

entire metabolic assembly was enclosed in a large (10 x l8-inch)

cylinder as described under 'EXPERIMENTAL.' After 28 days'

development of the culture the incubation was terminated and the

distribution of carbon-l4 in various fractions was determined.

The results of the analyses are recorded in Table 22,

In the eight cases to which the data of Table 1 refer

the incorporation of carbon-14 into oligosaccharides was negli- lif gible. This statement is based upon the release of C labeled

monosaccharides from the concentrated medium upon hydrolysis in

0,4 normal sulfuric acid. It was of interest to know if P.

charlesii had produced non-labeled oligosaccharides under the

experimental conditions. The areas (of the chromatograms of the

medium hydrolyzates), which corresponded to the position, to

which galactose migrates, were eluted from the chromatograms and

the eluates concentrated under reduced pressure. Two types of

patterns were observed when the concentrated eluates were sub

jected to various carbohydrate analyses; the galactose from the'

succinate labeling experiment contained about 40 C.P.M. per

micromole of galactose and a total of 16 micromoles of galactose

were isolated; the galactose isolated from the other labeling 14 experiments ranged in quantity from 6 (for the succinate 2,3-C l4 experiment) to 21 micromoles (for the acetate-l-C ) experiment and the isolated galactose contained negligible radioactivity. TABLE 22 INCORPORATION OF VARIOUS CARBON-14 LABELED CARBOXYLIC ACIDS BY P. CHARLESII

Radio Age of Culture Radioactivity Recovered in -jL Activity when Cl^-Subetrate ------:------— C Labeled Added Added Myceliunr Medium Sodium Carbonate Substrate jAc Days CPM x 10“* CPM x 10“6 CPM x 10”6

14 Acetate-l-C 50 16 64.16 11.17 119.95

Acetate-2-C^ 50 16 75.76 8.45 195.07 14 Malonate-l-C 50 16 340.44 8.45 144.88 14 Malonate-2-C 25* 0 8210.00 14.21 17.74

Succ^nate-1,4- 25 0 384.50 8.30 52.28

Succi.nate-2,3- 25 0 391.5** 10.13 55.89 C1* 14 Sue cinate-u-C 50 16 277.50 6.96 98.73

Fuma^ate-2, J>- 25 0 291.03 10.16 51.^3

* Two flasks were employed. 211

At the time these experiments were conducted the. relation ship between the total quantity of organic-carbon substrates available and the quantity of galactocarolose synthesized by P. charlesii was not appreciated. It is conceivable that the l6-day old mould had exhausted its available carbon supply at the time lif of addition of C labeled acids. Under these conditions the labeled acids which were added at 16 days were rapidly converted to cell-structural material and used in energy yielding processes*

In its state of '‘starvation’1 P. charlesii would be expected to convert little of its carbon supply to "shunt" or overflow metabolites.

The involvement of succinic acid in the citric acid cycle provides an easily accessible route for the complete oxidation of

this dicarboxylic acid. This fact would explain, at least in l^f part, the failure of succinate-2,3-C (added at 0 days of

growth) to label galactocarolose under conditions in which the

total amount of glucose available to P. charlesii was relatively

low.

(F) The Incorporation of Tartaric Acid-1,^f-C by Penicillium charlesii Grown in the Presence of Tartronate

Dihydroxymaleate is one of the possible first products

of the oxidation of tartaric acid. Further transformations of

dihydroxymaleic acid might involve the production of tartronic

acid semialdehyde or its oxidation product, tartronic acid, might

be a tartrate derived precursor of the hexose units of galacto

carolose. The conversion of tartrate to tartronate has been 5

Experiments were designed and conducted which involved variation of the dicarboxylic acid component of the medium, carbohydrate level, total nitrogen concentration, concentration of various carboxylic acids and an analysis of the effect of variation of these parameters on the synthesis of oligosac charides*

The effect on oligosaccharide synthesis of substitution

(in the growth medium of P* charlesii) of the enantiomorphs of tartaric acid was examined. Experiments were conducted which provided information as to the time-course of tartaric acid metabolism*

During the course of these investigations several unusual compounds were isolated from the mycelium of Penicillium charlesii and efforts were directed toward the characterization of these substances*

It was conceived that some information related to oligo saccharide formation in P. charlesii might be gained through use of disrupted mycelium. Cell-free extracts of the mould, grown

for various periods of time, have been used in attempts to demon strate the occurrence of certain enzymatic reactions hypothesized

to be related to oligosaccharide biosynthesis* 212

shown to occur in strain ?4a of Neurospora crassa (5*0 • The authors suggested that the conversion may have involved fixation

of carbon dioxide.

It would be of interest to determine if tartronic acid

is an intermediate in the conversion of tartaric acid to cell

metabolites of P. charlesii. An experiment was designed which l^j. involved the metabolism of tartaric acid-1,4-C in the presence

of tartronate. The Raulin-Thom growth medium was modified to

contain tartronate to a final concentration of 32.4 micromoles 14 ■ per milliliter. Tartaric acid-1,4-C was an essentially carrier-

free addition to a final concentration of 2 microcuries per

milliliter.

The final volume of the growth medium was 25 niilliliters

which was contained in a 125 ml Erlenmeyer flask. Respired

carbon dioxide was trapped in sodium hydroxide. At the end of

the 28-day growth period the mycelium, residual growth medium,

and sodium carbonate were analyzed for carbon-14 content according

to procedures described under 'EXPERIMENTAL.' The. distribution

of carbon-l4 in the various fractions is recorded in Table 23.

The concentrated growth medium was chromatographed as

cending in the Butanol:Pyridine:Water (6:4:3) solvent system.

The distribution of carbon-l4 label on the chromatogram is

represented in Figure 40. The area designated was eluted

from the chromatogram with cold distilled water and the resulting

eluate treated with 0.4 normal sulfuric acid for 90 minutes at

95°C. The neutralyzed eluate-hydrolyzate was chromatographed in Fig. ^O.--Distribution of carbon-l4 labeled components of the growth medium concentrate.

The upper scan (designated M) represents the medium concentrate

The lower scan (designated G) represents a galactose-C^ standard

The abscissa represents the distance from the origin that a component moved

213 t I o Q O

■1

1 DISTANCE FROM ORIGIN

aS o o o

Fig. 40 4T2 215 TABLE 23 DISTRIBUTION OF CARBON-14 IN VARIOUS FRACTIONS DERIVED FROM STUDY OF INCORPORATION OF TARTRATE-1, 4-C1^ IN THE PRESENCE OF TARTRONATE

Radioactivity Recovered in Fraction Quantity ------of Fraction Specific Activity Total Activity . _----- j~ t- Fraction Ml Grams CPM/mlX10"° CPM/mgXIO CPMxlO

Medium 8.4 5.003 42.032 concentrate

"Clif02 " 210 0.317 66.600 sodium hydroxide trap

Mycelium 0.330 1.0393 3.429 14 BaC 0^ 4.897 0.0489

Total 112.161 the Butanol:Pyridine;Water (6:4:3) solvent system. When the dried chromatogram of the S^ hydrolyzate was scanned for radio active areas the pattern represented in Figure 4l was obtained.

It was apparent that no carbon-l4 labeled monosaccharides were released through treatment of S^ with dilute acid. Area A, which contained no significant radioactivity (but which cor responded to the position where galactose would migrate if the latter had been present) and area S^ were eluted from the chro matogram. Eluate A was concentrated under reduced pressure and saved for further analyses. Eluate S^ was concentrated and treated with 3*0 normal sulfuric acid at 95°0 for 90 minutes. Fig. 4l.— Chromatogram of 0.4 normal acid hydrolyzate of fraction S^.

The upper scan (designated N) represents the 0.4 normal acid hydrolyzate

The lower scan (designated G) represents a galactose-C standard

The abscissa represents the distance from the origin that a component moved

216 £ a. d O Q Q cn

O Qd

DISTANCE FROM ORIGIN

Fig. 4l 217 218

The hydrolyzate was neutralyzed with solid barium carbonate and the neutralyzate freed of salts according to procedures described under ‘EXPERIMENTAL.'

The concentrated neutralyzate of was chromatographed ascending in the Butanol-based solvent described above. A representation of a scan of a strip of the chromatogram (of the

neutralyzate) is given by Figure 42. Figure 42 shows that no carbon-l4 was detectable in the monosaccharides which would be released by 3.0 normal sulfuric acid hydrolysis.

The failure to demonstrate an acid catalyzed release of carbon-l4 labeled monosaccharides suggested that tartaric acid-1, 14 4-C was not significantly incorporated into oligosaccharides under the conditions of this experiment. An alternative assump- 14 tion was that the amount of tartrate-C incorporated into the oligosaccharides was too low to be detected through the procedures which were employed. That little oligosaccharide was synthesized under the experimental conditions was another possible explanation of the observations. Support for this latter possibility was af forded by the data recorded in Table 24.

The analysis of eluate A suggested that little galactocarolose was present in the modified growth medium at the end of the 28-day growth period of P. charlesii. This observation may reflect that galactocarolose was not formed in significant quantity or that the galactocarolose, which was synthesized, was

subsequently catabolized by P. charlesii prior to termination of

the experiment. The data do not afford a distinction between

these two possibilities. Fig. 42.— Chromatogram of 3.0 normal acid hydrolyzate of fraction

N Represents chromatogram of hydrolyzate of S^.

TG Represents chromatogram of tartrate and galactose standards respectively.

The abscissa represents the distance from the origin that a component moved.

219 ro ro o

Z\ T V F ig . if2 ▼ DISTANCE FROM ORIGIN (INCHES) # 0 I g 3 4 5 6 7 0 9 IQ II 12 13 14 ID IG 17 18 19 20

<— WdO 0002 — > Wd^ 0 00 J -4 . 221

TABLE 2*f CARBON-1^ AND CARBOHYDRATE CONTENT OF CONCENTRATED ELUATES ______OF FRACTIONS S? AND A______

Carbohydrate Analyses of Fraction Volume (Values Expressed as Micromoles Radio of Per Ml) activity Fraction Recovered Total Total Reducing Glu- Galac- "... ML Sugar Hexose Sugar cose toseFraction CPM x 10”°

A 10*0 1A5 2. AS 1.37 None 1*18 _ BA 1*52 1.50 None None 37 A3 S3

It has frequently been noted that cultures of Penicillium charlesii do not produce significant amounts of galactocarolose when these cultures are grown upon less than 50 ml of Raulin-

Thom growth medium. That the latter observation was not a re flection of a loss of capacity of the cultures to produce galacto carolose was demonstrated by use of spores from a culture which had grown for 3 weeks upon 25 ml of Raulin-Thom medium as in- noculum for 150 ml of growth medium. The culture which was pro duced on 150 ml of growth medium synthesized galactocarolose whereas no galactocarolose was present in the;25 ml culture medium at the end of 4- weeks. Additionally, when spores of P. charlesii taken from agar plates were used to innoculate 150 ml and 25 ml, respectively, of growth medium the 150 ml-culture produced galactocarolose (8.6 micromoles) while negligible (0*5 micromoles) galactocarolose was isolated from the 25 ml culture medium*

These observations suggested that the absolute amounts of substrates (rather than their concentrations) available to REVIEW OF THE LITERATURE

(A) Isolation and Characterization of P. charlesii and Some of Its Metabolic Products

During the past thirty years existing knowledge of fungal metabolism has been greatly augmented through research performed at the London School of Hygiene and Tropical Medicine* Of par ticular note are the contributions made by the group which functioned under the directorship of Dr* Harold Raistrick; a myriad of fungi of various classifications were isolated, studied, and described* In many instances exhaustive Btudies were made of the immense number of mould products that could be isolated from growth broth and mycelium of the particular organisms studied*

One such organism, Penicillium charlesii* was isolated from mouldy

Italian maize by Dr* G.H.V* Charles* A brief description of the organism has been published (8)* The complete name for this mould is P. charlesii G. Smith*

P* charlesii, when grown upon the Czapek-Dox or the Raulin-

Thom growth medium, was shown to produce numerous heterocyclic carboxylic acids and two unusual oligosaccharides (9)* This latter report is now a classic contribution in the area of mycologic physiology.

In a subsequent report Clutterbuck and co-workers de scribed the molecular constitution of two heterocyclic carboxylic acids to which these authors gave the names carolic acid and 222

!!• charlesii influence significantly the quantity of galacto carolose synthesized*

The question of the nature of the carbon-l4 labeled compound(s) present in the medium concentrate (finally recovered as eluate S^) was examined by chromatographic and enzymatic methods.

Fraction S,, which contained essentially all of the radioactivity of the initial medium concentrate (Table 24), was subjected to chromatography in five different solvent systems*

The results for four of these systems are represented in Figures

4-3 and 44. These figures suggested that tartaric acid was the major component of the medium concentrate.

Approximately one-third of the total volume of was

resolved into the two components (shown in Figure 4) through use

of the 70 per cent phenol system. The major peak, resulting from

this chromatography on Whatman number 3 mm paper, was eluted with

distilled water and the solution evaporated to dryness under

reduced pressure at 20°C. The residue was dissolved in 20 ml of

distilled water and the resulting solution evaporated to dryness;

this procedure was repeated four times. The final residue was

dissolved in distilled water to a final volume of 3*0 ml which

contained 3.84 x 10 counts per minute per milliliter. This

latter solution was designated S^.

. The DL-Tartaric acid dehydrase of Pseudomonas sp. Shilo

was prepared and assayed according to the procedure of Shilo (44).

An aliquot of S^ was used as substrate for the crude tartaric

dehydrase; non-labeled DL-Tartrate was added to the assay system, Fig. 43.— Chromatography of fraction S, in various solvent systems.

Chromatogram Designation Solvent System Employed

0 •n-Butyric acid:n-Butanol:Water (2:2:1)

P n-Amylalcohol:5 M Formic Acid (1:1)

N Represents chromatogram of fraction l*f T Represents a tartrate-C standard

223 y ' V t- I o

4 ■>

Q. Q <3 •«»

. ; > DISTANCE■ FROM .-ORIGIN

Fig. V? Fig. ¥ t ,— Chromatography of fraction S., in various solvent systems.

Chromatogram Designation Solvent System Employed

P The organic phase of Ethylacetate: Pyridine:Water (36:10:11.5)

Q 70 per cent (aqueous) Phenol

225 226 y

Fig. 44 Fig. ORIGIN FROM DISTANCE DISTANCE DISTANCE DISTANCE FROM ORIGIN ------

4r~ltiO'2 0 0 9 1 — ^ <— Wdt) Q O O t — ^ Q Q O l — ^ ^ i!JlfO O O O I ^ 227 as the disodium salt, to give a final specific activity of

16,600 counts per minute per micromole of tartrate. At the end of the incubation period of 45 minutes the reaction mixture was deproteinized through the addition of trichloroacetic acid to a final concentration of 2.5 per cent. The acidified reaction mixture was centrifuged at 10,000 x g for 20 minutes. The supernatant and residue were separated by decantation and the keto acid of the supernatant was converted to the 2,4 dinitrophenylhydrazone and the latter extracted with xylene according to the procedure of Friedeman and Haugen (152).

The 2,4-dinitrophenylhydrazone was chromatographed de scending in the n-Butanol:Ethanol:0.5 normal Ammonium Hydroxide

(7sls2) solvent system described by Hawary and Thompson (153)»

The carbon-l4 labeled oxaloacetate-2,4-dinitrophenylhydrazone, from S^, migrated identically with authentic oxaloacetate-2,4- dinitrophenylhydrazone. l4 It was concluded that tartaric acid-1,4-C was the major radioactive compound present in fraction and the concentrate of the growth medium (upon which P. charlesii had grown for 28 days) which originally contained glucose, tartronate, and "carrier 14 free" tartrate-1,4-C as sources of organic carbon.

The question was raised as to the nature of the factors l4 which limited the metabolism of tartaric acid-1,4-C in the present experiment. Clearly the recovery in the 28-day old growth 14 medium of nearly 40 per cent of the added tartaric acid 1,4-C represents a departure from previous experiences involving tar- 228 trate metabolism in P. charlesii. Additionally in this experiment l4 about 3 per cent of the added tartrate-C was incorporated into the mycelium of the mould. In a companion experiment it was shown that approximately 9 per cent of the radioactivity initially l4 present in the growth medium as tartrate-1,4-C was recovered from the medium upon which Penicillium charlesii had grown for

28 days and approximately 10 per cent of the added tartrate-1,4- 14 C had been incorporated into the 28-day old mycelium.

It is quite probable that the observations of the present section result from some function of tartronate in the growth medium. If tartronic acid is a conversion product of tartrate in Penicillium charlesii then the addition of the former acid to the growth medium might be expected to result in a diminished utilization of tartaric acid by the fungus. Additionally, the similarities in the structures of these two acids suggests that tartronic acid might interfere with tartrate metabolism even if tartaric acid were not converted to tartronate in P. charlesii.

It is conceivable that tartronate might interfere with the trans port and/or intracellular metabolism of tartaric acid. A stereospecific transport system for tartrate could conceivably experience significant inhibition by a lower homolog (of tartaric acid) such as tartronic acid. Tartronic aciipl has been shown to inhibit the intracellularly-located tartrate dehydrase of Pseudomonas sp.

Shilo (154), the lactic dehydrogenase of mammalian brain slices

(155)» and E. coli (156), the malic dehydrogenase of pig heart

(157)» and of malic acid oxidation in pigeon liver extracts (158). 229

It would be desirable to eliminate one or two of the three possible effects of tartronate on tartrate metabolism which were outlined above. There are, at the time of this writing, no experimental data which allow a choice between the three suggestions.

14- (G) Incorporation of Glucose-u-C by Penicillium charlesii in the Presence of Tartronic Acid

Tartaric acid may be considered one member of a family of homologous alpha-hydroxydicarboxylic acids of which tartronic acid is the parent. The homologous series extends by the in sertion of hydroxymethylene groups between the carboxyl-group of tartronate and the alpha substituent.

A possible pathway for tartaric acid metabolism by P. charlesii has been suggested which involves the conversion of tartrate to dihydroxymaleate in a sequence which continues through transformation of dihydroxymaleate to tartronic acid semialdehyde. Additionally, tartronic acid semialdehyde might be converted to tartronic acid and it is conceivable that either of these two acids might be subsequently transformed into various metabolites by P. charlesii.

It has been observed that an oligogalactoside was produced by P. charlesii when tartronate was substituted for tartrate in the Raulin-Thom growth medium. It was also observed that the i 14- utilization of carrier-free tartaric acid-1,4-C by P. charlesii was inhibited when the mould was grown in the presence of 0.032 molar tartronate. Very little carbon-l4 from tartaric acid-1,4— 230 14 C was incorporated into galactocarolose in this latter case*

Alternative hypotheses of inhibition of transport and metabolism arid competition in metabolism were discussed.

If tartronic acid competes with tartaric acid by pro viding a precursor for galactocarolose then a dilution of the 14 radioactivity of glucose-u-C incorporated into the hexose units

of galactocarolose should be observed when tartronate replaces

tartrate in the Raulin-Thom medium upon which P. charlesii is

grown. An experiment designed to test this possibility was con

ducted and the results are recorded in what follows.

Penicillium charlesii was grown for 28 days upon 25 ml

of the Raulin-Thom medium which had been modified to contain

32 micromoles per milliliter of tartronate substituted for tar iff trate and a supplement of 50 microcuries of glucose-u-C • The

vessel in which the culture was allowed to proliferate was a

125-ml Erlenmeyer flask. Respired carbon dioxide was trapped in

sodium hydroxide. At the end of the 28-day growth period the

mycelium, residual medium, and carbon-l4 labeled carbonate were

treated and analyzed according to procedures defined under

'EXPERIMENTAL.'

The quantities of carbon-l4 recovered in the mycelium,

residual medium, and sodium carbonate are recorded in Table 25.

Only one radioactive area was observed when the concen

trated medium was chromatographed in 80 per cent phenol, the

organic phase of the Ethylacetate:Pyridine:Water (36:10:11,5)

solvent system, n-Butanol:Pyridine:Water (6:4:3) or Methanol: 231

TABLE 25 RADIOACTIVITY RECOVERED IN MYCELIUM, RESIDUAL GROWTH-MEDIUM, AND SODIUM CARBONATE WHEN P. CHARLESII METABOLIZED GLUCOSE-u-C1^ IN THE PRESENCE OF TARTRONATE

Radioactivity Recovered in Fraction Total Quantity of Fraction Specific Activity Total Activity

Fraction Ml Gms CPM/ml 10”4 CPM/mg 10"4 Counts x 10“^

Medium 6.0 83.020 19.86 concentrate

Sodium 200 39.034 78.07 carbonate

Mycelium 0.345 2.24 30.76

Barium 4.443 0.43 carbonate

Formic AcidsWater (80:15:5); the single area of radioactivity was immobile in these four solvent systems. Figure 45 records the nature of the various patterns obtained for the chromatography of the medium-concentrate and hydrolyzates in the Butanol:Pyridine:

Water (6:4:3) solvent system. The section of the figure labeled

N represents the chromatogram obtained for the concentrated medium. The single peak in section N Was designated S^ and was eluted with distilled water at 4°C. The eluate of S^ was con centrated and subjected to hydrolysis by 004 normal sulfuric acid at 95°C for 90 minutes. The hydrolyzate of was neutralyzed, freed of salts, concentrated under reduced pressure and the re sulting solution chromatographed on Whatman number 3 paper; the pattern of distribution of carbon-l4 from the hydrolyzate of 7 carolinie acid (2)* These and more recent investigations have shown that carolic acid and carolinie acid are the major hetero cyclic acids produced by Penicillium charlesii.

One of the oligosaccharides was found to be a homogeneous poly-D-mannopyranoside and was named mannocarolose. Exhaustive chemical studies which included fractional precipitation from the growth medium, methylation, acetylation, hydrolysis, and functional group analyses revealed that the mannopyranosyl units were mutually linked (X -1,6. Determinations by three different methods sug gested that the average chain of mannocarolose isolated consisted of 8 to 9 0(-D-mannopyranosyl residues (3).

Similarly comprehensive chemical analyses of the. second oligosaccharide disclosed it to be a water soluble "white,

amorphous, hygroscopic powder" • . • which was laevorotatory

(C°^3^g0 -80°) and rapidly hydrolyzed by very dilute acid.

Methylation and acetylation studies were interpreted to suggest

that the oligosaccharide contained only D-Galactofuranosyl

residues of -1,5-linkage. Molecular weight determinations sug

gested that the mean-length of the oligosaccharide chain waB 9 to

10 galactose units (4-). This communication composed the first

report of the occurrence in nature of the polygalactofuranoside.

(B) Occurrence in Nature of Oligosaccharides and Polysaccharides Containing Galactose

The very wide occurrence of oligosaccharides and polysac

charides which contain galactose has been reviewed by Whistler

and Smart (10). Among these is the galactogen of Helix pomatla T 1} Fig. ^ ‘““Distribution of C labeled components in growth medium concentrate and acid hydrolyzates.

Key

N Chromatogram of medium concentrate

0 Chromatogram of the 0.^ normal sulfuric acid hydrolyzate of fraction

P Chromatogram of 3*0 normal sulfuric acid hydrolyzate of fraction S^ 14 Q Galactose-C and glucose-C standards— obtained on two separate chromatograms.

The peak symmetrical at 6.3 inches from the origin is galactose while the faster moving component is glucose.

232 233

TA o i o o o

o o o 7

C..1 Q. d o o ©

0 1 2 3 4 5 6 7 8 9 10 II 12 13 14 IS 13 17 IQ !9

-a- ■■ a *-- » i « % . . -a 1---- !---- 1---- 1____ !_____ » ' DISTANCE FROM ORIGIN (INCHES)

Fig. 45 234

is represented by section 0 of Figure 45. Areas S2 and A were eluted from.the chromatogram and concentrated under reduced pressure. Fraction S2 was subjected to hydrolysis in 3*0 normal sulfuric acid at•95°C for 90 minutes. The cooled solution was neutralyzed with barium carbonate and the resulting barium-

sulfate removed after centrifugation. The neutralyzed solution was chromatographed to yield the pattern of carbon-l4 distribution

represented in section P of Figure 45. The peak designated S^ in

Figure 45 was eluted from the chromatogram and the eluate con

centrated under reduced pressure.

The amount of carbon-l4 and the results of other analyses

of fractions A and S, are recorded in Table 26. 3

TABLE 26 CARBON-14 CONTENT AND CARBOHYDRATE ANALYSES ______OF FRACTIONS A AND ______

Carbohydrate Analyses of Fraction (Values Expressed as Micromoles Per Ml) Radioactivity Present Total Total Total Reducing Fraction Volume Sugar Hexose Sugar Glucose CPM/M1

A 8.0 2.55 2.58 1.81 0,01 54,000 7*0 1.00 1.32 1.40 None 63,000 S3

The carbohydrate of fraction A was. .exclusively galactose.

It is perhaps significant that very little "galactocarolose" was

synthesized under the . conditions of growth that were employed in

the present experiment. This result has been observed whenever

P. charlesii was grown on volumes of growth medium which were 235 less than 50 milliliters. This observation has been interpreted to suggest that the quantity of galactocarolose synthesized by

P. charlesii is a function of the total amount of organic carbon compounds present in the growth medium rather than the concen tration of organic carbon compounds present;

When fraction A was chromatographed in 80 per cent phenol or the organic phase of the Ethylacetate:Pyridine:Water (36:10:

11«5) solvent system a single radioactive peak was obtained and the migration rate of this peak was identical with authentic galactose. The galactose content of fraction A was quantitated according to the procedures defined under ’EXPERIMENTAL.'

Table 27 affords a comparison of the specific activity of the glucose used initially in the growth medium with the

specific activity of the galactose isolated from galactocarolose.

TABLE 27 SPECIFIC ACTIVITY OF THE INITIAL GLUCOSE OF GROWTH MEDIUM WITH SPECIFIC ACTIVITY OF GALACTOSE ISOLATED FROM GALACTOCAROLOSE

Specific Activity Specific Activity at Initiation at Termination of Experiment of Experiment

Compound CPM/ jAmole CPM/ jjmole

Glucose 21,000 —

Galactose — 20,400

It was observed that the "carrier” acid exerted a pro

nounced effect on the pattern of distribution in the recovered

mycelium* residual medium, and respired CO2 of carbon-l4 from 236 14 l4 glucose-u-C or tartaric acid-1,4-C which was added to the

growth medium. Table 28 offers a comparison of the distributions

observed in 6 of the cases that have been studied.

(H) The Incorporation of Glucose-u-C^ by Penicillium charlesii Grown in the Presence of Tartaric Acid

Penicillium charlesii. when grown upon the Raulin-Thom medium, has been shown to convert glucose carbon to galactose

units of galactocarolose. A comparison of the initial specific l4 i4 14 activities of glucose-l-C , glucose-2-C , or glucose-u-C

supplied in the growth medium to the specific activity of the

galactose units isolated from galactocarolose suggested that the

glucose carbon chain had not remained intact during the glucose

to galactose transformation. The data obtained were interpreted

to suggest that glucose provided a 2-carbon unit which was in

corporated into the hexose units of galactocarolose (5)» The

glucose to galactose transformation probably did not occur

through a process of the type catalyzed by a galactwaldenase,

the ubiquitous enzyme which was first described by Leloir (103).

It would be of interest to determine the exact location

of carbon-l4 in the galactose units of the galactocarolose formed l4 when P. charlesii is allowed to metabolize glucose-u-C . An

experiment designed to achieve this objective involved the growth

of Penicillium charlesii on 150 ml of the Raulin-Thom medium l4 which contained approximately 60 microcuries of glucose-u-C .

The carbon dioxide which was released during the incu

bation was trapped in sodium hydroxide. At the end of the 28-

day growth period the mycelium, residual medium, and sodium- TABLE 28 EFFECT OF NATURE ON CARBOXYLIC ACID "CARRIER" ON ISOTOPE DISTRIBUTION FROM VARIOUS CARBON-14 PRECURSORS

Activity Reco.vered in Fractions

Sodium Source of Total Mediumi Mycelium Carbonate Total Organic Activity Radioactivity Carbon and Per Flask % of Total Wt. % of Total % of Total Recovered Radio activity >lc Total Grains Activity Activity CPM x 10“°

14 Glucose-u-C 50 16.40 0.229 10.79 72.73 + tartrate

Glucose + ^ 50 7.32 0.240 8.05 84.86 tartrate-C

Glucose + tar 50 3.06 0.330 37.37 59.38 112.16 tronate + ^ tartrate-C 14 Glucose-u-C 50 20.66 0.3^5 52.23 20.66 + tartronate

14 Glucose-u-C 50 46.23 2.163 14.54 39.24 101.72 + DHM 14 Glucose-u-C 2(50) 29.12 2.216 15.84 55.03 200.75 + tartrate 238 hydroxide-trap were analyzed for carbon-l4 content according to techniques described under 'EXPERIMENTAL.' Table 29 records the distribution of carbon-l4 among the various fractions analyzed upon termination of the experiment.

TABLE 29 RADIOACTIVITY RECOVERED IN MYCELIUM, RESIDUAL GROWTH MEDIUM AND SODIUM CARBONATE WHEN P. CHARLESII METABOLIZED GLUCOSE-u-C14 IN THE PRESENCE OF TARTARIC ACID

Total Quantity Radioactivity Recovered of in Fraction Fr ac ti on ------u Total , Fraction Ml Grams CPM/ml x 10-6 CPM/mg x 10' CPM x 10

Medium 19.0 3.081 58.539 concentrate lii "C^Og" 250 110.492 sodium hydroxide trap

Mycelium 4.432 0.716 31.725 lif BaC 0, 46.250 0.082 3 Total 200.756

14 Table 29 indicates that significant incorporation of C into the C^O^ and mycelium fractions has occurred. The medium concentrate was reduced to approximately one-third the volume recorded in Table 29 and the resulting dark brown solution was stripped upon Whatman number 3 bub chromatography paper. After complete drying, the paper chromatogram was developed, ascending, in the ButanoliPyridine:Water (6:4:3) solvent system. A 1.5-inch strip of the developed and dried chromatogram was analyzed for 239 distribution of carbon-14 labeled components as described under

'EXPERIMENTAL.' A representation of the scan which resulted is given in Figure 46. It was noteworthy that the chromatogram contained almost no free glucose; under the conditions of chro matography that were employed free glucose would migrate approxi mately 5*8 inches from the origin. The major component which remained at the starting line in the chromatography was eluted with cold distilled water at 4°C. After concentration of the eluate, the carbohydrate components were subjected to hydrolysis in 0.4 normal sulfuric acid at 95°C for 90 minutes. The cooled hydrolyzate was neutralyzed with solid barium carbonate and the completely neutral solution freed of salts according to the pro cedures described under 'EXPERIMENTAL.'

The concentrated neutralyzate was chromatographed as cending on Whatman number 3 mm paper in the n-Butanol:Pyridine:

Water (6:4:3) solvent system. When a strip of the dried chro matogram was scanned for radioactivity as described above, the pattern of carbon-14 distribution on the. paper was as represented in Figure 47. Three major components and one minor component

were observed. The three areas represented in Figure 47 were

eluted with cold distilled water and the resulting eluates were

concentrated under reduced pressure at about 20° to 22°C.. The

carbon-l4 content and results of carbohydrate analyses on

fractions A^ and are recorded in Table 30.

Analysis of fractions A^ and B^ revealed that both were

free of mannose. Chromatography of these two fractions in the ' Fig. 46.--Distribution of 0 -labeled components of medium concentrate after chromatography in the Butanol:Pyridine Water (6:4:3) solvent system.

Key

Sn Denotes areas on chromatogram which reduce silver nitrate

Bz Denotes areas on chromatogram which were positive in the benzidine-periodate spray procedure o O O o 00 1 DISTANCE FROM <5rIGIN

fig * *6

H% r

which has received recent attention by Weiniand and co-workers

(11). The characterization of this compound revealed the presence

of some L-galactose (12) although D-galactopyranosyl units pre

dominate*

The isolation from beef lung of a galactan which ac

companies but is separable from the heparin fraction has been

reported by Wolfrom and co-workers (13)* The material contained,

primarily, D-galactopyranosyl units but some L-galactose was also

thought to be present*

The disaccharide, lactose, is the most widely known and

studied of oligosaccharides which contain galactose* Galactose

is known to occur in cerebrosides and gangliosides of the nervous

tissue of vertebrates, and is a commonly found constituent of

glycoproteins* L-galactose is a common constituent of the blood

group substances and is the major component of numerous galactans

which find especially wide distribution in plants and micro

organisms.

Galactose occurs as its uronic acid in the pectins (l4)

and as a constituent of the galactomannans of legumes* Larch

wood contains a polysaccharide material whose major hexose com

ponent is galactose* This hexose occurs in agar in both the

D-galactopyranosyl and the L-3»6-anhydrogalacto-pyranosyl forms*

Galactose is also a constituent of the plant gums and mucilages*

The furanosidic forms of aldohexoses are quite rare of occurrence in nature whereas this statement is not true of 14 Fig. 47.--Distribution of C -labeled components of 0.4 normal sulfuric acid hydrolyzate of fraction S^.

Key

Sn Denotes areas on chromatogram which reduce ammoniacal silver nitrate

Bz Denotes areas on chromatogram which give positive reaction in the benzidine-periodate spray procedure

242 5T § Q

A->

DISTANCE FROM 0R16IN >

M . .

.Fig. k7

rv> 1 vn 244

TABLE 30 CARBON-14 CONTENT AND RESULTS OF CARBOHYDRATE ANALYSES ON FRACTIONS AND Bx

Total Carbohydrate Analyses of Fraction Volume (Values Expressed as of Micromoles/Hi) Radioactivity Fraction in Fraction Total Reducing Fraction MlSugar Hexose Sugar Glucose CPM/M1

9.2 12.2 12,0 9.75 None 166,000 ■*1 9.0 28.0 26.1 24.50 1.00 158,000 Bi organic phase of the Ethyl Acetate:Pyridine:Water (36:10:11*5) solvent system showed that fractions A^ and B^ were free of pentoses. Analysis of the two fractions for galactose according to the method of Fisher and co-workers (86) suggested that galactose was the only monosaccharide present. Fraction A^ con tained a significant quantity of a carbohydrate component which migrated with an R^ft^ value which was similar in magnitude to standard disaccharides. Additionally, fraction A^ contained a 9 small amount of a non-carbohydrate residue which was not identi fied.

Fraction Sg, which resulted after chromatography of the neutralyzate from the 0.4 normal acid hydrolysis, was concentrated . and the resulting solution was made 3*0 normal with respect to sulfuric acid and heated at 95°C for 90 minutes in a boiling water bath. The solution was cooled to room temperature and

neutralyzed. The neutralyzed solution was freed of salts and 245 concentrated as defined under 'EXPERIMENTAL.1 The concentrate was stripped upon Whatman number 3 MM chromatography paper and the dried paper developed in the n-Butanol:Pyridine:Water (6:4:3) solvent system. The distribution of carbon-14 labeled components on the chromatogram is represented in Figure 48. Two major com ponents were present. Similarly, when the 3*0 normal acid hydrolyzate of S^ was chromatographed in the organic phase of the Ethylacetate:Pyridine:Water (36:10:11*5) solvent system or in 80 per cent phenol only two radioactive areas were observed.

The result represented in Figure 48 suggested that area contained glucose and/or galactoae. Fraction. 83 and A., were eluted, concentrated, and subjected to the analyses summarized in Table

51-

TABLE 31 CARBOHYDRATE ANALYSES AND CARBON-14 CONTENT OF FRACTIONS S, AND A- 2 £•______Carbohydrate Analyses of Fractions (Values Expressed a6 Micromoles Radio Total Per Ml) activity Volume Recorded Total Total Reducing Fraction Ml Sugar Hexose Sugar Glucose CPM/M1

11.1 7.25 3.0 2.5 None 178,430 S3

30.0 29.2 23.6 36,850 A2 12.5 32.5

The identification of glucose as a component of fraction

A^ is based upon the use of the Glucostat reagent and the coupled system composed of hexokinase and glucose-6-phosphate dehydro- 14 Fig. 48.--Distribution of C -labeled components of 3.0 normal sulfuric acid hydrolyzate of fraction S

Key

Sn Denotes areas on chromatogram that reduce ammoniacal silver nitrate

Bz Denotes areas that give positive reaction in the benzidine-periodate spray procedure

246 -* : > DISUNCE FRo M origin

Sn %

Fig. kS

IV 248 genase. The methods employed in these determinations were described under 'EXPERIMENTAL.'

Although the glucose oxidase of the Glucostat preparation will attack mannose (112) and hexokinase will attack and phos- phorylate numerous sugars in the presence of adenosine-5 '-triphos phate (113) the reaction catalyzed by glucose-6-phosphate dehydro genase appears to be specific for glucose-6-phosphate (114). The results of the analytical procedures involving glucostat. and

glucose-6-phosphate dehydrogenase argue for the suggestion that

glucose is the major carbohydrate present in fraction

The observation of glucose as a component of the 3«0 normal acid hydrolyzate raised the question as to the origin of

the hexose. Were it present in the concentrate of the 28-day

old growth medium, free-glucose would have been observed in the

initial chromatography, represented in Figure 46. If glucose had

been present in fraction as a bound glucofuranoside the glucose

would have been released from fraction by the 0.4 normal acid

treatment. Hexofuranosides are quite labile in dilute acid (104).

It is significant that no glucose was present in fractions

and B^«

It might be suggested that the glucose observed in

fraction was present as a glucopyranoside in fractions and

The significance of the presence of bound-glucose in the

concentrate of the growth medium is not known at present.

Table 32 represents a summary of data representing the

percentage distribution of carbon-l4 between the various fractions

analyzed at the end of the 28-day growth period. 249

TABLE 32 i 4 DISTRIBUTION OF CARBON-14 OF GLUCOSE-u-C IN THE MYCELIUM MEDIUM AND C02 PRODUCED BY P. CHARLESII

Total Radioactivity in Fraction Percentage of Total Fraction CPM x 10“6 Recovered Radioactivity

Medium conc.* 60.122 28.35 14 NagC 0^ 123.158 58.14

Mycelium 28,562 15.51

All fractions 211.842 100

* Denotes concentrated growth medium.

14 (I) The Incorporation of Glucose-u-C by Penicillium charlesii Grown in the Presence of Dihydroxymaleic Acid

One possible pathway for tartaric acid breakdown and conversion to galactocarolose by P. charlesii involves the for mation of dihydroxymaleic acid (abbreviated DHM in part of what follows) as an intermediate. This latter compound has been shown to be the first product of the oxidation of tartaric acid in the presence of extracts of beef heart (37)* There appear to be no reports of the conversion of tartaric acid to dihydroxy maleic acid by enzymes isolable from fungi.

Decarboxylation of dihydroxymaleic acid would yield tartronic acid semialdehyde which is a widely-occurring precursor of glyceric acid in plants and in microorganisms, (115). The conversion of tartrate to tartronic acid-semialdehyde might provide a means by which tartrate carbon is incorporated into 250 cellular materials and the tartrate-derived precursor of galactocarolose.

When P. charlesii was grown in the presence of the Raulin- l*f Thom medium which was supplemented with glucose-u-C appreciable incorporation of isotopic carbon into galactocarolose was shown to occur (5» 23)» If dihydroxymaleic acid is an intermediate in the conversion of tartaric acid to galactocarolose then substi tution of dihydroxymaleate for tartrate in the Raulin-Thom medium might yield useful information which relates to the tar trate-derived precursor of galactocarolose.

Several experiments might be undertaken to clarify the relationship between tartrate and dihydroxymaleate as precursors of the galactose units of galactocarolose. These experiments include the examination of the distribution of radioactivity in galactocarolose and other cell constituents and metabolites when P. charlesii is grown upon the Raulin-Thom growth medium which contains the following combinations of carbon-l^f labeled and non-labeled organic carbon sources. l4 (a) Glucose-u-C + dihydroxymaleate l^f (b) Glucose-u-C + tartrate

+ dihydroxymaleate l4 (e) Glucose + dihydroxymaleate-C

(f) Glucose + dihydroxymaleate-C l^f (carrier free)

+ tartrate 251 14 The unavailability of dihydroxymaleate-C precludes experiments (e) and (f). Experiments (a), (b), and (c) have been carried out whereas experiment (d) has not been undertaken.

The results of experiment (a) form the topic of this section.

The total volume of the growth medium used in this ex periment was 150 ml and a wide mouth, 500-ml Erlenmeyer flask served as the culture vessel. 14 Respired C 0^ was trapped in 200 ml of 10 per cent potassium hydroxide. The entire experimental system was enclosed in a large jar (10 x 18 inches) which was sealed in such a

fashion that C02~containing air was excluded. At the end of the

28-day growth period the medium and mycelium were separated as

described under experimental. Considerable difficulty was en

countered during the concentration of the ..medium and much of the

difficulty was due to the syrup-like nature of the concentrate.

This highly viscous residue has often been observed upon concen

tration of the dihydroxymaleate-containing growth medium of P.

charlesii. The smelliest volume of medium concentrate obtainable

was 20 ml. The operations performed on the mycelium and potas

sium carbonate are those described under 'EXPERIMENTAL.'

The total activity recovered in the various fractions l^j. (C Opt medium concentrate, mycelium) is recorded in Table 55*

Particular note was made of the high (47 per cent) per

centage of the recovered activity that was in the growth medium

at the end of the 28-day growth period of P. charlesii. 9 aldopentoses and ketopentoses. Fructose is found as a constituent of sucrose, levan, inulin, and irisin; fructose is present in the furanose form in these four compounds. Examination of a large number of fructose-containing glycosides has afforded the widely held generalization that in naturally occurring fructosides the ketohexose is always present in the furanose form. An enzyme system from Leuconostoc messenteroides reacts with sucrose with the formation of leucrose, a disaccharide which has been shown to be 5-0-oC-D-glucopyranosyl-D-fructopyranose (15)•

(D) The Occurrence of Glycosides which Contain Galactofuranosyl Moieties

Information concerning the occurrence in nature of manno- furanosides and glucofuranoeides is lacking and only six well- documented instances have been noted of the presence of galacto furanosyl residues in naturally occurring glycosides*

(1) Umbilicin

The earliest reports on the occurrence of the unusual galactoside of lichens are due to Wachmeister (16). Definitive work on the structure of this galactoside was reported by Lind- berg and Wickberg (17)* Optical rotation periodate consumption, methylation and benzoylation studies suggested that the compound was D-arabitol--D-Galactofuranoside. The isolation from the hydrolyzate of fully methylated Umbilicin of 2,3,5»6-tetramethyl-

D-Galactose provided evidence that the galactose residue must have been in the tvucanoee form and linked to arabitol through the one-position of the hexose. The confirmation of the structure of 252

TABLE 33 DISTRIBUTION OF C1*1- RECOVERED WHEN P. CHARLESII WAS ALLOWED TO INCORPORATE GLUCOSE-u-C14 IN THE PRESENCE OF DIHYDROXYMALEIC ACID

Total Quantity Radio of Total activity Fraction Specific Activity Activity Recovered as Ml Mg CPM/M1 x 10 CPM/Mg x 10"4 CPM x 10"^

Medium 20 237.86 ^7.02 concentrate

Potassium 220 17.32 39.92 carbonate

Mycelium ^325.5 0.3^95 1^.78

Barium 0.0^-83 carbonate

Total 101.72

Some difficulty was encountered when the medium concen

trate was applied to Whatman number 3 MM paper for initial,

resolution of the carbohydrate components. The syrup-like nature

of the concentrate was responsible for difficulty in drying areas

on the chromatography paper to which the viscous concentrate was

applied.

The pattern of resolution of isotopic compounds was a

function of the quantity of the concentrate originally applied

to the chromatography. The pattern of distribution after chro

matography in the n-Butanol:Pyridine:Water (6:^:3) solvent system

is represented in Figure 4-9* The areas indicated by the letters

S, T, U, V, and W were defined as shown and eluted from the chro

matogram in strips and at 4°C. Similarly a portion of Whatman lA Fig. 49.--Distribution of C label on chromatograph of, medium concentrate when P. charlesii incorporated glucose-u- in the presence of dihydroxymaleic acid.

253 3000cpm

20 22 3000 cpm 1.0 a.

0 2 4 6 8 10 12 14 16 18 20 22

DISTANCE FROM THE ORIGIN (INCHES)

F ig . kS ro VJl -p-

■% 255 number 3 MM paper containing no added carbohydrate was treated with the Butanol:Pyridine{Water (6:4:3) solvent system and a section of the dried paper weighing the same amount as the heaviest strip (S^, in this case) was eluted as a blank cor rection. As is discussed later, the blank contained negligible traces of carbohydrate.

Operations performed on the various eluted and concen trated fractions are described in the following sections which were defined as indicated below.

Section of Text Fractions

(A) Starting line S^

(B) T and U

(A) Analysis of fraction S1

Concentration of fraction S^ yielded a syrup which was similar in appearance to the initial medium concentrate. When the concentrate was treated with 0.4 normal sulfuric acid at

45°C for 90 minutes and neutralyzed the concentrated desalted mixture gave the pattern of distribution (of labeled compounds represented in Figure 50» The areas indicated were eluted from

the chromatogram and concentrated to small volumes. Results of

some analyses of fractions A and B are represented in Table 34. ‘

The area designated B (fraction B) corresponded to.that

region where authentic galactose migrated under the conditions

of chromatography that were employed. This fraction contained

negligible traces of material which reacted with hexokinase and l*f Fig. 50.--Distribution of C label on chromatogram of 0.4 normal sulfuric hydrolyzate of S^,

256 1000 cpm < lOOOcpm; 0.5 0.5 1.0 0 0 2 2 4 4 6 8 6 ITNE RM ORIGIN (INCHES) FROM DISTANCE , >' B, 8 10 10 F ig . . ig F 50 21 16 14 12 12 14 16 GALACTOSE-C

10 18 20 20

22 22 257 258

TABLE 34 PRELIMINARY ANALYSES OF FRACTIONS A AND B DERIVED FROM HYDROLYSIS OF S^^

Carbohydrate Analyses of Fraction Radio Total (Values Expressed as Micromoles Per Ml) activity Volume Present Totsil Totsil Reducing u a e o e u a Fato Ml Sugar Hexose SugarFraction Glucose Galactose CPM/Ml

7.0 102 86.0 97.0 148,530 *1 12.7 85.4 7.0 B1 140 140 136.5 7.5 131.2 162,030 glucose-6-phosphate dehydrogenase under the conditions of assay defined under 'EXPERIMENTAL.1 Fraction B was not resolved into two or more components upon chromatography in the organic phase of the Ethyl Acetate:Pyridine:Water (36:10:11*5) solvent system.

The quantitative reaction of the carbohydrate of this fraction in the Fischer procedure for galactose (86) and stoichiometric reaction in the presence of crude galactose dehydrogenase of

Pseudomonas sacch. afforded the suggestion that galactose was the only monosaccharide present.

Fraction A contained negligible glucose but not all of the carbohydrate present existed in monosaccharide form. The

Fischer procedure indicated that little non-galactose carbo hydrate was present. On the other hand when sin aliquot of this I fraction was analyzed with crude galactose dehydrogenase only

40 per cent of the ceirbohydrate present reacted as galactose.

It is possible that fraction A contained di and tri gsilactosides but this was not proven. Fig. 51•--Distribution of carbon-l4-labeled components after hydrolysis of fraction id 3*0 normal sulfuric acid.

259 CPM I X 10 XI <-S itne rm rgn (Inches) Origin From Distance g. 51 . ig F

20 20

22 22 260 261

After concentration, hydrolysis in 3.0 normal sulfuric acid neutralization and chromatography, fraction S^ gave rise to the pattern of distribution of carbon-1^ labeled components that is represented in Figure 51. The small amount of radio activity symmetrical about a point 11.25 inches removed from the origin corresponded to the position to which mannose would migrate under the conditions of chromatography. The fractions designated A2 and were eluted from the chromatogram with dis tilled water. The elution was performed at ^°C. The distribution of carbon-l4 and results of several other analyses are recorded in Table 35*

TABLE 35 DISTRIBUTION OF C1^ LABEL AND RESULTS OF CARBOHYDRATE ANALYSES OF FRACTIONS A- AND S, £ 5 Total Volume Carbohydrate Analyses of Fraction Radio of (Values Expressed as Micromoles/Ml) activity Fraction ------Present ------Total Total Reducing Galac Fraction Ml Sugar Hexose Sugar Glucose tose CPM/M1

A2 . 10.0 15.82 15.32 16.21 1.35 15.01 3^,27^

S ' 13.7 1.^0 2.30 1.26 None None *f2,500' 5

Neither of the two fractions contained significant quantities of carbohydrate identifiable as glucose. Fraction

Sj was dark brown in color and contained a significant quantity of salt. This fraction appeared to contain very little sac charide material and was not further investigated. 10

Umbilicin as 1-0-(3-D-arabitol)-^ -D-Galactofuranose was obtained by synthesis.

(2) The extracellular polysaccharide of Gibberella fu.iikuroi

Galactofuranosyl residues have been shown to represent a

significant percentage of the carbohydrate portion of a protein-

linked extracellular polysaccharide produced by Gibberella

fu.jikuroi. "Growth of Gibberella fujikuroi (Fusarium moniliforme)

on a glucose medium produced an extracellular polysaccharide con

taining D-glucose, D-mannose, D-galactose, D-glucuronic acid

(molar ratio 1.0tl«l:1.3:0.6), and possibly D-mannuronic acid.

Protein was retained tenaciously by the polysaccharide and several

deproteinzation methods reduced the nitrogen content only slightly.

Methylation studies showed that the polysaccharide was highly

branched with D-glucose and D-mannose forming the non-reducing

ends in the molecule. Host of the D-mannose and D-galactose units

were joined by 1 — > 2 and 1 > 6 linkages with some branching

also at and Cg positions; D-galactose occurred exclusively in

the furanose form. D-Glucose units were joined by 1 — > 2 and

1 — y 3 linkages. The D-glucuronic acid residues were mainly non

terminal and were attached to both D-galactose and D^mannose units.

Periodate oxidation studies supported the foregoing conclusions."

CAuthors abstract (127)3.

(3) The galactomannan of Trichophyton granulosum

The antigenic properties of certain of the polysaccharides

isolated from several dermatophytes has stimulated research 262

(B) Analysis of Fractions T and U

The location of fractions T and U as a result of the initial chromatography (Figure ^9) suggested that these two areas did not contain free, underivatized monosaccharides as major components. The location on the chromatogram of T and U raised the question as to whether the two fractions contained di, tri, and/or tetra-saccharides or unusual monosaccharides which were precursors or degradation products of galactocarolose. That these two areas contained significant* carbon-l^f labeled material and carbohydrate was indicated by the analyses recorded in Table

36.

TABLE 36 1*+ DISTRIBUTION OF C LABEL AND RESULTS OF CARBOHYDRATE ANALYSES OF FRACTIONS T AND U

Carbohydrate Analyses of Fraction Radio- Total (Values Expressed as Micromoles Per Ml) activity Volume Total Present ----- Total Reducing Total Pen- Methyl- Glu------Fraction Ml Sugar Sugar Hexose tose pentose cose CPM/M1

T 15.0 12^.8 87.5 75.0 6.60 5 38. k 233,025

U 1^.6 38.6 39.5 25 • - 5.5 33.6 176,993

The analyses recorded above clearly indicated that both

fractions contained significant quantities of non-hexose carbo hydrate. It was anticipated that further insight into the exact nature of the components of these two areas would be gained by

subjecting the fractions to the chromatographic, chemical hydrolytic and enzymatic procedures described in what follows. 263

Two milliliters of each fraction were hydrolyzed in capped tubes for 90 minutes at 97°C in 0.2 normal and 0.4 normal sulfuric acid respectively. After being cooled to room temper ature the tubes were opened and the contents neutralyzed with solid barium carbonate, the resulting barium sulfate removed by centrifugation and the supernatants stripped upon Whatman number

3 MM chromatography paper and chromatographed descending in the organic phase of the Ethyl Acetate:Pyridine:Water (36:10:11.5) solvent system. Figures 52 and 53 represent the patterns of distribution of labeled components that were observed.

These chromatographic patterns suggested that both fractions T and TJ contained several labeled components. An increase in the concentration of acid (from 0.2 to 0.*f normal) used in the hydrolysis appeared to increase the fraction which was immobile under the conditions of chromatography. The various peaks obtained as a result of chromatography of the hydrblyzates were labeled 1, 2, and 3 respectively as indicated in Figure 52 and Figure 53 • It was assumed that corresponding areas from the

0.2 and O.^f normal acid treated fraction T contained similar l^f components; corresponding-C -containing areas (for example, fraction 1 from the 0.2 normal acid treatment and fraction 1 from the 0.^ normal acid treatment) were pooled and eluted at

4°C with double distilled water. An identical assumption was made for the acid treated fraction TI; similarly, elution was performed at 4°C with double distilled water. The distribution of radioactivity and results of other analyses are represented in Table 37* Fig. 52.-— Chromatograjn of fraction T subjected to hydrolysis in 0.2 normal and 0.4 normal sulfuric acid.

Key

N No hydrolysis

0 Hydrolysis in 0.2N sulfuric acid

P Hydrolysis in. 0.4N sulfuric acid

Q Standard galactose (Gal) and glucose (Glu)

264 lOOOcpro-^* «£—IOOOcprn-T> lOOOcpm-S’ «a— lOOOcptn— RCIN NOHYDROLYSIS T FRACTION 0 2 4 6 ITNE RM ORIGIN (INCHES) FROM DISTANCE 8 Gal Fig. 52' 10 214 12 618 16 265 Fig. 53.“-Chromatogram of fraction U subjected to hydrolysis in 0.2 normal sulfuric acid and 0.4 normal sul furic acid.

Key

N No hydrolysis

0 Hydrolysis in 0.2N sulfuric acid

P Hydroylsis in 0.4N sulfuric acid

Q Standard galactose (Gal) and glucose (Glu)

266 :-1000cpm-?» <— lOOOcpr.:-^

TABLE 37 14- DISTRIBUTION OF C LABEL AND RESULTS OF CARBOHYDRATE ANALYSES OF FRACTIONS RESULTING FROM HYDROLYSIS OF T AND U

Volume Carbohydrate Analyses of Fraction Radio of (Expressed as Micromoles Per Ml) activity Fraction Total Reducing CPM n _-4 -rrzr x 10 Fraction Ml Sugar Hexose Sugar Glucose Ml

5.0 0.720 0.341 0.600 - 5.011 Ti 4.0 19.00 20.00 22.50 8.174 . ' 20.35 4.o 0.575 0.090 0.180 - 4.616 T3 0.200 U1 3.4 0.500 0.250 - 3.772

D2 3.5 36.50 24.60 22.00 28.31 19.914 4.5 0.100 0.080 0.180 - 2.081 n3

Although the data of Table 37 suggested that glucose was

the major component of both fractions T and U, these data could

also be interpreted to suggest that both fractions contained

carbohydrate material which was not hexose. This latter sug

gestion was confirmed by two different procedures; (l) chro

matographic analysis of fractions T and U in six different sol

vent systems provided evidence that each fraction contained five

to six component carbohydrates, and (2 ) chemical analyses for

aldonic acids, ketohexose, deoxyhexose, deoxypentose, uronic

acid, hexose, pentose, and glucose provided evidence that several

of these carbohydrate species were present.

The chromatographic and chemical analyses of fractions T

and U suggested that both contained ketohexose, methyl pentose, and pentose in significant quantity. Ribose and xylose appeared to be present. The ketopentose and methylpentose have not been identified. Figure 54 represents the results of chromatography of fraction T in the Ethyl Acetate:Pyridine:Water (36:10:11.5) solvent system. The chromatography was descending and the sol vent flowed off the lower edge of the chromatogram for three

hours. At least one component of T was lost by this procedure.

The a r e a s indicated were eluted at 4°C with double-distilled

water and the distribution of radioactivity in the various

fractions recorded in Table 33.

TABLE 38 Ik DISTRIBUTION OF C LABEL IN CHROMATOGRAPHICALLY RESOLVED COMPONENTS OF FRACTION T

Total Total Activity Volume in Fraction

Fraction Ml CPM x 10-5

ta 8.5 6.257

®B 8.5 9.362 Tc 8.0 8.042

Perhaps of some significance is the fraction (area) A

in Figure 54. Figure 54 and Table 38 suggested that this

material, which does not appear to be present in acid treated

fraction, is not glucose. Fraction T^ may represent a component

which was destroyed by the acid treatment. That better resolution

of the fractions was not achieved (Figure 5*0 may be a reflection T Zj. Fig* — Distribution of C -labeled components of fraction T after chromatography in an Ethyl AcetatetPyridine: Water solvent system.

Glu and Man represent standard glucose and mannose respectively 3000 cpm > < lOOOcpm 0.5 0.5 1.0 I. ITNE RM ORIGIN FROMDISTANCE (INCHES) Fig. & — —< B— <— <—A— l X Man X Glu y — —?“ c— 20 22 11 interests related to the purification and molecular definition of numerous of these unusual carbohydrates* Bishop and co-workers have isolated a galactomannan from powdered cells of Trichophyton granulosum* The galactomannan, which could be resolved by chro matography on diethylaminoethylcellulose, was shown to be protein free and contained 16 per cent galactose and 84 per cent mannose*

D-mannose was present exclusively as mannopyranosyl residues which were highly branched. The D-galactose residues were terminal non reducing furanoside units* According to the authors • • • ”the polysaccharide from T* granulosum is the first galactomannan isolated from a microorganism but some of its structural features can be related to those occurring in other microbial polysaccha rides." (126).

(4) The complex oligosaccharide of Type 34 Pneumococcua

Brown and Robinson (128) were able to prepare the type specific substance from Type 34 Pneumococcus and it was later shown by Shabarova and co-workers (129) that this type specific material contained phosphorus in addition to ribitol, glucose, and galactose* More recently Roberts and co-workers have performed extensive chemical analyses of the oligosaccharide from the specific substance of Type 34 Pneumococcus and these authors have reported a glucoses galactose:phosphate ratio of 1:2:1* Additionally the galactose residues present in the intact substance could be con verted to arabofuranose units by sequential periodate oxidation and borohydride reduction* It was suggested that the type specific 272 of the time of extended flow of the chromatographic solvent suid

the fact that the mixture (and not the organic phase) of the

Ethyl Acetate:Pyridine:Water (36:10:11.5) solvent system was

employed in the present case.

When fraction U was chromatographed in the organic phase

of the Ethyl Acetate:Pyridine:Water (36:10:11.5) solvent system

and a strip of the dried chromatogram was scanned for radio

activity, the patterns of Figure 55 were obtained. The indicated

areas were eluted and the distribution of carbon-1^ labeled com

pounds recorded in Table 39*

TABLE 39 DISTRIBUTION OF C1**" LABEL IN CHROMATOGRAPHICALLY RESOLVED COMPONENTS OF FRACTION U

Total Volume Total Activity of in Fraction Fraction

Fraction Ml CPM x 10~5

7.8 2.083 "a 7.8 1.686 "b 13.0 Dc 6.325

It was of interest to note that several areas of the

chromatographed fractions which reduced ammoniacal silver nitrate

to give very dark spots contained very little or no radioactivity.

This observation is graphically represented in Figure 56 and

Figure 57* The chromatogram was obtained by spotting aliquots

of the indicated fractions on Whatman number 3 MM paper and Fig. 55.— Distribution of C X*T labeled components of fraction U after chromatography in the organic phase of an ethylacetate:pyridine:water solvent system.

Glu and Man represent standard glucose and mannose respectively £ oQ. o o o to

Ol o O

6lu \ / Man

0 2 4 6 8 10 12 14 16 18 20 22 DISTANCE FROM ORIGIN (INCHES)

Fig. 55

ro -o •p- 1 if Fig. 56 .--Distribution of C labeled components of fraction T after chromatography in 80 per cent phenol.

Sn Denotes areas on chromatogram that reacted with ammoniacal silver nitrate

Bz Denotes areas on chromatogram that reacted in the periodate-benzidine procedure

275 Y -lOOOcpm /i 22 { i 20 ( i ) i ) )( c?

Sn Denotes areas on chromatogram that reduced ammoniacal silver nitrate

Bz Denotes areas on chromatogram that reacted in the benzidine-periodate procedure

277 22 20 18 16 14 12 10 8 6 4 1 2 0 DISTANCE FROM ORIGIN (INCHES)

( ) C = 3

Fig. 57 279 development of the dried paper with 80 per cent phenol in the descending direction. The "reducing" areas were visualized with ammoniacal silver nitrate according to the procedure in reference under 'EXPERIMENTAL.'

The latter observation suggested that significant quan tities of unlabeled dihydroxymaleic acid of the growth medium may have been incorporated into components that will reduce am moniacal silver nitrate.

The data of this subsection are interpreted to confirm the assumption that fractions T and U obtained from the initial chromatography of the concentrated growth medium contained com ponents other than simple aldohexoses. Glucose was found to be the primary monosaccharide present in the mixture of carbohydrates and on the basis of chromatography in several solvent systems the aldopentoses, ribose, and xylose were identified.

A methylpentose (6 deoxyhexose) was observed to be present in relatively high concentration when the dihydroxymaleate- supplemented Raulin-Thom medium was concentrated and chromato graphed. The exact nature of this deoxysugar has not been de termined.

The mobility of fractions (areas) V and W in the original chromatography (Figure *f9) suggested that these two fractions consisted primarily of monosaccharides. Chromatographic, chemical, and enzymatic analyses suggested that fraction V was simple, 280 consisting of one major and two or three very minor components.

Similar analyses indicated that fraction W consisted of a mixture of about seven or eight chroraatographically distinguishable com ponents. The distribution of radioactivity and results of other analyses of fractions V and W are recorded in Table 40.

TABLE 40 1^ DISTRIBUTION OF C LABEL3EL AND RIRESULTS OF CARBOHYDRATE ANALYSES OF FRACTIONS V AND W

Volume Carbohydrate Analyses of Fraction Fraction ^ a^-ues Expressed as Micromoles/Ml) Present^ Total Total Reducing Fraction Ml Sugar Hexose Sugar Glucose CPM/M1

V 11.8 11.1 9.5 9.6 9.6 374,760

W 20.5 22.4 23.4 20.5 13.7 322,927

When analyzed independently of other observations the data of Table 40 suggested thqt further analyses of fractions V and W would not be necessary. Table 40 expresses a ratio of 1,0 for hexose"' a va^ue 1*17 ,in fraction V.

The corresponding ratio for ^^exose^1* does not differ signifi cantly from 1.0 for fraction W. Other colorimetric analyses, however, revealed that fractions V and W were not so simple as

Table 40 predicts. Table 4l indicates that both fractions V and

W contained significant methylpentose and smaller amounts of aldopentose.

That both fraction V and fraction W were quite hetero geneous with respect to carbohydrate components, was confirmed by subsequent chromatographic analyses in five different solvent . 281

TABLE kl CARBOHYDRATE ANALYSES OF FRACTIONS V AND W

Volume Carbohydrate Analyses of Fraction of (Values Expressed as Micromoles Per Ml) Fraction Total Total Deoxy- Methyl- Fraction Ml Sugar Pentose Pentose Pentose

V 11.8 11.1 4-.00 1.00 10.00

W 20.5 ZZ.k 2.00 1.00 7.55 systems. The results obtained when chromatography was conducted,

descending, in 80 per cent phenol are represented in Figures 58

and 59.

Figures 60 and 6l delineate the results obtained when

fractions V and W, respectively, were subjected to descending

chromatography in the organic phase of the Ethyl Acetate:Pyridine:

Water (36:10:11.5) solvent system using Whatman number 3 MM chro matography paper. The indicated areas were eluted from the two

chromatograms with double-distilled water at 4°C. The distribution

of carbon-l^f in the various concentrated fractions is recorded in

Table kZ.

The R _ value for area C of fractions U, V, and W glucose * * is approximately 2.00. It would be of interest to determine if

the C areas from the three fractions contain a common component

as the Bglucose values suggest.

It was of interest to note the presence of significant

quantities of deoxysugar in fractions V and W. This observation

is without precedent in analyses of the partially expended Raulin-

Thom medium upon which Penicillium charlesii has been allowed to ACKNOWLEDGMENTS

I wish to acknowledge the very generous counsel and assistance given me by my graduate adviser, Dr. John E. Gander, without whose patience, guidance, instruction, and knowledgeable approach to the subject this work would have been impossible.

Sincere thanks are accorded the many members of the instructional staff of The Ohio State University whose highly- valued instructions are difficultly enumerated and defined,

I express my appreciation to Miss Margie Rausch for invaluable assistance rendered in preparation of illustrations

contained in the text.

A particular note of thanks is extended Dr. J. E. Varner

who provided me with an introduction to the discipline of bio

chemistry, and whose approach to and understanding of this

subject, though impossible to emulate, have provided unique

challenges and references for future thought, study, and

experimental endeavor.

I express my sincere gratitude for fellowships awarded

me by the Mershon Foundation of The Ohio State University and

the National Institutes of Health of the Department of Health,

Education, and Welfare of the United States.

ii 12 substance contained at least one galactofuranose unit for each unit of ribitol phosphate present and that the galactofuranosyl moiety contained a 2 or 3-0-phosphoryl substituent which was linked through a phosphodiester bridge to the 1(3) hydroxyl group of ribitol (73)*

(5) The polysaccharide of Cladophora rupestris

The presence of an unusual galactose-containing polysac charide in the green sea weed Cladophora rupestris has been re ported by Fischer and Percival (18). The material, when isolated with boiling water or dilute acid was found to contain etherally linked sulfate and the following aldoses, arabinose, galactose, xylose, rhamnose, and glucose in the molar ratio of 3*7:2,8:1,0:

0,4:0.2.

When the desulfopolysaceharide was methylated and the methylation product hydrolyzed, galactose and the following galactose derivatives could be isolated and identified; 2,3*4,6- tetra-O-methyl galactose; 2,3,3‘~t?i**0’*methyl-D-Galactose; 2,4-di-

0-methyl-D-galactose, and 2-0-methyl-D-galactose,

The presence in the hydrolyzate of tetra-O-methyl-D- galactose suggested that a portion of the galactose functioned as end-units in the polysaccharide while the 2,3,5~tri-0-methyl compound reflected the galactofuranose units linked 1,6 in the native polymer. The authors suggested that "the monomethyl derivative and the free galactose residues situated in a main chain and at branch points, as such residues, if they carried sulfate groups would have at least one hydroxyl group free for methylation," Fig. 58.— Distribution of C -labeled components of fraction V after chromatography in 80 per cent phenol.

282 J

6 CLO o o O

. I L 22 20 DISTANCE FROM ORIGIN (INCHES)

BZ £ 3 O

□ D O

Figo 58 283 • 14 Fig. 59.— Distribution of C -labeled components of fraction after chromatography in 80 per cent phenol.

Sn Denotes areas that reduced ammoniacal silver nitrate

Bz Denotes areas on chromatogram that reacted with periodate-benzidine procedure

284 VW/W^VVw\/^A/V\y<\^''AAvLA/vv^ I I « I > I 1 — I I I I I L . 22 20 18 16 14 12 10 8 6 4 DISTANCE FROM ORIGIN (INCHES)

Q O o )()()( ) Fig. 60.— Distribution of C -labeled components of fraction V after chromatography in the organic phase of an Ethyl Acetate:Pyridine:Water (36:10:11.5) solvent system.

Glu and Man* represent standard glucose and mannose respectively

286 •< lOOOcpm lOOOcpm >

CD ro o n 2l Fig. 6l.--Distribution of C -labeled components of fraction W after chromatography in the organic phase of an Ethyl Acetate:Pyridine:Water (36:10:11.5) solvent system.

Glu and Man represent standard glucose and mannose respectively

288 lOOOcpm < 300cpm 0 2 4 6 ITNE RM RGN (INCHES) ORIGIN FROM DISTANCE ManGlu 8 g. 61 . ig F 10 12 —C <— 14 16 —D— <— 8 20 18 ><-E' 22 vo ro oo 290

TABLE 42 DISTRIBUTION OF CARBON-14 LABEL IN CHROMATOGRAPHICALLY RESOLVED COMPONENTS OF FRACTIONS V AND W

Total Volume Total Radioactivity of Fraction of Fraction. -«5 Fraction Ml CPM. x 10

r vc 11.5 11.68 8.0 WB 2.07 wc '7*5 % 3.59 V 6.0 1.53

WE 6.3 3.99 grow. Fraction T contained a high percentage of its total carbo hydrate as deoxysugar. A comparison of the amounts of deoxysugar present in each of the initial fractions is afforded through

Table 43.

The possibility that a deoxysugar may be involved in the biogenesis of galactocarolose provides basis for the interest which derives from partial analysis of fractions T, U, V, and W of this section. That a deoxysugar accumulated in the DHM- supplemented Raulin-Thom medium was, of itself, a fortuitous observation. The quantity in which the deoxysugar was present suggested that it must have been of some functional significance in the metabolism of P. charlesii. An assumption of involvement of a deoxysugar in galactocarolose biogenesis would make reason able the interpretation (of the present results) that dihydroxymaleate stimulated the accumulation of a possible precursor of the hexose units of galactocarolose. 291

TABLE 45 COMPARISON OF CARBOHYDRATE COMPOSITION OF FRACTIONS T, U, V, AND W FROM CHROMATOGRAPHY OF DHM-SUPPLEMENTED RAULIN-THOM MEDIUM

Carbohydrate Analyses of Fraction Total (Values Expressed as Micromoles Per Ml) Volume ------:— ------Total Total Total Deoxy- Fraction Ml Sugar Hexose Pentose Pentose Glucose

T 14.5 124.80 75.00 6.60 45.40

U 14.6 58.60 25.00 5.80 5.50

V 11.8 11.10 9.50 4.00 10.00 9.60

W 20.5 22.40 25.40 2.00 7.55 13.70

It is also plausible that the deoxysugar is not related

to the biosynthesis of galactocarolose and that it (the deoxy-

sugar) arose as the result of a metabolic lesion which was

specifically effected by dihydroxymaleate or a conversion-product

of: dihydroxymaleate.

An hypothesis might be advanced that dihydroxymaleate

serves as an efficient precursor of the deoxysugar and that the

accumulation of the deoxysugar was a reflection of the precursive

capacity of dihydroxymaleate present in the growth medium of

Penicillium charlesii. A consequence of this precursory role for

dihydroxymaleate might be the accumulation of galactocarolose

(if, as mentioned above, the deoxysugar were a precursor of

galactocarolose) or some other conversion product of the deoxy

sugar. Alternatively, the deoxysugar-conversion-product of di

hydroxymaleate might accumulate regardless of its involvement or (6) The galactan of Penicillium charlesii

The structure of the polygalactofuranoside, galacto carolose, was defined through the effortB of P. W. Clutterbuck and co-workers (3)» This oligosaccharide was discussed earlier in this section.

(7) Di, tri, tetra, and pentagalactofuranosides

Gorin and Spencer (19) have reported chemical studies which involved di, tri, tetra, and penta-saccharides homogeneous of galactofuranosyl units which were linked -1,5* These oligo saccharides were, however, derived from galactocarolose by con

trolled acid hydrolysis. The observations made by the Canadian workers has confirmed the structure of galactocarolose which was initially proposed by Clutterbuck and co-workers (3)*

It would appear, therefore, that galactocarolose is the

only oligogalactofuranoside (homogeneous of galactose) that has

been reported to be present in nature. The uniqueness of galacto

carolose is accentuated by the fact that several Penicillia which

are related to Penicillium charlesii G. Smith— the producer of

galactocarolose— effect the synthesis of galactose-containing

oligosaccharides the galactose units of which are in the pyranose

form. Thus, Penicillium luteum produces luteic acid (a polymer

which contains glucose and malonic acid) and a neutral, dextro

rotatory [ ^ + 28.6°] polysaccharide which contains galacto-

pyranosyl residues (20)j the manner of combination of these

residues was not specified. • Penicillium varians produces a 292 non-involvement in the biogenesis of galactocarolose or some other metabolite.

The detection of two pentoses and significant quantities of glucose in the growth medium was, similarly, without precedent 14 in studies involving the metabolism of C labeled compounds by

P. charlesii. Free pentoses and hexoses have often been found

in the extracts of the mycelium of P. charlesii but no pentoses

and very little glucose (after about 8 days of growth of P.

charlesii) have been observed to occur in the growth medium.

Table records‘the comparison of the specific activity

of the glucose present at the initiation of growth of P. charlesii

on the DHM-supplemented Raulin-Thom growth medium and the specific

activity of the galactose isolated from the galactocarolose.

TABLE kk l4 COMPARISON OF SPECIFIC ACTIVITY OF GLUCOSE-u-C AT INITIATION OF EXPERIMENT AND GALACTOSE ISOLATED FROM GALACTOCAROLOSE

Total Specific Activity of Compound Activity Present At Initiation At Termination of Experiment of Experiment Compound p c CPM/ ^Jimole CPK/p mole

Glucose 50 1986 -

Galactose - - - 725

(J) Studies on the Dihydroxymaleate Dependent Oxidation of NADH- Catalyzed by Extracts of P. charlesii

Cell free extracts of higher plants (116) and mammalian

tissue (117) have been shown to catalyze interconversion of tar

taric acid and dihydroxymaleic acid but there have been no 293 published reports of the occurrence of this reaction in extracts of fungi or bacteria. The enzyme which catalyzes the tartrate- dihydroxymaleate interconversion has been referred to as tartaric acid dehydrogenase.

The mammalian enzyme (118) and the enzyme from plants

(119) appear to be most active in dihydroxymaleate reduction when reduced-nicotinamide adenine dinucleotide, rather than reduced-nicotinamide adenine dinucleotide phosphate, serves as the reductant.

Extracts of Penicillium charlesii have been shown to catalyze a dihydroxymaleate-dependent oxidation of reduced- nicotinamide adenine dinucleotide. Some of the observations on

the course of this reaction are described in what follows*

The general methods for the preparation of extracts of

Penicillium charlesii were described under 'EXPERIMENTAL.* The

extracts used in the studies recorded in this section were pre

pared by a slight modification of the procedures defined under

•EXPERIMENTAL.*

The thoroughly washed and pressed mycelium was ground to

a paste with a mortar and pestle and acid-washed sand as an

abrasive. The sand was prepared for use by washing extensively

with hydrochloric acid, distilled water, and 0*01 molar ethyl-

enediaminetetraacetic acid in successive operations* The mortar,

pestle, and sand were precooled at 3°C for 15 minutes before use.

The paste which'resulted from the grinding of the mycelium was

extracted with 0.2 molar trishydroxymethylamino methane•hydro

chloride buffer, pH 8.3. Ten ml of the buffer were used for each gram of mycelium extracted. The mixture resulting from the extraction was centrifuged at J000 x g for 15 minutes at 0°C.

The supernatant solution was removed by pipet and the sedimented material was discarded. The supernatant solution wa6 subjected to centrifugation at 3000 x g for 10 minutes at 0°C, the super natant separated and the supernatant used as the source of the crude enzymes. The extract was assayed for dihydroxymaleic acid reductase by following the time-course of oxidation of nicotin amide adenine dinucleotide at 3^00 angstroms in a Zeiss spectro photometer. The assays were performed on a 3.0 ml final volume of reaction mixture in quartz or silica cuvettes with light path of 1.0 cm.

The following abbreviations were employed:

Abbreviations Compound in Reference

NAD, NADH2 Nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide, respectively

NADP, NADPH2 Nicotinamide adenine dinucleotide phosphate and reduced-nicotinamide adenine dinucleotide phosphate respectively

DHM Dihydroxymaleic acid

DHMRg Dihydroxymaleic acid reductase

TRIS Trishydroxym e t hylaminomet hane

FAD Flavin adenine dinucleotide

EDTA Ethylenediaminetetraacetate, disodium salt 295

(a) Demonstration of the dihydroxymaleic acid dependent oxidation of DPNH

The complete reaction system contained the following components.

TABLE 45

Volume Added Quantity Added Component Ml jttmoles

TRIS * HC1 pH 8.3 2.50 500

DHM 0.10 20

n a d h 2 0.10 0.30

h 2o 0.20

Homogenate 0.10

Figure 62 demonstrates the dependency of the NADH2 oxidation on the presence of dihydroxymaleate and the crude extract. Curve C indicates that the extract when heated at 95°C

for 10 minutes lost its capacity to catalyze the reaction.

Similarly a portion of the extract lost its ca-talytic capacity when the extract was made to 5 per cent with respect to tri chloroacetic acid.

That the.capacity of the extract to catalyze NADH2

oxidation was. lost when the extract was heated or treated with

trichloroacetic acid suggested that the component of the extract

responsible for catalysis was enzymic in nature. Fig. 62.— Oxidation of N A D ^ by Penicillium charlesii extracts in the presence of dihydroxymaleate(DHMT.

Curve Modifications of Reaction System

A None

B . r Omit* NADH^

C Substitute heated extract for the fresh homogenate

D , Omit DHM A A O.D. AT 340 nyi 0.5 0.6 0.9 0.8 0.7 0.4 0.2 0.3 0.1 2 3 IE F REACTION(MINUTES) OF TIME 4 ?ig. 5 62 6 7 8 297

o o 298

(b) The effect of volume of the extract on the time-course of oxidation of NADH^

It was of interest to determine the relationship between the volume of the crude extract added to the reaction mixture and the time-course of the oxidation of NADH^. The experimental assay conditions were similar to those described in section (a)

except that in the present case the volume of the homogenate

varied from 0 to 0 .^ ml and the volume of distilled water (added

to give a final volume of 3*0 ml of reaction mixture) varied from

0,4 ml to 0 ml.

The results of the experiment are recorded in Figure 63.

It is seen that whereas the rates of oxidation of NADEL, over the

first 2 minutes vary almost linearly with extract volume, line

arity was not maintained beyond 3*0 minutes of reaction. The

significance of this figure lies in its demonstration of the

dependence of the rate of oxidation on the amount of added

enzyme(s)•

The tartaric acid dehydrogenase isolated from mammalian

tissues was initially associated with a particulate fraction of

the tissue homogenate but the enzyme could be solubilized with

facility (ll8 ). On the other hand, the tartaric dehydrogenase

of higher plants appears to be "soluble” in the initial homogen-

ates. Fig. 63»— The effect of ■volume of added extract on the time course of *'the DHM-dependent oxidation of NADH^.

Curve Volume of Extract Added to System Defined in Sections (a) and (b) Ml

A 0.40 B 0.30 C 0.15 D 0.05 E 0.02 F None

299 O f REActi ION (telHu 'S. 301

It was of interest to determine if the enzyme from P.

charlesii which catalyzes the oxidation of NADH2 in the presence * of DHM was associated, in the crude extract, with particles that

could be sedimented in centrifugal fields of moderate intensity.

Six milliliters of the crude extract were centrifuged

for 20 minutes at 10,000 x g in a refrigerated centrifuge which

was operated at 0°C. The supernatant (designated Sup^) and the

sediment were separated with a pipet. The sediment was suspended ’

in 2.0 ml of 0.2 molar TRIS*HC1 buffer (pH 8.3) and the suspension

centrifuged at 10,000 x g. The supernatant was separated and

discarded after assay revealed that it did not catalyze NADH^

oxidation in the presence of DHM; the sediment was suspended in

1*5 ml of 0.2 molar TRIS*HC1 buffer (pH 8 .3 ) and the suspension

designated 'SecL^.1

7 Three milliliters of Sup2 were centrifuged for 20 minutes

at 35*000 x g in the refrigerated centrifuge operated at 0°C.

The supernatant solution was removed by pipet and designated

Sup^; the sediment was washed once with 0.2 molar TRIS'HCl (pH

8.3 ) and the sediment which resulted after centrifugation for

20 minutes at 35*000 x g was dissolved in 1.5 ml of the 0.2

molar TRIS*HC1 buffer. This latter solution was named Sed2#

The absorption properties and protein content of these

fractions which resulted from the differential centrifugation

are recorded in Table ^6 . When each of these fractions was assayed

for capacity to oxidize NADH2 in the presence of DHM the data

which were obtained could be represented as in Figure 6k, 14-

complex polysaccharide which Is composed of D-glucopyranose,

D-galactopyranose, and D-idose or D-altrose In the molar ratio

of 1:6 to 8:1 (21). The mould Penicillium capreolinium produces

a polysaccharide which contains galactose, glucose, mannose, and malonic acid (22).

There appear to be no published accounts of studies

related to the biosynthesis of the galactofuranose-contalning

polysaccharides of Cladophora rupestris. Gibberella fujikuroi.

Trichophyton granulosum, of the type-specific substance of Type

34- Pneumococcus or the arabitolgalactofuranoside of lichens.

Despite or perhaps because of its uniqueness, galactocarolose

has not been the subject of extensive biochemical studies. The

isolation and structural definition of galactocarolose by Clutter

buck and co-workers was not succeeded by experiments designed to

determine the pathway of synthesis of the polygalactofuranoside

or its biochemical function.

The first report of an attempt to establish the pathway

of biogenesis for galactocarolose is.due to Gander (3). Studies

were reported which related the incorporation of carbon-14- from l4- i4. i h acetate-l-C , glucose-1-C , glucose-2-C , glucose-u-C , and

tartaric acid-1,k- (J \ into galactocarolose. When Penicillium

charlesii was allowed to grow on the Raul in-Thom, medium (103) 14- which was supplemented with glucose-u-C , the specific activity

of the galactose isolated from galactocarolose was approximately

one-third the specific activity of the glucose initially added

to the growth medium. Similarly, when the labeled supplement was Fig. 64.— The oxidation of NADHg by differentially centrifuged fractions of a P. charlesii extract.

Curve Enzyme Component

A Initial homogenate B Sup^ C Sup2 D SecL^ E Sed2 F None

502 303

0 .9 0

0.70

O 0.50 to_-

0.30

0.10

TIME OF REACTION (MINUTES)

Fig. 6b 304

TABLE 46 ABSORPTION PROPERTIES AND PROTEIN CONTENT OF FRACTIONS OF EXTRACT OF P. CHARLESII

Protein Concen- Total Ultraviolet Light Absorption v oxuxzie vTavXO& Protein O.D.* Va • Uri • ‘* 260 Fraction Ml Mg/Ml Mg 300 m 280 m 250

Initial 6.0 24.0 144.0 0.159 0.401 1.683 Homog enate Sup2 3.0 12.8 38.4 0.117 0.403 1.675

Sed^ 1.5 30.0 45.0 0.069 0.112 1.051

Sup3 3.0 12.0 36 0.098 0.339 1.737 0.096 0.126 Sed2 1.5 15.5 23.25 1.103

*For 0.2 ml of solution in 2.8 ml 0.2 M TRIS'HCl pH 8.3.

The components of the assay system and the experimental con

ditions in this present experiment were identical to those

employed in section (a). Equal volumes of the "Initial Homog

enate" Sup2 or Sup^ were used in the experiment rather than equal

quantities of protein from these fractions. On this basis the

data of Table 1 and curves A, B, and C of Figure 3 suggest that

insignificant quantities of the enzyme catalyzing NADH2 oxidation

were sedemented under the conditions employed; since the protein

concentrations of Sup, and Sup^ are about one-half the protein 3 £ concentration of the initial homogenate and the rates of reaction

in the presence of these fractions are approximately equal, it

appears that the specific activity of the initial homogenate was 305 one-half the specific activity of Sup^ and Supg. The catalytic activity of Sed^ may reflect incomplete washing of this fraction*

These data appear to afford the conclusion that the enzyme in extracts of P. charlesii which catalyzes the DHM- dependent'oxidation of NADH^ was not associated with particulate material which can be sedimented in centrifugal fields of 35*000 x g or less. Since essentially all of the enzyme activity of the

P. charlesii extract was non-sedimentable at centrifugal fields of 35*000 x g the high speed supernatant (Sup^) was used in all the experiments described in the remainder of this section.

(d) The effect of concentration of DHM on the time-course of oxidation of NADH^ by extracts of P. charlesii

The effect of the concentration of dihydroxymaleic acid on the time-course of N A D ^ oxidation was investigated. The results of a series of experiments regarding this relationship are recorded in Figure 65, The experimental protocol employed in the present case was identical with that defined in section

(a) except that the volume of DHM added to the reaction system was varied from 0 to 0,*t ml* while the volume of distilled water was varied from 0,k ml to 0 ml.

The data of Figure 65 suggested that relatively high concentrations of DHM are required to fully saturate the enzyme.

It has been observed on several occasions that the reaction

(oxidation of DPNH) slows down markedly after the first 5 to 6 minutes. This result has not been extensively investigated but Fig. 65«--The effect of the concentration of DHM in the reaction mixture on the time-course of oxidation of NADH^.

Curve Final Concentration of DHM in rxn Mixture (Expressed as Micromoles/Ml)

A 5.33 B ^.00 c 2.67 D 1.33 E 0.60 F None G 5*33 in presence of heated extract

306 DPNH OXIDIZED (MILLIMICROMOLES) 500 900 700 300 100 2 IE F REACTION(MINUTES) OF TIME 4 F ig . . ig F 65 6 8

Oil 307 it suggests that a product of the reaction is inhibiting the

NADH2 oxidation or that one of the products is undergoing further transformations* It was considered possible that some of the NAD which formed was being reduced in an enzymatic process which involved the conversion of dihydroxymaleate to diketosuccinate. It has been demonstrated, however, that when the enzyme preparation was incubated with NAD in the presence of DHM negligible reduction of NAD occurred.

(e) The effect of EDTA on the time-course of DHM-dependent oxidation of NADH^

It was reported (120) that magnesium ion catalyzes a non-enzymatic decarboxylation of DHM to hydroxypyruvate and that this non-enzymic reaction is inhibited in the presence of EDTA.

The author also reported that an EDTA-Mg complex activated the enzyme catalyzed oxidation of hydroxymaleate to diketosuccinate*

These reports made it desirable to determine whether the seques tering agent affects the dihydroxymaleate reductase of P. charlesii.

A series of experiments were performed which involved additions of EDTA to a reaction system similar to that defined in section (a). EDTA was added as the disodium salt prior to ad dition of substrate to the cuvette in which the reaction (re duction of DHM) took place. The results of these experiments are represented graphically in Figure 66. Note should be made of the linearity, over the first 9 minutes of reaction, of curve F which represents NADH^ oxidation in the absence of added EDTA; the rate of reaction in this system gradually decreased beyond Fig* 66.— The effect of EDTA on the reduction of dihydroxymaleate.

309 N A D H a OXIDIZED ( miIlKnicromoles €00 70'0 0 0 9 200 0 0 4 0 0 3 500- 100 0 z TIME 4 3 F ECIN ( REACTION OF g. 66 . ig F 5 6 7 i. ) min. 9 8

310 311

11 minutes. Curves A through E suggested that a slight stimu lation, over the control F, of the enzymic oxidation of NADHg was affected by EDTA but that this effect was not directly pro portional to the concentration of added EDTA.

It was further observed that beyond 10 minutes time of reaction the rate of DHM reduction was much higher in the control system, represented by curve F of Figure 66, than in the systems which contained EDTA. This effect might be explained in terms of an EDTA-stimulated reduction of NAD (formed when DHM is re

duced) at a rate slightly lower than the rate of NADH2 oxidation.

According to this explanation a quasi steady state for the

NADH2— :-- «>NAD transformation might be reached and since the

experimental method measures accumulation of NAD as a function

of time an "apparent" retardation of the rate of dihydroxymaleate

reduction would be observed. That this explanation is not ap

plicable to the present observations was suggested by the failure

of the extract to reduce NAD in the presence of DHM when EDTA is

either added to or omitted from the assay system.

As mentioned in the previous section, the enzyme prepa

ration used in these studies apparently does not contain a di

hydroxymaleate dehydrogenase. This latter enzyme, if present,

would catalyze the reaction which may be represented as:

COOH COOH I I HO-C C=0 | + N A D ’— ------> | + NADHp HO-C C=0 I I COOH COOH 1 5 14 tartaric acid-1 ,4-C the specific activity of the galactose isolated was approximately one-half the specific activity of the initial tartrate* On the other hand the galactose units of galactocarolose isolated when P* charlesii metabolized Acetate-1- 14 C was slightly higher than the acetate initially employed. The results of these experiments were interpreted as support for an hypothesis that the galactofuranose precursor of the hexose units

of galactocarolose derived through the condensation of and

carbon fragments*

Recently, Bentley and co-workers (23) confirmed the re ported incorporation of tartaric acid into the oligosaccharides

of P. charlesii*

The nature of the galactofuranose units of galactocaro

lose suggest that the former must be synthesized through a unique

sequence of events. Of interest in this connection is the sug

gestion of a condensation between a tartrate derived metabolite

and a fragment which derives from glucose*

(E) The Biosynthesis of Oligosaccharides

In a review on the "Biosynthesis of Complex Saccharides"

it was pointed out (24) that three general types of biochemical

reactions are recognized which lead to oligosaccharide formation.

The processes include transglycosylation, the action of phos-

phorylases, and the action of nucleoside diphosphoglycoside

transglycosylases* 312

It was observed that the reaction system containing di hydroxymaleate and EDTA becomes yellow upon standing in the presence or the absence of the enzyme (dihydroxymaleate reductase)

from P. charlesii. This change in color of the system is ac companied by a decrease in the absorption at 295 millimicrons

(due to the dienol group of DHM) with a gradual increase in ab sorption at longer wavelengths. It is conceivable that a complex

of EDTA and a divalent metal ion (this latter being present in

the enzyme preparation) might stabilize dihydroxymaleate in the

dienol form and the combination of the EDTA-metal complex with

DHM could give rise to a species which absorbs light at wave

lengths longer than 295 millimicrons. If the equilibrium between

the keto and dienol forms of dihydroxymaleate is displaced

toward the dienol form by the formation of a DHM-EDTA-metal

complex and dihydroxymaleate reductase requires the keto form of

the acid, then the non-enzymic process involving complex for

mation would have the effect of removing the substrate of the

reductase from the solution. What results, therefore, is a com

petition between two processes for the substrate common to both—

dihydroxymaleic acid.

The adequacy of this explanation of the results (stated

in Figure 66) is questioned when consideration is given to the

relative concentrations of dihydroxymaleic acid and EDTA present

in the experimental systems.

It appears reasonable to conclude that under the experi

mental conditions employed EDTA did not significantly stimulate 313 the reduction of dihydroxymaleic acid by the enzyme system from

P. charlesii,

(f) On the "specificity" of the oxidant in the soluble-dehydrogenase catalyzed oxidation of NADH.,

The question arose as to whether or not the enzyme studied in the preceding sections was specific for reduction of dihydroxy maleate. It was conceivable that the enzyme activity resided in a non-specific hydroxyacid dehydrogenase or kqtoacid reductase.

Several compounds were tested with the enzyme in the presence of NAD or NADH^ and in the presence of NADP or NADP^.

Some of the results of these experiments are represented in

Figure 67. .

Pyruvic acid and oxaloacetic acid were added to the assay system as the sodium salt and tris‘hydrochloride salts respectively.

Because of the instability of these two compounds fresh solutions

of oxaloacetate and pyruvate were made a few minutes prior to

their use in assay, L-ascorbic acid was added as the tris*

hydrochloride salt.

Although the dienol group of L-ascorbic acid is very

similar to that in dihydroxymaleic acid the former compound was

not reduced by the extract in the present study. Significantly,

the extract reduced oxaloacetate only poorly and pyruvate not at

all. The results obtained in the case of oxaloacetate substi

tution are significant in that malic dehydrogenase has often been

observed as the most active hydroxyacid dehydrogenase in extracts

of P. charlesii. Fig. 67*— Specificity of the oxidant in the enzyme catalyzed oxidation of NADEL,*

Curve Additions to the Reaction System

A Dihydroxymaleate (DHM) B Oxaloacetate C Pyruvate D Ascorbate £ None A O.D. At 340m jj 90 .9 0 0.70 0.30 0.50 0.10 IE F REACTION (MINUTES) OF TIME Fig. 6? 315 316

(g) Reduction of NAD by extracts of P. charlesii in the presence of tartaric acid

The enzyme preparation which was used in studies recorded in previous sections has been shown to catalyze the reduction of

NAD in the presence of tartaric acid. The enzyme catalyzed reduction of NAD appeared to specifically require tartrate; lactate, £ -hydroxybutyrate and L-ascorbate were not oxidized and negligible reduction of NAD occurred in the presence of tartronate. Malic acid was oxidized, but at a rate which was much lower than the rate of tartrate oxidation. A record of these

observations is represented in Figure 68.

The rate of tartrate oxidation is significantly lower

than the rate of reduction of dihydroxymaleate in the presence

of equivalent amounts of enzyme protein.

(h) Other properties of the enzyme

Neither the enzyme catalyzed reduction of dihydroxy

maleate nor the oxidation of tartrate was significantly affected

by the addition of coenzyme A, ATP, NADP, NADPHg, or FAD. Di

hydroxymaleate and tartrate appear to be specifically required

for the NADH^ oxidation and NAD reduction respectively. Although

detailed thermodynamic analyses of the system have not been

carried out, the preliminary observations indicated that the

equilibrium of the reaction favors reduction of dihydroxymaleate.

The specific activity of dihydroxymaleate reductase

isolable from P. charlesii mycelium of various ages appears to Fig. 68.— Oxidation of tartrate by crude extract of P. charlesii.

Curve Deletions from the complete System

A None B MgCl2 C NAD ' D Crude enzyme preparation E DL-Tartrate F DL-Tartrate , (DL-Tartrate was replaced by L-Malate)

517 A 0. D. AT 340mjj 0.30 0.40 0.20 0.10 OXIDATION OF DL-TARTRATE BY CRUDE HOMOGEN ATE HOMOGEN CRUDE BY DL-TARTRATE OF OXIDATION 2 4 6 TIME REACTION OF (MINUTES) F P.CHARLES!! OF 8 i. 68 Fig. 10 12 14 16 18

mo 318 approach a maximum value at 12 days. More specifically, the

12-day old mycelium yields an extract of higher specific activity

for DHM reduction than mycelium of any other age tested. Since

this latter observation has not been accurately quantitated its significance has not been defined. DISCUSSION

A casual review of publications related to metabolism in fungi reveals that little is known about the details of metabolic processes occurring in certain of the Penicillia. Much credit is due the work recorded by Charles Thom and students in this hemisphere, to members of the European groups headed by

F. F. Van Beyma and G* Banier, respectively, and to the group of mycologists led by George Smith and G* E. Turfit in London*

These four groups are responsible for numerous classic contri butions to the isolation, characterization, and maintenance of hundreds of species of Penicillia*

A vast quantity of human effort has been devoted to the isolation and characterization of various fungi but relatively few studies have been devoted to the physiology, biochemistry, and genetics of the genus Penicillium. The group of chemists and mycologists headed by H. Raistrick at the London School of

Hygiene and Tropical Medicine has contributed significantly to one area of mycologic physiology* This group has been concerned primarily with the isolation and structural definition of hundreds of the myriad of complex organic compounds synthesized by various speci of fungi. The Raistrick school appears to have been pri marily interested in the nature-structure of fungal metabolites and not in the how and why of the biosynthesis of these compounds*

320 Contemporary biochemists, mycologists, and physiologists have thus become heir to a great wealth of information related to the structures of hundreds of metabolic products of various Penicillia but discouragingly little information related to the functions of

and biosynthetic pathways for these metabolites. This statement

adequately summarizes the status of Penicillium charlesii G. Smith

and numerous of its metabolic products. P. charlesii has been

shown to produce several heterocyclic carboxylic acids and two

oligosaccharide© (l). The complete definitions of the structures

of the heterocyclic acids (2) and the oligosaccharides (3» *0

have been reported. Almost 30 years elapsed between the time of

publication of descriptions of these metabolites and first re

ports of studies which engaged the problems attending possible

biosynthetic pathways for these metabolites. The work of E.

Bentley and co-workers (23) afforded suggestions of a sequence

of processes which might lead to the enzyme catalyzed formation

of carolic and carlosic acids. Although informative, the reports

from the latter workers have not been extended to studies of the

details of the molecular features of the transformations involved

in carolic and carlosic acid formation by P. charlesii. In

regards to the oligosaccharides produced by P. charlesii, there

have been no reports of studies directed toward the oligoman-

noside, mannocarolose, and only two reports have been forthcoming

which have dealt with the biosynthesis of the second of the two

oligosaccharides, galactocarolose (5, 23). 16

(1) Transglycosylation

The process is effected by enzymes termed transglycosylases and may be represented according to the notation of Eassid (24-):

Rt - o-R" + R 1'' - OH ---- * R' - 0 - R ' 1' + R" - OH ^---- Glycosyl Acceptor Product By-product Donor

Some of the well-studied enzymes of this class include amylomaltase (25), levan sucrase (26), and dextron sucrase (27)*

(2) Action of phosphorylases

The general reaction catalyzed by the phosphorylases can be represented as below:

Glycosyl-O-Phosphate + XO-R 1 iV> Glycosyl-O-R + X-Phosphate

Glycosyl Donor Glycosyl Product Acceptor

In this representation X is usually hydrogen* Sucrose phosphorylase (28) glycogen phosphorylase (29) and starch phosphorylase (30) are well-studied members of this class of enzymes*

(3) Nucleoside diphosphorylglycoside transglycosylases

The reaction catalyzed by enzymes of this category is

that typified below:

Nucleoside-O-P-O-P-O-Glyc • + X-OH ---- • ■ Nucleoside-O-P-O-P-O + Glyc*-0rox

In this representation X may be hydrogen, phosphoryl, or an alcohol fragment or polymer* 322

Aside from the two latter reports and a publication which involved a rather limited study of the mould, the general metabolism of P. charlesii and the production of oligosaccharides by this mould remain virgin areas of mycologic research.

There exists a significant body of unknown factors which relate to the metabolism of P. charlesii and oligosaccharide

formation by the mould. The organism is often cultured upon the

Raulin-Thom growth medium but little has been published which relates the metabolism of this mould to variations in concen

trations of components of the growth medium. Tartaric acid, a product of carbohydrate metabolism in certain fruits, plants, and microorganisms, is a component of the Raulin-Thom medium but the functions of this C^-dihydroxydicarboxylic acid have been largely ignored or unexamined. The first description of the

Raulin-Thom medium is due to P. W. Clutterbuck and co-workers

(105). The authors examined the production of numerous metabo lites of the Penicillium brevi-compactum series and compared the

formation of these metabolites when the organism was grown upon

the Raulin-Thom and Czapek-Dox media. Clutterbuck and co-workers

experienced considerable difficulty in reproducing observations

made when the mould speci were cultured on the Czapek-Dox medium,

(which does not contain tartrate) and

. . .it was found necessary to decide whether the metabolic products (the empirical formulae of which were C^H^Og, C^H^Og, C^H^C^, C^H^C^, and CgHgO^) arise from tartaric acid, or are true metabolism products of glucose. An experiment on 323

the Czapek-Dox solution (glucose being the only carbon source) was therefore carried out in tubes • • • • The three metabolism products acid), and C^gH^Or,, were shown to be present and the failure to detect the substances and CgHgOg was probably due to the smallness of the amount of product.

The authors concluded that

the products C^H^Og, C^H^Og, and are therefore certainly, and the products C^qH^qO^ and CgHgOg are probably, metabolic products of glucose and the presence of tartaric acid is not essential for their formation.

This questionable conclusion derives from an unexpected source. Nevertheless, studies performed more recently than those of Clutterbuck and co-workers have not significantly increased the understanding of "how tartrate functions'1 in the

Raulin-Thom medium.

Tartrate can be postulated to function as a buffer system maintaining an acidic pH during the early stages of growth of cultures maintained upon the Raulin-Thom medium. Additionally, as implied by Clutterbuck and co-workers (105) tartrate could be an inert component of the medium. It was also thought that tar trate could serve as a substrate for P. charlesii; evidence to this effect was presented by Gander (5 ) and subsequently con firmed by Bentley and co-workers (23). It was reported that tartaric acid carbon was incorporated into galactocarolose, one of the oligosaccharides produced by P. charlesii. This obser vation negated an hypothesis that tartaric acid and its ammonium 32k salt of the Raulin-Thom medium functioned only as an inert buffer system. Additionally the report that tartrate dilutes carbon-l4

from glucose-u- C14 which was incorporated into galactocarolose

suggested that the glucose carbon chain was not converted intact

into the galactose units of galactocarolose.

An approach to a study of the functions of tartaric acid

of the Raulin-Thom medium in general metabolism of P. charlesii

and production of galactocarolose involved the substitution of

various dicarboxylic acids for tartaric acid in the medium. A

specific and absolute requirement for tartrate in oligosaccharide

synthesis suggests that a reduction of formation of oligosac

charide would be observed when tartrate is not present in the

growth medium.

Several dicarboxylic acids and malonate were sub

stituted for tartrate in the Raulin-Thom growth medium. The

acids included maleic, malic, succinic, fumaric, and dihydroxy

maleic. When the Raulin-Thom medium, modified to contain one of

these acids substituted for tartrate, provided nutrients for P.

charlesii the organism was observed to grow without appreciable

lag. The slow rate of growth observed under the conditions of

test was attributed to the low levels of nitrogen available to .

the organism.

Significant amounts of glucose were present in the growth

media at the end of the incubations. Oligosaccharide synthesis

occurred in the presence of all the dicarboxylic acids tested.

It was observed that oligosaccharide and mycelium synthesis in 325 the presence of tartrate was much higher than in the presence of other dicarboxylic acids. The ratio of oligosaccharide to mycelium synthesized was highest when dihydroxymaleate was sub stituted for tartrate in the Raulin-Thom medium.

Several experiments were performed which involved vari ation of the ammonium salt concentration of tartaric acid and various dicarboxylic acids substituted for tartaric acid in the

Raulin-Thom medium. It was observed that the highest levels of mycelium were obtained in the systems which contained the highest levels of ammonium ion in the initial growth medium. On the other hand it was observed that maximal synthesis of oligosac charides obtained in the systems which contained lowest levels of ammonium ion in the initial growth medium. "Efficiency" of oligosaccharide synthesis was arbitrarily defined as the ratio of oligosaccharide synthesized to the amount of mycelium formed and it was found that this ratio was highest when the absolute value was large for the initial growth medium.

Since the concentration of the ammonium salts of the di carboxylic acids were being varied the experimental results discussed above could be interpreted in terms of an inhibition of oligosaccharide formation by high levels of the dicarboxylate ions. Alternatively the results may have reflected the total amount of organic carbon and/or nitrogen available for synthetic processes. These two possibilities could be distinguished by experiments which involved growth of P. charlesii on fixed levels of medium carbon while the ammonium ion concentration was varied. 326

Were the oligosaccharides of P. charlesii produced as by-products or as a result of a shunt process it might be anticipated that large quantities of oligosaccharide would be synthesized under

conditions that involved a high carbon-sourcetlow ammonium-level medium. In a series of experiments which involved growth of P. [glucose] - charlesii on the Raulin-Thom medium, the ratio ^ nw' \

was varied from 5.2 to 30 through 4 or 5 intermediate values.

Ammonium carbonate served as external source of added ammonia in

the presence of various dicarboxylic acids. Proliferation and

other observable metabolic changes in the mould were essentially

"normal" when the growth medium contained up to 33 micromoles of

ammonium ion per ml. When the concentration of ammonium ion in

the growth medium was higher than 33 micromoles per ml, the

metabolism of glucose by the mould was retarded during the early

stages of growth; after a 4 to 6-day lag period glucose metabolism

was quite rapid. The slow rate of metabolism in the latter cases

was probably due to the high pH of the medium which contained am

monium ion in concentrations higher than 33 micromoles per ml*

Maximum "efficiency" of oligosaccharide formation, in the presence

of fumarate, was obtained when the ammonium ion concentration was

l4.1 micromoles per ml# and this efficiency was not significantly

altered when the concentration of ammonium ion in the medium was

increased to 28 micromoles per ml. Maximum "efficiency" of oligo

saccharide formation for systems containing tartrate or malonate

was obtained when the growth media contained 36*8 micromoles per

ml. When the growth medium contained dihydroxymaleic acid 327 maximum efficiency of oligosaccharide formation was obtained in the system which contained k6,5 micromoles of ammonium ion per ml. In all cases studied the absolute amount of oligosaccharide synthesized by P. charlesii appeared to vary inversely with the amount of nitrogen in the initial growth medium. This general ization did not hold for systems which contained less than 14.1 micromoles of ammonium ion per ml of growth medium. These ob servations suggested that the absolute levei of nitrogen, avail able to proliferating cultures of P. charlesii, exerted a pro nounced effect on the amount of oligosaccharides synthesized.

Large quantities of mycelium and small amounts of oligosaccharide were formed when growth occurred in the presence of high concen trations of ammonium ion. On the other hand, when the initial ammonium ion concentration was low, relatively small amounts of mycelium and large quantities of oligosaccharide were produced.

The data can be interpreted in terms of the broad characteristics of-shunt metabolites that are produced by numerous moulds. It is quite possible that oligosaccharide formation by

P. charlesii reflects one alternate process for the deposition of excess organic carbon substrates such as glucose and dicarboxylic acids.

That P. charlesii produced galactocarolose when the mould was cultured upon a medium which contained one of several di carboxylic acids, substituted for tartrate, suggested that tar trate was not specifically required for production of galactocaro lose or that the mould was endowed with the capacity to synthesize, 328 from glucose or other sources, the tartrate transformation- product which is incorporated into the hexose units of galacto carolose* The former view is supported by the observation that galactocarolose is synthesized when the mould is grown on the

Czapek-Dox medium which does not contain tartrate. The latter hypothesis is also probable and can be subjected to experimental test with carbon-l4 labeled substrates in the growth-medium.

Experimental approaches to this latter hypothesis will be dis cussed as part of the concluding remarks of this section.

Fumarate was observed to stimulate mycelium and oligo- 1^ saccharide synthesis by P. charlesii. Fumarate-2,3-C and l^f fumarate-l,4-C added to the growth medium at the time of innoculation were shown to be inefficient precursors of the hexose units of galactocarolose. Thus, the observed effect of fumarate on mycelium and oligosaccharide synthesis were largely indirect.

The'importance Cf fumaric acid as a component of the citric acid cycle and as a precursor of important cell components suggested that fumarate may have functioned in the growth medium of P. charlesii by "sparing” the glucose of the medium for processes associated with oligosaccharide formation.

Studies involving the substitution of malonate for tar trate in the growth medium of P. charlesii showed that this C., - 3 dicarboxylic acid stimulates oligosaccharide and mycelium pro duction but both these to a lesser extent than was observed in the presence of fumarate. Studies on the incorporation of l4 14 malonate-l-C and malonate-2-C showed that malonate carbon 329 does not efficiently precurse the oligosaccharides of P. charlesii.

The stimulation qf oligosaccharide and mycelium production by malonate was probably indirect. It is well known that malonate severely inhibits succinic dehydrogenase which is a key enzyme component of a vast majority of aerobic microorganisms. Ad ditionally, malonate is known to be an important precursor of

fats (121), lipids, and numerous other substances produced by

fungi. Malonate, by serving to supply precursors for, certain

synthetic routes and by acting aB an antimetabolite may have

served as a metabolic dam which functioned to increase the pool

of glucose-derived precursors of oligosaccharides.

Several unprecedented observations were made when P.

charlesii was grown in the presence of dihydroxymaleic acid

which replaced tartrate in the growth medium. •The mycelium

generally did not form the characteristic green covering on its

top surface and the culture was quite rigid and compact. The

rate of growth of P. charlesii was quite similar to growth ob

served when tartrate was the dicarboxylic acid component of the

growth medium. Pronounced increases in total mycelial material

synthesized were not observed when the dihydroxymaleate-growth

medium contained high concentrations of.ammonium ion. On the

other hand, maximum efficiency of oligosaccharide production

appeared to obtain when the mean concentration of ammonium ion

in the "DHM-medium" was *f6.5 micromoles per ml. In contrast to

the other systems and series which were studied, numerous mono

saccharides were detected in the concentrated growth media from-

the dihydroxymaleate systems. 350

Carbon-14- labeled dihydroxymaleate was not available and a direct measure of its incorporation into oligosaccharides could not be performed. However, dihydroxymaleate was observed 14 to dilute carbon-14- of glucose-u-C which was incorporated into galactocarolose. This observation suggested that dihydroxymaleate functions directly in the stimulation of oligosaccharide pro duction. That dihydroxymaleate did not serve as an effective stimulant of mycelium production was quite noteworthy. Con sideration was given to the view that, in vivo, one of the optically active forms of tartrate is preferentially converted to dihydroxymaleate and that this transformation provides eventual precursors of oligosaccharides. Similarly, the second optically active form may be preferentially converted to some metabolite, such as oxaloacetate which is an efficient energy source and precursor of cellular other than carbohydrate. These two suggestions could explain adequately the results obtained— and dis cussed subsequently— when P. charlesii was grown in the presence of D or L-tartaric acid. Evidence to be discussed supports the suggestion that dihydroxymaleate is one of the possible first- products of metabolism of tartrate in P. charlesii. The simi larities in structure which exist between dihydroxymaleate, flunarate, malate, oxaloacetate, and alpha oxoglutarate suggest that dihydroxymaleate might serve as an antimetabolite of these important dicarboxylic acids. However, no data is available which would influence a decision in regards to this latter pos sibility. 331 Consideration was also given to the possibility that only one of the enantiomorphs of tartaric acid is actually involved in the biosynthesis of oligosaccharides by P. charlesii. Experiments were performed which involved growth of P. charlesii on the Raulin-

Thom medium which contained D and/or L-tartrate at a final con centration of 32 micromoles per ml. The final concentration of ammonium ion was 18.9 micromoles per ml in the three systems.

Significant differences were-obtained when the mould growth- medium contained L-tartrate as compared to D-tartrate or DL- tartrate. The amount of galactocarolose isolated from the L- tartrate growth-medium was approximately twice the amount of oligosaccharide formed when P. charlesii was cultured in the presence of D-tartrate or the DL mixture. Additionally, mould growth, upon the medium which contained D-tartrate, was quite irregular and the morphology of the mycelium was quite atypical.

The absolute amounts of mycelium synthesized in the presence of

D, L, or DL-tartrate were essentially identical. The data which related absolute amounts of galactocarolose synthesized to the configuration of the tartrate employed in the growth medium sug gested, but did not prove, that only the L-form of tartrate con tributed significantly to the tartrate derived precursor of galactocarolose. Doubt that D-tartrate is significantly metabo lized by the organism was overcome by studies which involved DL-

tartaric acid-1,4— o14. When the growth medium containing tartaric 14- acid-1,4— C , (50 microcuries added at the time of innoculation of

the system) upon which P..charlesii had grown for 28 days was 17

The enzyme uridinediphosphoryl-D-glucose-glycogen trans- - glucosylase is one of the widely studied members of this category.

A wide variety of N-heterocyclic bases have been shown to be glyoosyl "carriers” or "donors" in reactions of this type and

an even larger number of sugar residues has been reported to be

involved in the "activated" pyrophosphoryl diester linkage of

such nucleosidediphosphoglycosides.

Nucleosidediphosphoglycosides occur widely in nature and

these have been shown to be involved in the formation of a wide

variety of oligosaccharides. Such nucleotides have been shown

to participate in the synthesis of glycosidic linkages found in

numerous and diverse compounds of biological significance. Among

the latter compounds are the followings the capsular polysaccharide

of Type I Pneumococcus (3l)> the cellobiose linkage of glycosy

lated DNA of coliphages (52), sucrose (35)» lactose (3*0* tre

halose (35)j and rutin (56).

The third of the three types of processes outlined above,

for the formation of oligosaccharides, may be the most important.

A view to this effect has been advanced by Robbins and Lipmann

(6) for the UDPG-Glycogen transglycosylase system.

A brief theoretical discussion of the possible involve

ment of a nucleoside in the biogenesis of galactocarolose has

been discussed (?)• This latter work reported the isolation from

the mycelium of Penicillium charlesii of a nucleoside-sugar which

contained uracil linked through an N-onium bond to a carbohydrate.

The observation that the nucleoside contained no phosphate and concentrated and analyzed by chromatographic procedures, no tar taric acid could be demonstrated. Thus, both .D and L tartrate were metabolized by the organism. It is possible that the D and

L-forms of tartrate are metabolized by different and independent pathways but there is no direct evidence which bears upon this concept. The relationship between D and L-tartrate in metabolism of P. charlesii can be approached through use of the carbon-l4 labeled optically active isomers of this acid. The avail ability of specifically labeled succinate and the possible con version of succinate to DL tartaric acid or meso tartaric acid through transepoxysuccinic acid and cis epoxysuccinic acid, respectively (122) suggests that optically active D or L tar iff taric acid-C can be readily made available for studies in volving P. charlesii.

Reference to DL-tartrate as a "secondary" carbon source in the Raulin-Thom growth medium is a reflection of its concen tration (32 micromoles per ml) relative to the concentration of glucose (277 micromoles per ml) in the growth medium. The question arose as to whether or not tartrate served only a

"secondary" function in the metabolism of P. charlesii, tartrate being metabolized only after most or all of the glucose of the medium has been exhausted. An approach to answering this question involved a time-study of tartrate uptake by the fungus and a qualitative examination of the molecular forms in which tartrate- 14 C was stored in the mould mycelium. It was found that when the

Raulin-Thom growth medium, upon which P. charlesii proliferated, 333 n Z}. was supplemented with tartaric acid-1,^-C the tartrate was rapidly removed from the growth medium after a "lag" period of

2 days. Tartrate uptake continued over the entire 28-day growth period. Measureable carbon-l4 was detected in respired CO^ after l^j. 2 days of growth. The rate of tartrate-1,^-C uptake was quite

rapid over the first 5 days of growth of the mould. When the

glucose content of the medium had been reduced by 50 per cent of

the initial value, the tartaric acid concentration of the medium

had suffered an identical dimunition. The conclusion was reached

that complete removal of glucose from the growth medium of P.

charlesii was not required for tartaric acid uptake to occur.

The uptake of tartaric acid and of glucose occurred as collateral

events in metabolism of P. charlesii.

Further insight into the interrelationships between

glucose and tartrate was supplied by examination of the carbon-1^

content of respired when the Raulin-Thom medium was supple- l^f mented with DL-tartaric acid-1,4-C • Although the DL-tartrate-

1,4-C l^f was rapidly removed from the growth medium the catabolism 1 /j. to C Og was quite slow over the first 6 days of growth of the Ik culture. The release of C 0^ was most rapid during those stages

of growth at which most of the glucose had been removed from the

medium. The results suggested that tartrate was rapidly removed

from the medium in the presence of high concentrations of medium

glucose but that tartrate was rapidly catabolized to CC^ only

after most of the glucose of the growth medium had been metabo

lized. It appeared that significant quantities of tartrate were being removed from the growth medium and that little of the con centrated tartrate was metabolized to carbon dioxide during the first 10 days of growth of the mould*

Two experiments were performed to qualitatively establish l^j. the form(s) in which the carbon-l^f of tartrate-C was present in the mycelium of Penicillium charlesii* The organism was al lowed to grow for 10 or 10*5 days on the Raulin-Thom medium which i l4 was supplemented with tartaric acid-1,4-C . The mycelium was extracted according to standard techniques and column and paper chromatography of the extracts suggested that intramycelial carbon-l4 from tartrate-1,4-C had been transformed into numerous labeled components. Negligible amounts of free-tartaric acid-1,^f- ik C were extracted from the 10 or 10.5-day old mycelium. A very significant quantity of radioactivity derived from tartrate-1,k- lif C could not be extracted from the 10,5-day old mycelium with hot perchloric acid. These observations afforded the conclusion lif that tartrate-C had been transformed into mycelial structural materials as well as numerous soluble cell components.

An approach to establishment of the nature of small unit compounds which serve as precursors of galactocarolose involved the addition to the growth medium of P. charlesii of several carbon-lA- labeled substances. Acetate-l-C l^f and/or acetate-2-C l^f , 14 i/f malonate-l-C , and succinate-u-C were added to the growth l4 media after 16 days of growth of the mould while malonate-2-C , l^f l^f lA- malonate-u-C , succinate-1,4-C , succinate-2,3-C , and fumarate-

2,3-C were present in the growth medium from the time of innoculation. The organism was grown on 25 ml volumes of medium.

The incorporation of the labeled compounds into galactocarolose was negligible in all the systems to which the preceding state ments refer. The conclusion of negligible incorporation of radioactivity into oligosaccharides was based upon released C l^f labeled-monosaccharides upon treatment of aliquots of the con centrated growth media with 0.4 normal or 3*0 normal sulfuric acid, respectively. It was shown that the cultures used in these studies were fully capable of synthesizing oligosaccharides when growth was effected in the presence of larger volumes of growth medium. The observations afforded the conclusion that a close relationship existed between the total quantity of organic carbon substrates available and the quantity of oligosaccharides synthesized by P. charlesii. The 16-day old mycelium had ex hausted its available carbon supply at the time of addition of the carbon-14 labeled acids. Under these conditions the labeled acids which were added at 16 days were rapidly converted to cell structural material and used in energy yielding processes. In its state of inanition P. charlesii converted little of its carbon supply to metabolites which were not used immediately as building-blocks in synthetic processes or as sources of energy for maintenance and growth of the organism. Additionally, the involvement of succinic and fumaric acid in the citric acid cycle and the accessibility to this cycle of acetate suggested readily

.available means for the complete oxidation of these labeled com pounds. Subsequent experiments, which involved studies of 336 14 14- incorporation of fumarate-2,3-G and malonate-2-C when P, charlesii was grown upon 150 ml of the Raulin-Thom medium, have shown that fumarate-2,3- c1* and malonate-2-C^ are poor precur sors of galactocarolose. Significant quantities of galactocaro lose were isolated from the growth media in the latter cases but the incorporation intp the oligogalactoside of fumarate-2,3- 14 x^f C or malonate-2-C was negligible.

Studies were performed of the incorporation into galacto- 4- carolose of glucose-u-C in the presence of tartronic acid, tar taric acid, and dihydroxymaleic acid,

Tartronic acid was considered a possible product of sequential transformation of tartaric acid and it was desirable to know if this C^ dicarboxylic acid were in equilibrium with or convertible to the tartrate-derived precursor of galactocarolose,

Tartronic acid was substituted for tartrate in.the Raulin-Thom 14- growth medium which had been supplemented with glucose-u-C to a final isotope concentration of approximately 2 microcuries per ml. Comparison of the specific activity of the glucose present in the medium at the beginning of the experiment with the specific

activity of the galactose isolated from galactocarolose suggested

that tartronate had failed to dilute glucose in the synthesis of

galactocarolose. Additionally, preliminary experiments have

shown that tartronate interferes with the uptake of tartrate-1, 14- 4--C by P, charlesii from the Raulin-Thom medium. In this 14- latter case measurable quantities of tartaric acid-1,4—C were

incorporated into galactocarolose. The tentative conclusion has 337 been made that tartronate is not incorporated into galactocaro lose and that this dicarboxylic acid does not appreciably

dilute the carbon-l4 labeled tartrate-derived precursor of

galactocarolose. lA- The incorporation of glucose-u-C into galactocarolose

was studied by Gander (5). The report stated that when the iZf organism was allowed to metabolize glucose-u-C in the presence

of tartrate, the specific activity of the galactose isolated

from galactocarolose was one-third the specific activity of the

glucose present in the medium at the time of initiation of growth

of P. charlesii.

An experiment similar to that described by Gander (5 )

was performed. P. charlesii was grown upon the Raulin-Thom lif medium which contained glucose-u-C as supplement. The galacto

carolose isolated after 28 days of growth of the mould was

hydrolyzed and the specific activity of the galactose determined.

The specific activity of the isolated galactose was but slightly

greater than one-third the specific activity of the glucose

present upon initiation of growth of the culture. This obser

vation essentially confirmed the earlier findings of Gander.

Similarly, the results were interpreted to suggest that possibly

two of the six carbons of glucose are incorporated into galacto

carolose.

Dihydroxymaleate was shown to exert pronounced effects

on the metabolism of P. charlesii. Experiments which involved

the substitution of this dicarboxylic acid for tartrate in the growth medium showed that galactocarolose was synthesized in the presence of dihydroxymaleate# When dihydroxymaleate replaced

tartrate, the organic carbon sources of the medium were converted

to galactocarolose with greater efficiency than under any other

conditions studied. Growth of P. charlesii in the presence of

dihydroxymaleate resulted in the elaboration into the growth

medium of a significant quantity of non-glucose carbohydrate#

Note was made of the presence in the concentrated growth medium

of two pentoses and an unidentified carbohydrate component. It

was of interest to learn if the effect on metabolism of P. charlesii

by dihydroxymaleate was direct or indirect. Experiments were

performed to establish whether dihydroxymaleate or one of its

conversion products served as a precursor of metabolites of P.

charlesii.

Penicillium charlesii was allowed to metabolize glucose- 1^ u-C in the presence of dihydroxymaleate which replaced tartrate

in the Raulin-Thom medium. After 28 days the growth medium was

separated from the mycelium and the resulting filtered medium was

concentrated to a small volume under reduced pressure. The

galactocarolose, present in the concentrated growth medium, was

isolated and hydrolyzed to free galactose. The specific activity

of the galactose was approximately one-third the specific ac

tivity of the initial glucose. Several carbon-l4 labeled mono

saccharides were observed to be present in the growth medium.

At least two pentoses, glucose and a methylpentose were identified.

Furthermore, appreciable non-carbon-l^-labeled carbohydrate was 339 observed, to be present. These observations were without precedent in studies involving P. charlesii and suggested that unusual patterns of metabolism by P. charlesii had obtained in the presence of dihydroxymaleate.

The data were interpreted to suggest that dihydroxy maleate, upon substitution for tartrate in the Raulin-Thom medium, affects galactocarolose synthesis directly by serving as a pre cursor of the galactose units of the oligosaccharide. Additional, indirect affects on P. charlesii metabolism by dihydroxymaleate have been considered but presently available data do not afford clear cut decisions in this regard.

The similarities in the extents of dilution of glucose- li+ u-C by tartrate and dihydroxymaleate afforded the suggestion that these two acids might serve as precursors of a common unit which is incorporated into galactocarolose. Support was given this hypothesis by the observation of -.an enzyme system extract- able from Penicillium charlesii, grown upon the Raulin-Thom medium, which effected a reduction of dihydroxymaleate in the presence of reduced nicotinamide adenine-dinucleotide (NADH^),

The "initial11 rate of dihydroxymaleate reduction by the crude enzyme system corresponded to the oxidation of 750 millimicromoles of NADH^ per hour per milligram of protein. Oxaloacetate, pyru vate, tartronate, and ascorbate failed to substitute for dihydroxy maleate in the reaction catalyzed by the crude preparation. Ad

ditionally this crude enzyme preparation catalyzed the slow oxidation of DL-tartrate in the presence of oxidized nicotinamide 3^0 adenine dinucleotide (NAD); neither flavin adenine dinucleotide

(FAD) nor nicotinamide adenine dinucleotide (NADP) would sub stitute for NAD in the oxidation of tartrate. Dihydroxymaleate ascorbate, lactate, malate, and tartronate failed to substitute for tartrate in the reaction catalyzed by the crude enzyme prepa ration in the presence of NAD.

The data and observations in the preceding pages serve as prelude to studies which may lead to complete definition of the role played by tartrate and other carboxylic acids in the synthesis of galactocarolose. It is clear that tartrate may serve one or several functions in the Raulin-Thom medium. Present observations, the. reports by Gander (5 ) and Bentley and co workers (23)— the latter that tartaric acid is incorporated into the oligosaccharides but negligibly into the tetronic acids and that succinate, propionate, acetate, and malonate are incor porated fairly readily into the tetronic acids but only poorly into the oligosaccharides of P. charlesii— provide a basis for discussion of a possible metabolic sequence for formation of galactocarolose•

The biosynthesis and accumulation in the growth medium of galactocarolose are phenomena which may be closely associated with the function of the oligogalactoside and this latter is herewith considered.

The view has been advanced by the Raistrick school that

galactocarolose serves as an intermediate in the carbon catabolism

of various substrates upon which P. charlesii is allowed to grow. 3^1

The heterocyclic compounds carlic, carlosic, carolic, and

carolinic acids are viewed as products of degradation of the polygalactofuranoside•

Clutterbuck has recorded that • the °9 aoiis haTe been shown to be derivatives of tetronic acid, the acids

having the general formula A, the C^Q acids the formula B

COOH CH. CH ----0,

H R h ' ^ r

HO-CH. HO-CH,

HOCH HOCH

\.«0 i^OH ^ jOH ,0H

K/ ^ O E bH

A comparison with the structure of galactofuranose (C)

or better still with that of the corresponding lactone obtained

by oxidation of the methylated material with bromine (D),

indicates the close similarity of structures B and D. .This close

relationship makes it appear probable that this polysaccharide 18 released formaldehyde upon titration with periodic acid suggested that the unidentified compound was a uracilnucleoside of a hexofuranose. After its release from the nucleoside the carbohydrate was shown to react with ATP in a process catalyzed by a partially purified galactokinase to yield a phosphorylated product. Chro matography of the sugar phosphate revealed that it was not identical with either galactose-l-phosphate or glucose-6-phos-

$ phate.

More recently it has been demonstrated that carbon-14- l^f from tartaric acid-1, C was incorporated into the unidentified uracil-nucleoside to a much lower extent than would be expected for a precursor of galactocarolose (36). The function of this, nucleoside in Penicillium charlesii remains undefined.

(F) Functions and Transformation of Tartaric Acid in Biological Systems

Aside from the previously mentioned reports of Gander and of Bentley and co-workers little information has been published which relates to the metabolism of the tartaric acids and their significance as precursors of major cell metabolites. Neverthe less, the biochemical literature contains numerous references to studies which have involved tartrate as a stimulant or as a de pressant of some biochemical function, a product of some catabolic process or as a substrate for any one of several enzymes. A brief summary of such studies involving tartrate is outlined below. 3^2 is an intermediate in the microbiological synthesis of these acids” (123)•

Workers in the Raistrick school observed that numerous

fungi produce oligo and polysaccharides from "• • . simple molecules containing only a few carbon atoms . • *" (125) and suggested that "the oligosaccharides and polysaccharides are

subsequently further metabolized to products which bear structural resemblance to the components of the oligo and polysaccharide."

This view has been criticized by Foster (reference 124, p. 4?9) who indicated "... its weakness lies in the lack of general

applicability so far, and in the fact that of the multifarious products of mould metabolism known, few bear chemical similarity

to a parent carbohydrate as depicted above."

A second view quoted by Maynard (7) "• » • suggests that

the various (tetronic) acids are intermediates in the synthesis V of the galactofuranose units, rather than degradation products

of these units . . . ." The results of the experiments performed by Bentley and co-workers (23), published after the suggestion

quoted from reference 7 , appear to suggest that the tetronic

acids are not important intermediates in the biosynthesis of l4 galactocarolose. Tartaric acid-1,4-C , which was shown to be

incorporated into the oligogalactoside, galactocarolose (5 ), was

very inefficiently incorporated into tetronic acids (23)•

Additionally, malonate has been shown to be an excellent

precursor of the tetronic acids but present observations suggest

that malonate is not readily incorporated into galactocarolose. 3^3 Compounds such as galactocarolose have been viewed as products of shunt metabolism (12*f). According to this view

". • • enzyme mechanisms normally involved in complete oxidation of the substrate become saturated and the substrate molecules

are then excreted and accumulate as such or they are shunted off

to secondary or subsidiary enzyme systems which are able to effect

only relatively minor changes in the substance, which then ac

cumulates in the transformed state. The latter mechanism is by

far the most important common. The limiting or bottleneck enzyme

systems are never those concerned with the initial stages of

carbohydrate dissimilation but are those which act on the sub

strate only after it has been brought through the stage of split

products . . • » As regards a raison d'etre of galactocarolose, this latter

view is difficultly challenged by data which has been compiled

during the course of present studies on galactocarolose synthesis

by P. charlesii. In fact most of the observations which have been

made can be interpreted in terms of galactocarolose biosynthesis

occurring as a result of an overflow or shunt process.

It is not inconceivable that galactocarolose serves some

important substrate or structural function in P. charlesii. Some

consideration has been given the view that galactocarolose serves

as a "storage" oligosaccharide, the mould producing significant

quantities of this material during growth stages at which the

medium is rich in substrates such as glucose and dicarboxylic

acids. According to this concept the oligosaccharide is extruded into the growth medium where because of its unusual structure it remains inert to attack by chemical and/or microbiological agents which reside in or on the culture fluid* When all of the

"primary" and "secondary" carbon sources of the growth medium have been exhausted by Penicillium charlesii* the galactocarolose is reconcentrated within the mycelium where it is used for synthetic and energy-yielding processes. It might be pointed

out that attempts to demonstrate hydrolysis and/or phosphorolysis

of partially purified galactocarolose by extracts of P. charlesii

(mycelium of various ages) have been uniformly unsuccessful*

This negative observation does not necessarily preclude the

hypothesized storage function for galactocarolose. It has been

observed that the oligosaccharide content of cultures of P.

charlesii* older than three to four weeks, gradually decreases

and this observation suggested that the mould possessed enzyme

systems which effected the complete conversion of the oligosac

charides to other metabolites.

Galactocarolose could conceivably be involved as. a con

stituent of the cell wall or some other structural unit of

Penicillium charlesii. The unusual nature of this oligosac

charide suggests that it might enddw a structural unit with the

capacity to resist attack by foreign agents, chemical and bio

logical* Unusual glycoside and mucopeptide linkages are known

to occur in the walls of all bacteria (l46). A significant amount

of experimental effort has been devoted to the delineation of the

structure and function of these mucopeptide components (1^7, 1^8, 3^5

1^9,150). A recent publication has described the isolation and examination of an intracellularly-located teichoic acid from

Staphylococcus aureus H (151).

The accumulation of galactocarolose in the growth medium of P. charlesii might be a reflection of an oversynthesis of this oligogalactofuranosyl component of a structural material.

On the other hand the accumulated galactocarolose may represent a portion of a structural component which was not incorporated because of some metabolic lesion.

The lack of knowledge related to the exact chemical com position and structure of wall materials of P. charlesii and the lack of availability of methods for the isolation of cell walls of fungi in a manner which yields these cell walls free of other mycelial components precludes final acceptance or re jection of this latter concept.

Whatever be the function of galactocarolose a proposed biogenetic pathway for this oligosaccharide must allow for the considerations that:

a) Succinate, fumarate, and malonate are not efficiently

incorporated into galactocarolose whereas the former

three dicarboxylic acids are significantly incorporated

into the tetronic acids of the mould.

b) Tartrate is incorporated into galactocarolose and tar

trate is not incorporated, to a significant extent,

into the tetronic acids produced by P. charlesii. 3^6

c) Acetate-l-C and Acetate-2-C are readily incorporated

s into galactocarolose.

d) Dihydroxymaleic acid appears to act as a precursor of a

portion of the hexose units of galactocarolose.

One suggested pathway visualizes the participation of a compound such as a nucleoside diphosphoryl derivative of 5-keto-

6-deoxy-L-arabohexose. A possible sequence for the conversion of this hypothetical compound to oligosaccharide is represented in Figure 69 while Figures 70 and 71 represent two possible sequences through which tartrate and glucose-derived precursors might be incorporated into the nucleotideglycoside.

The sequence of processes shown in Figure 69 represents phosphorylation, pyrophosphorolysis of a nucleoside triphosphate, enolization, an antimarkovnikov hydration and polymerization.

It is of interest that a 5-keto-6-deoxy-arabohexose has been identified as a. component of the antibiotic Hygromycin A (130) and the biosynthesis of this sugar has been studied by Elbein and co-workers (131). Numerous authors have postulated the occurrence of nucleoside diphosphoryl derivatives of A—keto-6- deoxy-hexoses as intermediates in the' synthesis of L-fucose and of L-rhamnose (132, 133* 13^, 135* 136).

Pathway A represents a sequence which involves the con version of an intact tartrate chain into carbons 1 through k of the eventual galactofuranosyl moiety of galactocarolose. L- threonic acid is pictured as an early intermediate in the con version of tartrate to the oligosaccharide. L-threonic acid or Fig. 69.--A hypothetical sequence for conversion of 5-keto-6-deoxy-L-arabohexose to galactocarolose.

3^7 OH 0 P 0 9 Base C‘H " CH Ribpse HQOH HOOH P HOC-H A HOC-H B P -0 -9 -H -OC-H . 0 0 Q--0 CH3 CH3

VHT 0=6

:xzr

Base Bpse Rifeose Ribose i i

? P e p P o _ 0 -v Galactocarolose

XZEL

OJ 4=- OC

x m . Fig. 69 Fig. 70»--A hypothetical biosynthetic sequence for 5-keto-6-deoxy-L-arabohexose— pathway A •

3^9 9 0 9 ‘ O C R C-OH C-OH C-OH CH HC-OH. ► HC-OH HC-OH ______IT HOC-H HOC-H h o 6-h

COOH CH20H CHPPO 3

n r IS?

0 q H C-OH — C-H H C-OH HC-OH H < fO fc j HOC-H HOC-H HOC-H HOC-H C-H 0=0 c h o • C H j 35 a 'V r r

Fig. 70 5-Keto -6 - Deoxy L-Arobohexose Fig. 71.--A hypothetic biosynthetic sequence for 5-keto-6-deoxy-L-arabohexose— pathway B.

351 1 9

(1) Mammalian and avian metabolism of tartaric acids

The enzymatic oxidation of tartrate by mitochondria from a number of animal tissues has been demonstrated by Kun and co- workers (37)* The protein which catalyzed this oxidation was particulate but could be solubilized with facility. The enzymic oxidation of tartrate was absolutely dependent upon added nicotinamide adenine dinucleotide. Nicotinamide adenine dinucleotide phosphate (NADP) would not function as a cofactor in the process. Products of the reaction were reduced nicotin amide adenine dinucleotide and oxaloglycolic (dihydroxymaleic) acid. The reversibility of the reaction was readily demonstrated in the presence of reduced-nicotinamide adenine dinucleotide and oxaloglycolic acid.

Schofield (38) has described a crude preparation from an acetone powder of pigeon liver which oxidizes tartaric acid in an oxygen independent process when either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was present in the experimental system. The substrate affinity constant of the enzyme responsible for tartrate oxidation was relatively high, being 1.4 x 10 M as compared to an affinity constant of 6.2 x 10”^ M for a malic dehydrogenase present in the preparation. The product of tartrate oxidation in this system was not defined. ftHO HOOCCOH O-COH OCOH . 0 OCOH OCOH OCOH ^ H0 (J;H *1y0H *C0H COH Y tCHO c c o o h .HodH ? „ h o c h HOCH "— ~ QOH ~*HQOH 6 0 do (J>o eo

0=C0H 0=C0H CHO CH-'3 6 h3 ch3 ch3

3 1 33E SEC 3 E

go oh g o o H — C--0 H 0 0 C-OOH HgoH h Coh HOOH w h o -c h HOC-H h o -Ch HO-g-H 0 g*H “ p-o ? wlla CH, S£

U> VJ1 ZE3ZH szn r m . w

rig. 71 5-Keio-6-Deoxy L-Arcsbohexose \ the 4-0-phosphoryl derivative might undergo condensation with active acetate to yield 5-keto-6-deoxy-L-arabohexonic acid or the corresponding 4-lactone (VII), Reduction of the lactone would yield 5-keto-6-deoxy-L-arabohexose (VIII). Pathway A includes the consideration that the specific activity of the

galactose, isolated from galactocarolose, will be identical to 14 the specific activity of the tartrate-C initially present in

the growth medium of P. charlesii. An argument against this

sequence derives from the report of Gander (5) which related the 14 incorporation of tartrate-1,4-C into galactocarolose.

Figure 71 represents a second, more complicated sequence

which leads to formation of the postulated 5-keto-6-deoxy-L-

arabohexose. 'Tartaric acid is imagined to be converted to di-

hydroxymaleic acid. Becarboxylation of dihydroxymaleic acid

to tartronic acid semialdehyde could be followed by condensation

of the latter with an activated C^ unit to yield 2-acetyltartronic

acid semialdehyde which might undergo dehydrogenation to yield

XIII and decarboxylation of this latter gives rise to XIV. The

decarboxylation of XIII could conceivably give rise to XIV which

has only one-fourth the specific activity of the initial tar

trate; the equivalent carboxyl groups might or might not be *

treated indiscriminately by an enzyme. If the non-labeled

carbonyl group were preferentially removed the species XIV would

have one-half the specific activity of the initial tartaric acid.

The condensation with XIV of a second mole of tartronic acid

semialdehyde would yield the 7-ca5:bon saccharide, 2-carboxy-5- keto-6-deoxy-L-arabohexose XVI* Oxidation of XVI to the dicarboxylic acid would yield XVII which could give rise to 5-keto-

6-deoxy-L-arabonic acid, XVIII. The reduction of XVIII would yield 5-keto-6-deoxy-L-arabohexose, VIII. Alternatively, the removal of the labeled carboxyl group of XVI would yield VIII with a specific activity one-fourth to one-half the specific activity of the tartrate initially employed. Operation of this sequence precludes significant incorporation of succinate, malate, oxaloacetate, lactate, or of phosphorylated acids such as phosphoglycerate, .phosphoenolpyruvate, activated malonate, or propionate into galactocarolose. The failure of in corporation of tartronate into galactocarolose might be ex plained on the basis of incapacity of P. charlesii to perform a tartronate to tartronic acid semialdehyde transformation or if such a transformation does take place the resulting pool of 5 tartronic acid semialdehyde is isolated from the pool of the tartrate-derived semialdehyde, XI, which might be a precursor of galactocarolose*

Pathways A and B, and other sequences not described or discussed above, are conceivable and evidence to demonstrate the exclusive operation of one of these sequences might be de rived from degradation of the hexose units of galactocarolose which has been labeled by several of the carbon-l^f labeled com pounds postulated as intermediates in Figures 70 and 71*

The failure of incorporation of the glucose carbon chain, intact, into a galactoside has been noted in the case of lactose 355 (137» 138). Several accounts have been made of the incorporation of small-unit precursors into the galactose and glucose moities of lactose (138, 139» 1^0). Evidence suggests, that in bovine mammary tissue, the galactowaldenase catalyzed reaction (103) does not constitute the major process for the conversion of glucose to galactose (l^l). The observations have frequently been explained in terms of randomization of the glucose carbon chain through operation of the pentose phosphate shunt and other pathways that involve fragmentation of the Cg glucose chain*

It has been shown that Tetrahymena pyriformis, strain E,

can convert fats to glycogen (1^2 , 1^3 ) and these reports and subsequent studies have led Hogg and Kornberg to suggest that

the glyoxylate cycle (l*»4) plays an important role in glyconeo-

genesis in this protozoan (l**5)» It would appear, therefore,

that unique processes may be operative in the conversion of small unit compounds to glycogen T. pyriformis, strain E.

The conversion of small unit precursors to oligosaccharides

through quasi de novo processes may constitute important pathways

for the synthesis of certain glycosides in animals, plants, and microorganisms. Galactocarolose formation by P. charlesii may

represent an instance of such a quasi de novo process*

The sequence of reactions described as a hypothetical

pathway to galactocarolose formation is offered merely as a

suggestion and it is worthy of. declaration that the indicated

processes (Figures 69, 70, and 71) may or may not be realized

in vivo by P. charlesii. Whereas the suggested sequences 356 adequately explain the existing relevant data, this accumulated data is sufficiently meager and incomplete that any proposed pathway for galactocarolose formation is presently premature.

Formal regard and/or acceptance of the hypothesis offered herein (for a biosynthetic pathway for galactocarolose) must await the accumulation of a larger volume of relevant data than is presently available. SUMMARY

Investigation of the role of dicarboxylic acids in the production of oligosaccharides by P. charlesii have shown that several acids may substitute for tartrate in the Raulin-Thom growth medium. Production of mould mycelium was stimulated by the presence of malonate, fumarate, or succinate in the growth medium whereas the formation of oligosaccharides, by P. charlesii, was stimulated by dihydroxymaleic acid, fumarate, and to a lesser extent malonate. Experiments which employed carbon-14- labeled malonate, succinate, or fumarate in the growth medium revealed that these three dicarboxylic acids are very poor pre cursors of the oligosaccharides#

Dihydroxymaleate stimulated both oligosaccharide and mycelium production and evidence was obtained which suggested that a portion of the chain of dihydroxymaleate was incor porated into galactocarolose. Tartronic acid was not incorpo rated into galactocarolose. Tartronic acid inhibited the 14- metabolism of DL-Tartrate-C but the former acid did not sig- 14- nificantly depress the absolute incorporation of glucose-u-C into galactocarolose#

Cell-free extracts of P« charlesii, grown on the Raulin-

Thom medium, were shown to catalyze the oxidation of tartrate in

357 the presence of NAD and the reduction of dihydroxymaleate in the presence of NADH^, Consideration was afforded the possibility that dihydroxymaleate is one intermediate in the metabolism of tartrate by P, charlesii.

Several unusual organic compounds were isolated from the mycelium-of P. charlesii but the structure and functions of these compounds have not been completely defined,

P. charlesii was shown to rapidly concentrate tartrate of the growth medium when the latter was still relatively rich . in glucose. Studies have shown that very little of the carbon-1^ of tartrate-C (concentrated by P. charlesii) is present as free tartrate within- the mould mycelium. LITERATURE CITED

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(2) Tartaric acid metabolism in bacteria

A large number of bacteria have been screened for capacity

to attack tartaric acids. La Bivi&re (39) has shown that intact

cells and cell-free extracts of Pseudomonas putida rapidly con

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115. Kornberg, H. L., and S. E. Elsden in F. F. Nord(editor). Adv. in Enzymol., Vol. 23. P. 401. Interscience, New York Tl96l).

116. Stafford, H. A., A. Magaldi, and B. Vennesland. Science 120: 265 (195*0.

117* Kun, E., and M. Garcia Hernandez. Fed. Proc. 14: 240 (1955).

118. . J.B.C. 218: 201 (1956).

119. Stafford, H. A. Plant Physiol. £1: 135 (1956).

120. Kun, E. J.B.C. 221: 223 (1956).

121. Bressler, E., and S. Wakil. J.B.C. 236: 1643 (1961).

122. Krasna, A. I. J.B.C. 237: l4l8 (1962).

123. Clutterbuck, P. W., J. Chem. Ind. 55T-61T (1936). CQuoted in reference 124, p. 478TT

124. Foster, J. W. "Chemical Activities in Fungi," Academic Press, New York (1949).

125. Birkinshaw, J. H., and H. Eaistrick. Biochem. J. 27: 370, 375 (1933).

126. Bishop, C. T., F. Blank, and M. Hranisavljevic-Jakovljevic, Can. J. Chem. 40: 1816 (1962).

127. Siddiqui, I. E., and G. A. Adams. Can. J. Chem. 39: 1683 (1959).

128. Brown, E., and L. K. Eobinson. J. Immunol. 47: 7 (1943).

129. Shabarova, Z. A., J. G. Buchanan, and J. Baddiley. B.B.A. (1962)— announced in reference 73* 366

130. Mann, R. L,, and D. 0. Woolf. J.A.C;S. 72s 120 (1957)I M. Namiki, K. Isono, and S. Suzuki. J. Antibiotics 10: 160 (1957).

131. Elbein, A. D., R. L. Mann, H. E. Renis, W. M. Stark, Henry Koffler, and H. R. Garner. J.B.C. 236: 289 (1961).

132. Pazur, J. H., and E. W. Shuey. J.B.C. 236: 1780 (1961).

133. Glaser, L., and S. Kornfeld. J.B.C. 236: 1795 (1961).

134. Ginsberg, V, Fed. Proc. 19: 85 (i960).

135. Okazaki, R., T. Okazaki, J. L. Strominger, and A. M. Michelson, J.B.C. 237i 3014 (1962).

136. Ginsberg, V. J.B.C. 236: 2389 (l96l).

137. Watkins, W. M., and W. Z. Hassid. J.B.C. 237: 1432 (1962).

138. Wood, H. G., P. Schambye, and G. J. Peeters. J.B.C, 226: 1023 (1957).

139* Schambye, P., H. G. Wood, and M. Kleiber. J.B.C. 226: ,1011 (1957).,

140. Wood, H. G., P. Siu, and P. Schambye. A.B.B. 69: 390 (1957).

141. Hansen, R. G., H. G. Wood, G. J. Peeters, B. Jacobson, and J. Wilken. J.B.C. 2££ (4): 1034 (1962).

142. Wagner, C. "The Glycogen Metabolism of Tetrahymena pyriformis. Ph.D. thesis, University of Michigan, Ann Arbor, Michigan (1956).

143. Hogg, J. F., and C. Wagner. Fed. Proc. 13: 275 (1956).

144. Kornberg, H. L., and N. B. Madsen. Biochem. J. 68: 549 (1958).

145. Hogg, J. F., and H. L. Kornberg. Biochem. J. 86: 462 (1963).

146.‘ Work, E. Biochem. J. 82: 35 (1962).

147. Baddiley, J. Biochem. J. 82: 36 (1962).

148. Strominger, J. L. Fed. Proc. 21 (1): 134 (1962).

149. Wicken, A. J., S. D. Elliot, and J. Baddiley. J. Gen. Microbiol. %0i 111 (1963). 3 6 7

150. Wicken, A. J., and J. Baddiley. Biochem. J. 8£: 54 (1963).

151. Rajbhandary, U. L., and J. Baddiley. Biochem. J. 8?: 429 (1963).

152. Friedeman, T. E,, and G. E. Haugen. J.B.C. 14?: 415 (1943).

155. El Hawary, M.F.S., and.R.H.S'. Thompson. Biochem. J, 53s 340 (1953).

151*-. Shilo, M., and R. Y. Stanier. J. Gen. Microbiol. 16: 432 (1957). ,

155. Jowett, M., and J. H. Quastel. Biochem. J. £1: 275 (1937).

156. Quastel, J. H., and R. Woolridge. Biochem. J. 22: 689 (1928).

157. Green, D. Biochem. J, ^0: 2095 (1936).

158. Stafford, H. A., A. Magaldi, and B. Vennesland. Science 120: 265 (1954). AUTOBIOGRAPHY

I, John Maxwell Jordan, was born in Westfield, Alabama, on February 15* 1938* I received my elementary and secondary- school education in the public schools of the Jefferson County

(Alabama) school system. I enrolled at Central State College,

Wilberforce, Ohio, in September of 1955 and received a B.S. degree from this institution upon my graduation in June of

1959* I was enrolled in the Graduate School, Department of

Agricultural Biochemistry, of The Ohio State University in

September of 1959 and I was in residence at this university throughout the period September 1959 through August 1963*

368 21 cryptic to tartrate although they could still attack oxaloacetate*

The capacity of the stationary cells to attack tartrate was re gained when these cells were exposed to tartrate* It was also shown that meso tartaric acid inhibited the d-tartaric acid dehydrase in the cell extracts* This latter effect could not be demonstrated in whole cells which had been adapted to one of the tartaric acid enantiomers*

(3) -Metabolism of tartaric acid by moulds

The formation of tartaric acid by Aspergillus niger from glucose (1*5) from ^-ke to gluconate (46) has been reported*

The information supplied in these publications did not include definition of the stereochemical form of the tartaric acid enantiomer which was produced*

Tartaric acid has been shown to be converted, in quanti tative yield, to oxalic acid via a fluoride and fluoroacetate insensitive process by washed suspensions of Aspergillus niger strain 72-4 (47)* In this study it was reported that a large number and variety of substances including fructose, 5-keto-

gluconate, mannitol, citrate, cis aconitate, and succinate were

also converted to oxalic acid in high yield. The fluoride and

fluoroacetate insensitivity of the conversions was interpreted

as suggesting that the "Krebs cycle and glycolysis beyond the

state of trioses do not participate as pathways for oxalate

formation*" It was further suggested that tartrate might be an

intermediate in the conversion of oxaloacetate to oxalic acid, To the extent that presently reported experimental efforts have afforded meaningful results, gratitude is expressed to N. M. Hughes, M. H. Jordan, and J. L. Jordan with whom I identify the sources of concern, encouragement, and confidence which have served to sustain me throughout the course of my graduate studies. 22 the conversion being accomplished through a hydration of the enol form of oxaloacetate.

Aspergillus fumigatus has been shown to effect the for mation of trans-l-epoxysuccinic acid and it has been suggested that this process occurs through the action of an oxygenase on fumaric acid (48). The possibility that tartaric acid was similarly converted to the epoxy acid was not discussed. In this study A. fumigatus was grown upon the Raulin-Thom medium which does contain tartaric acid.

It was demonstrated by Martin and Foster (49) that trans- l-epoxysuccinic acid is converted to tartaric acid by Aspergillus fumigatus. Contrary to the chemical process, which yields a mixture of meso and D-tartrates in the ratio of 60/40 (50), the

fungal enzyme stereospecifically converts trans-l-epoxy succinic acid to meso tartaric acid with formation of 100 per cent of the internally compensated isomer.

The observation that trans-l-succinicepoxide grown cells

from several soil bacteria, Flavobacterium sp., Penicillium vermiculatum and a yeast attack meso-tartrate suggested that meso-tartrate might be an intermediate in the carbon and energy

yielding reactions associated with breakdown of trans-l-epoxy

succinic acid.

Penicillium atrovenetum. when grown on the Raulin-Thom medium has been shown to produce -nitro-propionic acid to a

maximum extent of 2.4 grams per liter of growth medium (51).

When the organism was grown upon the Czapek-Dox medium the yield 23 of the nitro acid was only 10 per cent of that obtained with the

Raulin-Thom medium. The suggestion that aspartic acid is directly convertible to jg-nitro propionic acid without loss of the alpha- amino nitrogen (52) was not confirmed by the studies of Hylin and

Matsumoto (53)* The latter authors suggested that the nitro acid arose through the condensation of fumaric acid with an oxidized form of ammonia*

When tartaric acid was tested as a stimulant for pro duction of mycelium and ^-nltropropionlc acid the absolute affect, in both cases, was quite similar to that obtained for fumaric acid. Tartaric acid labeled with carbon-l^f was not tested as a precursor of -nitropropionic acid*

Tartaric acid has been shown to be stimulatory to Neuro- spora crassa strain 7^-A (5*0* The authors suggested that the organism converts d or 1-tartaric acid to mesoxalic acid and tartronic acid in a process which may have involved fixation of carbon dioxide. The observed formation of tartronic acid could also be explained in terms of conversion of tartrate to glyoxylate, a process which might be succeeded by the action of glyoxylate carboligase and a tartronic semialdehyde dehydrogenase* Mesoxalic acid could be derived from tartronate by the action of a dehy

drogenase*

Figure 1 affords a summary of most of the defined re actions of biological systems which involve synthesis or other

transformations of tartrate* Fig* !•— -Biochemical processes involving tartrate.

The numbers in parenthesis represent references (notation in Bibliography) to pertinent literature.

Zk 9 $

TRANS -I-EPOXY SUCCINATE

g l u c o s e : r-: 5 - KE TOG LUCO NATE

Fungi C4t») Fungi

OXALATE 4JM- (.Zd y- DIHYDROXYMALEATE Fungi Mammalian Tis sue "p

(3S) Pigeon Liv^r

C39) Bacteria Bacteria

TARTRONATE (W) GLYCERATE .

OXALOACETATE ri|* 1

BIOCHEMICAL PROCESSES INVOLVING TARTRATE EXPERIMENTAL

(A) Maintenance and Growth of Cultures

(l) Pseudomonas sp. Shilo

Cultures of Pseudomonas sp. Shilo (44) were provided by

Dr. R. Y. Stanier of the University of California at Berkeley.

Prior to transfer the cultures were maintained on slants. When it was desirable to obtain cells for preparation of enzymes, a sterile loop was used to transfer inoculum from slants to 250 ml

Erlenmeyer flasks which contained 50 ml of the sterilized medium

suggested by Shilo (44), This latter growth medium was modified

to contain disodium tartrate at a final concentration of 0.5

per cent and diammonium sulfate to 1.0 per cent.

The flask was shaken at 30°C for 13 hours and the contents

transferred to a 1000 ml Erlenmeyer flask which contained 250 ml

of fresh growth medium. This inoculated medium was shaken in an

Eberbach horizontal shaker over a period of 13 hours and the

resulting mixture of cells and medium transferred to a 4.5 liter

heavy walled florence flask which contained 1000 ml of fresh

growth medium. Shaking on a rotating table for 15 hours at 30°C

yielded a culture which was used to inoculate (to 2 per cent)

30 liters of fresh, recently sterilized medium contained in 50-

liter bottles. In this latter instance growth was allowed to

occur at 30°C with vigorous aeration over a period of 15 hours.

26 27

The cells were collected in a Sharpies centrifuge, the cup of which had been pre-cooled at -10° C for 6 hours. The harvested cells were washed .three to five times with three volumes of in organic orthophosphate buffer of the same composition as that used in growth of the organism.

When lypholized and stored at -10°C the cells remained viable for at least 5 months.

(2) Pseudomonas saccharophillia Doudoroff

Cultures of Pseudomonas saccharophilla Doudoroff (American

Type Culture Collection catalog number 91140 were obtained from

Dr. Michael Doudoroff of the Department of Microbiology of the

University of California at Berkeley. The cultures were maintained and grown essentially according to the procedure of Wallenfels and Kurz (55)• Cells were harvested in a Sharpies centrifuge and washed three times in three volumes of the buffer of growth.

Washing was effected through suspending the harvested cells in

three volumes of buffer and centrifugation at 10,000 x g in a

Servall refrigerated centrifuge which was operated at 0°C.

When lypholized the cells remained viable for several months.

(5) Penicillium charlesii

Cultures of Penicillium charlesii G. Smith were obtained

from the American Type Culture Collection (A.T.C.C. strain 1887),

Washington, D. C«, and maintained in agar plates which had been

fortified with the Czapek-Dox medium containing 2 per cent agar. 28 •

Transfers to fresh agar plates were usually made after two to three weeks development of a culture. Mature spores were

transferred by platinum wire loop to 500 ml widemouth Erlenmeyer

flasks which contained 120 to 150 milliliters of sterilized

Raulin-Thom growth medium* Cultures were maintained at 22° to

24°C throughout the growth period* Exceptions to these conditions

are noted in the relevant section of the text*

The composition of the Raulin-Thom and the Czapek-Dox

growth media have been described elsewhere (105)*

(B) Enzymes: Sources, Extraction, Assay

(1) Tartaric acid dehydrase

Crude tartaric acid dehydrase was prepared from Pseudo

monas sp* Shilo by the originally published procedure (44). The

assay for enzyme activity depended upon the conversion of Oxalo-

acetate (the product of dehydration of the tartaric acids) to

•«* the relatively stable 2,4-Dinitrophenylhydrazone'’and the spectro

scopic and chromatographic detection of the latter compound*

(2) Galactose-dehydrogenase

* D-Galactose dehydrogenase was prepared from Pseudomonas

sac charophillia according to the procedure of Doudoroff (57) •

The partially purified enzyme, which showed no activity in the

presence of mannose or glucose, was assayed according to the

procedure of Su and Hassid (58)• 29

(2) Extraction and assay of enzymes from P. charlesii

Several procedures were employed in attempts to solu bilize certain enzymic activities believed to reside within the mycelium of P. charlesii. The method generally used involved grinding of the washed, pressed mycelium with an abrasive

(Alundum, Sea Sand, or Glass Beads) with a mortar and pestle and

further disruption of the suspended paste through ultrasonication.

The specifics of methodology are further defined in the relevant area of the text.

(l) Alcoholic extraction

Alcoholic extractions were accomplished with a Soxhlet

continuous extraction apparatus or through direct suspension of

the mycelium in boiling alcohol of composition defined in the

particular experiment in reference.

Prior to extraction the washed mycelium was pressed

between several layers of cheesecloth or filter paper to remove

most of the mycelial liquids that could be expressed. The pressed

mycelium was frozen in liquid nitrogen and powdered while in the

solid state. The paste, which resulted upon warming to room

temperature, was added directly to the Soxhlet extraction cup.

Direct extraction by alcohol under reflux was usually

preceded by a step involving powdering of the mycelium as de

scribed above. Direct extraction was effected through addition 3° of the mycelial paste to a round-bottom flask which was fitted with a reflux condenser. Alcohol and several small glass marbles were added to the flask and the mixture maintained at reflux for several hours.

(2) Acidic extraction of P. charlesii

Perchloric acid of various concentrations was used in the acid extraction of the mycelium of Penicillium charlesii.

The general procedure consisted of homogenization of the mycelium in cold perchloric (13 ml of perchloric.acid per gram of fungal tissue) with a Waring Blender, the cup of which had been pre cooled. The homogenized mould was subjected to sonication for

30 to kO minutes at 0° to 4°C and the resulting mixture stirred for at least 12 hours at -10°C. The soluble and insoluble com ponents of the extract were separated by centrifugation, the residue washed thoroughly and the washings added to the main supernatant• The supernatant plus washings was neutralized in the cold to pH 7.0 with potassium hydroxide. After standing at

- 1 0 ° C for sufficient periods to ensure complete precipitation the supernatant and precipitate were separated by centrifugation.

The neutralyzed extract was generally concentrated by lyophili- zation of the solvent.

(D) Chromatographic Procedures

(l) Carbohydrates

The following solvent systems were employed in various experiments. ! (a) n-Butanol:Pyridine:Water (6:4:3, V/V/V) (59)

(b) Ethyl Acetate:Pyridine:Water (36:10:11*5, V/V/V) (60)

(d) MethanolsFormic Acid:Water (80:15:5, V/V/V) (6l)

(e) Pyridine:Ethyl AcetatesWater (2:7*1, V/V/V) (62)

(f) Pyridine:Ethyl AcetatesAcetic AcidsWater (5:5:1*8,

V/V/V) (63)

;(g) 80 per cent phenol (aqueous)

(h) n-Butanol:Acetic AcidsWater (6*0

Reducing sugars were detected on paper chromatograms with an ammoniacal silver nitrate spray reagent which was composed of a 1:1 mixture by volume of 0.1 molar silver nitrate and 0.1 molar ammonium hydroxide. The silver nitrate and ammonium hydroxide solutions were mixed immediately prior to use. Non-reducing carbohydrates were detected on paper chromatograms with the periodate and benzidine spray reagents (65, 66).

(2) Sugar phosphates

The solvent systems which were employed in paper chroma tography of sugar phosphates included

(a) Methanol:Formic Acid:Water (80:15:5, V/V/V) (6l)

(b) Methanol:28 per cent Ammonia:Water (60:10:30)

Hydroxide (7:2:3, V/V/V)

Sugar phosphates were visualized on paper chromatograms through the procedure of Bandurski and Axelrod (6l). CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

ILLUSTRATIONS...... viii

TABLES ...... xiv

INTRODUCTION...... 1

REVIEW OF THE LITERATURE . 6

(A) Isolation and Characterization of P. charlesii and Some of Its Metabolic Products ...... 6

(B) Occurrence in Nature of Oligosaccharides and Polysaccharides Containing Galactose ...... 7

(D) The Occurrence of Glycosides which Contain Galactofuranosyl Moieties ...... 9

(1) Umbilicin ...... 9 (2) The Extracellular Polysaccharide of Gibberella fu.likuroi ...... 10 (3) The Galactomannan of Trichophyton granulosum ...... 10 (k) The Complex Oligosaccharide of Type 3^ Pneumococcus...... 11 •. (3 ) The Polysaccharide of Cladophora rupestris • 12 (6) The Galactan of Penicillium charlesii .... 13 (7) Di to Penta-Galactofuranosides...... 13

(E) The Biosynthesis of Oligosaccharides ...... 15

(1) Transglycosylation ...... 16 (2) Action of Phosphorylases ...... 16 (3) Nucleoside Diphosphoryl Glycoside Trans- glycosylases . . 16

iv 32

Unless noted otherwise mixtures of phosphorylated com pounds were resolved through ascending chromatography on Whatman number 1 or Whatman number 3 MM chromatography paper*

(3) Carboxylic acids

Several solvent systems were found to be useful in the resolution of mixtures of aliphatic carboxylic acids* The systems employed included n-Butyric acid:n-Butanol:Water (2:2:1,

V/V/V) (68), 70 per cent aqueous phenol, 80 per cent aqueous phenol, and n-Amylalcohol:5 M aqueous Formic acid (1:1, V/V)

(69).

Descending chromatography was used in the resolution of mixtures of carboxylic acids. Saturated ammonium vanadate and ammoniacal silver nitrate (0.1 molar silver nitrate and 0.1 molar ammonium hydroxide 1:1 volumes mixed immediately prior to use) were used as spray reagents for the detection of carboxylic acids on paper. The spray reagents and the techniques employed have been described by Aronoff (68).

(4-) Heterocyclic bases and acids

Heterocyclic compounds which were derivatives of pyridine were separated in several of the solvent systems employed by

Hendrick and co-workers (70)• The solvent systems found most satisfactory were n-Butanol:Water:Glacial Acetic Acid (30:10:2,

V/V/V) (71) and a mixture of water:concentrated HC1; diethyl etherimethanol (15:^:50:30, V/V/V/V) (72). 33

The heterocyclic compounds were located on the chroma tograms with a Mineral!ght ultraviolet lamp (253751) in a process similar to that described by Hendrick and co-workers (70)•

(£) Quantitation of Carbohydrates in Solution

(1) General Methods

Total sugar was determined by the sulfuric acid-phenol method (7*0 and the cysteine sulfuric acid procedure (75)•

Total hexose was determined by the procedure of Gurin and Hood

(76) and the cysteine-sulfuric acid method (75)•

Reducing sugar was determined by the method of Nelson

(77)* Ketohexoses were determined by the modification of the original procedure of Roe (78) and by the method of Dische and

Borenfreund (79)* Pentoses were quantitated by the orcinol method (80). Deoxypentoses were measured by the diphenylamine procedure (8l) and methyl pentoses according to Dische and

Shettles (82).

(2) Specific methods '

(a) Determination of glucose.— Glucose was specifically quantitated through use of two enzyme systems known to demon strate great specificity for this hsxose; Glucose oxidase was employed as a component of the commercially available Glucostat reagent (83) and a combination of crystalline yeast hexokinase and glucose-S-phosphate dehydrogenase was used in the rapid spectrophotometrie determination of glucose. 3*f

The procedure employing the Qlucostat reagent is that recommended in a circular which accompanies the enzyme preparation from Worthington.

A more sensitive, accurate, and reproducible means of determining glucose involved the coupling of hexokinase and glucose-6-phosphate dehydrogenase. Hexokinase converts glucose to the 6-0-phosphoryl glucose derivative in the presence of adenosine-3'-triphosphate. Glucose-6-phosphate dehydrogenase, in the presence of nicotinamide adenine dinucleotide phosphate, catalyzes the formation of 6-phosphogluconic acid and reduced- nicotinamide dinucleotide-phosphate (NADPH^); the formation of the latter compound can be followed spectrophotometrically as a result of its large extinction coefficient at 3^00&.

An aliquot of the sugar solution to be tested was placed in a quartz cuvette of 1.0 cm light path which contained the following components: tris*hydrochloride buffer pH 7*7, 250 ^4- moles; nicotinamide adenine dinucleotide phosphate (NADP), 3*0 )A- moles; adenosine-5!_-t?iphosphate, 2*0 moles; magnesium chloride, k moles; crystalline Hexokinase (Sigma, Type IV)', 10.2 units; ‘ glucose-6-phosphate dehydrogenase (Sigma, Type V), 0*05 units; the carbohydrate sample and double distilled water to a final volume of 3*0 ml. The aliquot of the sugar solution to be tested was so chosen that the final concentration of the carbohydrate was about 0.0*f Jblmoles per ml in a total volume of 3*0 ml.

Reaction was allowed to proceed for kO minutes at 23°C in a Zeiss model PMQ 11 spectrophotometer. The total optical 35 density in a control cuvette, which contained all the reaction components listed above except carbohydrate, was subtracted from the total optical density change at for the experimental- test cuvette. The total NADPH^ produced under conditions of the assay was determined by employing an extinction coefficient of

6.2 x 10^ for NADPHg (8*0. Control cuvettes containing standard quantities of D-glucose, confirmed that, under the conditions of assay, the procedure employed was quantitative and specific for added glucose.

(b) Determination of galactose.— The procedures employed in the specific determination of galactose included use of the galactose dehydrogenase of Pseudomonas saccharophillia (85) and the chemical procedure described by Fisher and co-workers (86).

The latter procedure was reported to detect 0.1 . ^Amole of galactose in the presence of 1.0 ^mole of glucose. The authors also reported that mannose interferes in the determination of galactose according to this method. Consequently, it was found desirable to resolve mixtures of galactose and mannose by paper % chromatography prior to assay for galactose according to the chemical procedureo

Enzymatic assay for galactose involved use of the D-

galactose dehydrogenase of Pseudomonas saccharophillia. Partially purified galactose dehydrogenase was prepared according to the procedure of Doudoroff (57)* Tlie method used in assay for

galactose was described by Su and Hassid (87). This latter pro

cedure was modified in that the total volume of the reaction mixture was 3.0 ml and changes in optical density were observed in a Zeiss model PMQ II spectrophotometer.

(c) Determination of mannose.— Galactose-free mannose was determined quantitatively by the method of Fisher and co workers (86).

(F) Metabolic Studies Involving Penicillium charlesii

(1) General growth procedures

Cultures of Penicillium charlesii were grown in 500 ml widemouth Erlenmeyer flasks which were fitted with cotton plugs.

When P. charlesii was grown upon a carbon-l4 labeled substrate lif and it was desired to quantitate C 0^ liberated, the Erlenmeyer flask containing inoculated growth medium was placed in a large cylindrical jar which also contained standard volumes of sodium hydroxide in 200 to 500 ml beakers. The cylindrical jar was sealed by a sheet of plate glass over its top; under these con- 14 ditions respired C 0^ was trapped in the sodium hydroxide solution. Except where noted to the contrary, all cultures were maintained at 22° to 24°C.

(2) Growth of Penicillium charlesii in "metabolic train"

Experiments requiring the exact determination of the 14 rates of uptake of tartaric acid-1,4-C from the growth medium 14 and conversion of this to C -carbon dioxide was performed

through use of the apparatus represented in Figure 2. The entire

apparatus was sterilized prior to use and the solutions of sodium Fig. 2.— "Metabolic Train" used in studies of F. charlesii.

(A) 1000 ml flask containing 500 ml cf 10 normal potassium hydroxide through which air was bubbled continuously at a controlled rate. The stream of air entering was both sterile and free of carbon dioxide.

(B) 500 ml bottles containing sterile cotton or glass wool which served to reduce the moisture content of the stream of air entering C.

(D) Bulb through which sterile, CO.-free air was introduced in a stream that was parallel to the surfaces of the culture.

(E) 250 ml Erlenmeyer flask (equipped with special side arms) containing growth medium and culture.

(F) Syringe— afforded removal of standard aliquots of growth medium under aseptic conditions.

(G) Respired "CO^" and inlet-overflow were swept out at this opening.

(H) 500 ml flask— served as a secondary trap.

(I) 500 ml flask containing 200 ml of 2.0 normal sodium hydroxide. A special sidearm allowed removal of disodium carbonate solution and avoided introduction of exogenous CO.,.

(J) Syringe, used in the removal of standard aliquots of di sodium carbonate, aqueous sodium hydroxide solution.

(K) Secondary trap— 1000 ml Erlenmeyer flask containing 500 ml of 1.0 normal sodium hydroxide. This trap was used to determine if C-^Op escaped the trap designated (I), ELask K also insured that no CO- from the atmosphere reached (I) if the pressure at A fell to or below atmospheric pressure.

(L) Syringe— used to remove dissolved sodium carbonate from the trap designated (I).

37 38

O r 39 hydroxide were prepared with double-distilled water which had been brought to boiling and cooled while sealed with aluminum foil* The connections between adjacent components of the metabolic train consisted of tygon tubing which was sterilized before use*

(3) Separation of medium and mycelium after termination of growth experiments

At the end of the growth period the medium and mycelium were separated by filtration through cheesecloth. The mycelium was washed several times with cold distilled water and the washings added to the filtered growth medium. The medium plus mycelial washings was concentrated to a very small volume under reduced pressure and at about 20° to 30°C. The concentrated solution was made 85 per cent with respect to absolute ethanol and the resulting mixture allowed to stand for 12 hours at -4°C.

The oligosaccharides precipitated as a result of these procedures and were collected by centrifugation for 15 minutes at 15•000 x g in a Servall refrigerated centrifuge which was operated at 0°C*

One of two alternative procedures was employed in further operations involving the oligosaccharides*

Alternative procedure (A)*— This procedure was employed when it was desired to qualitatively establish the nature of the oligosaccharides present* The precipitated oligosaccharides, after collection by centrifugation were washed in two successive operations with cold absolute ethanol* Separation of the oligo saccharides after the washings was again afforded by centrifugation. The oligosaccharide mixture was dissolved in a minimum volume of water and the solution stripped upon Whatman number 3 mm chromatography paper. The development of the chromatogram through use of a solvent system containing Methanol:Formic acidrWater

(80:15:5* V/V/V) was followed by drying and development in the same direction in the n-Butanol:Pyridine:Water (6:4:3* V/V/V) solvent system. The material at the origin was eluted with cold distilled water at 4°C. The eluate was concentrated under re

duced pressure to a small volume. An aliquot was made 0.3 Normal with respect to sulfuric acid and the resulting solution sealed

and heated in a test tube at 95°C for 90 minutes. Solid barium

carbonate was used to neutralyze the cooled hydrolyzate. The precipitated barium-sulfate was removed by centrifugation and

the neutralyzate treated with mixed resin [Dowex-l-OH and

Dowex-50-H, 1:1). The solution which resulted after removal of

resin was concentrated to dryness under reduced pressure at 15°

to‘20°C and the residue dissolved in a minimum volume of double

distilled water. A portion of the solution was chromatographed

in three different solvent systems: (a) n-Butanol:Pyridine:Water

(6:4:3), (b) Ethylacetate:Pyridine:Water (36:10:11.5), and (c)

80 per cent aqueous phenol.

Alternative procedure (B).— This procedure was identical

to that described above up to the step which precedes hydrolysis.

The concentrated eluate was treated with mixed resin as above and

the resulting resin-free solution assayed directly for carbohydrate. 1*1

(if) Nature of the oligo saccharides synthesized

Standard chromatographic and chemical techniques were

employed in order to establish that the oligosaccharides syn

thesized by P. charlesii under various growth conditions were

mannocarolose and galactocarolose. Galactose was also identified

and quantified by the galactose dehydrogenase of Pseudomonas

saccharophillia.

Hydrolysis of oligosaccharides*--An aliquot of known

volume and sugar concentration was hydrolyzed at 95°C for 90

minutes in a solution made 0*3 normal with respect to sulfuric

acid. The solution was neutralized with solid barium carbonate

and the resulting precipitate of barium sulfate was removed by

centrifugation. The supernatant solution containing the carbo

hydrate material was treated with five times its volume of mixed

(Dowex-l-OH and Dowex-50-H/l:l) resin and filtered by suction

.filtration. The neutral filtrate was concentrated to dryness

under reduced pressure at 20° to 22°C. The residue was dissolved

in a minimum volume of cold distilled water.

The solution was stripped and dried on an area 0.2 x 8.3

inches on Whatman number 3 MM paper and chromatographed through

one ascension in the Butanol:Pyridine:Water (6:4:3) system. The

chromatogram was dried at 23°C and a second ascension in the

organic phase of the Ethylacetate:Pyridine:Water (36:10:11.3)

solvent system. This double development procedure afforded the

separation of the three aldohexoses, galactose, glucose, and

mannose. Alternately, the quantitative recovery of galactose CONTENTS— (Continued) Page

(F) Functions and Transformations of Tartaric Acid in Biological Systems ...... 18

(1) Mammalian and Avian Metabolism of Tartaric A c i d s ...... 19 (2) Tartaric Acid Metabolism in Bacteria • • • • 20 (3) Metabolism of Tartaric Acid by Moulds .... 21

EXPERIMENTAL...... 26

(A) Maintenance and Growth, of Cu l t u r e s ...... 26

(1) Pseudomonas sp. Shilo ...... 26 (2) Pseudomonas saccharophillia Doudoroff ...» 27 (3) Penicillium charlesii...... 27

(B) Enzymes: Sources, Extraction, Assay ..••••• 28

(1) Tartaric Acid Dehydrase 28 (2) Galactose Dehydrogenase ...... ••••• 28 (3) Extraction and Assay of Enzymes from P. charlesii ...... 29

(D) Chromatographic Procedures .....;••••• 30

(1) Carbohydrates ...... 30 (2) Sugar Phosphates...... 31 (3) Carboxylic A c i d s ...... 32 (*f) Heterocyclic Bases and Acids ...... 32

(E) Quantitation of Carbohydrates in Solution .... 33

(1) General Methods 33 (2) Specific Methods ...... 33

(F) Metabolic Studies Involving Penicillium charlesii 36 •

(1) General Growth Procedures 36 (2) Growth of Penicillium charlesii in "Metabolic Train" 36 (3) Separation of Medium and Mycelium after Termination of Growth Experiments . 39

v 42

from the resin-treated neutralyzate was effected by paper chro matographic double development in the Butanol:Pyridine:Water

(6:4:3) system.

The mannocarolose present in the initial sugar solution

was not hydrolyzed by 0.3 normal sulfuric acid in the procedure

described above. Mannocarolose remained at the starting line in

the chromatographic procedure and was isolated by elution of the

starting-line-area of the chromatogram with cold double distilled

water. The concentrated eluate was hydrolyzed with 3*0 normal

sulfuric acid and the resulting hydrolyzate subjected to the pro

cedures described above for the 0.3 normal hydrolyzate. Concen

tration of the neutralyzed solution (from the 3»° N hydrolysis)

and chromatography revealed the presence of one major carbo

hydrate component which was mannose. Neither galactose nor

glucose was detected in the neutralyzate which results after

3.0 N acid hydrolysis. Galactose was the only hexose which was

detected in the 0.3 normal acid hydrolyzate.

(G) Quantitation of Radioisotopic Compounds

(l) General methods

Three types of instruments and procedures were employed

in the quantitation of compounds labeled with radioactive iso

topes: an end-window counter (Nuclear Chicago Model Dl8l) and a

thin window gas-flow counter (Nuclear Chicago Model l8lA) were

generally used to quantitate samples which were solids or easily

converted to solids; a liquid scintillation counter (Packard

Model 314 Tricarb Liquid Scintillation Counter) was employed in counting liquid and some solid samples; the detection of radio isotopic compounds on paper chromatograms was accomplished through use of an automatic strip scanner (Atomic Accessories

Model BSC-l80 chart and strip housing coupled to a Baird Atomic ratemeter model 432) •

The detectors of the end-window and thin-window gas flow counters were operated at 1120 and 1150 to 1200 volts respectively

The detector of the automatic strip counter was operated at 1125 volts. Chart and strip speed for the latter instrument was 0.75 inches per minute unless indicated otherwise in the text. The end-window and gas flow counters usually functioned at 15 to 19 per cent efficiency and the values reported using these instru ments have been corrected to 100 per cent efficiency unless stated otherwise. The strip counter usually counted samples at about 20 per cent efficiency and none of the values reported through its use have been corrected. \ (2) Preparation, transfer, and counting of samples 4

(a) Solid samples were counted at both infinite thickness and infinite thinness. The samples counted by the latter pro cedure were high in total and specific activities. Self ab sorption and correction curves were obtained for both processes.

Solid samples were plated on thin stainless steel planchets

(which were 7*93 cm in diameter by 0.23 cm in depth) and dried

on a rotating planchet holder with an infrared heat lamp placed

at an average distance of 8 to 10 inches above the sample surface. bk

Under these conditions the temperature at the surface of the planchet was 55° to 60°C. lif Solid samples of sodium carbonate-C were obtained when aliquots of solutions were plated and dried as described above*

Values obtained represent the average of no less than four inde pendent determinations each of which was counted to at least

10,000 counts* Solid sodium carbonate was occasionally counted in suspension in the liquid scintillation counter as described below* The former procedure was not subject to significant error if the dried sample of sodium carbonate (which contained traces of sodium hydroxide) was kept in a closed petri dish prior l^f to counting* It was observed that plated Na^C 0^/Na0H samples underwent changes in specific activity when allowed to stand in an open atmosphere*

Solid barium carbonate was transferred to the stainless steel planchets with the aid of ethanol and the alcohol was removed during the drying process*

Portions of mycelium of P. charlesii were prepared for

counting according to the following procedure; the mycelium was

thoroughly washed and dried according to the method outlined above and milligram quantities transferred to a tared stainless

steel planchet and counted*

(b) Samples in solution were quantitated for radioactivity

by transfer of aliquots to weighed planchets and counting of the

non-volatile residue (after drying according to the procedure

described for solutions of sodium carbonate) in the gas flow 45 counter or through direct counting of the solution in the Tricarb liquid scintillation counter*

The solvent-phosphor system used in liquid scintillation counting was composed of absolute ethanol:toluene (3*2, V/Vl) with

2.0 grams of l,4-bis-2-(5-phenyloxazolyl) benzene and 0*2 grams of 2,5-diphenyloxazole per liter* Liquid samples were dissolved directly in the scintillation solvent-phosphor system and counted in a total volume of 13 ml in 20 ml glass vials* For most systems assayed the efficiency of counting carbon-l4 samples was

37 to 40 per cent of theoretical. Efficiency of counting was determined by dissolving a defined quantity of a radioiostopic standard in 13 ml of the scintillation solvent phosphor system and counting at least 10,000 counts* Carbon-l4 labeled toluene and carbon-l4 labeled sodium carbonate were usually employed as standards* Values recorded through liquid scintillation counting were corrected to 100 per cent efficiency*

The mycelium was pressed firmly between several layers of cheesecloth and filter paper to remove expressible liquids*

This procedure was repeated until there-was no moisture in fresh

absorbent filter paper* The value which resulted when the pressed mycelium was weighed is designated "wet weight of mycelium*11

Thorough drying of the mycelium was accomplished by

placing the pressed pad in an oven at 70°C for 48 hours. At the

end of this 48-hour .period the mycelium was powdered with a mortar

and pestle. The powder was placed in the 70° oven and maintained

at this temperature for 24 hours* There was no decrease in weight 46 of the powdered mycelium when the second drying period was ex tended to 48 hours. The powder was weighed and the weight re corded as "dry weight of mycelium."

For quantitation of carbon-l4 quantities of the dried mycelium were weighed into tared stainless steel planchets and counted in a thin-window gas flow counter. Finely powdered samples were counted at infinite thickness. Alternatively the samples were corrected to 100 per cent of theoretical activity through use of self absorption curves which were obtained by counting powdered mycelium labeled by Acetate-2- C1^.

(5) The conversion of sodium or potassium carbonaterC-*-^ to barium carbonate-C^

The solution of sodium carbonate, in a glass beaker, was made 1 per cent with respect to ammonium chloride and brought to

4°C by immersion in an ice bath. All operations were performed

rapidly and absorption of CO^ from the atmosphere was minimized

by covering the top of the beaker with aluminum foil. Molar

barium chloride was added dropwise (with continuous stirring)

to the sodium carbonate solution. Addition of barium chloride

continued until precipitation was complete. The precipitate was

collected by centrifugation at 10,000 x g in a refrigerated

centrifuge. Occasionally, transfer operations were minimized

by performing the precipitation in 50 ml polyethylene centrifuge

tubes. The precipitate was washed with cold double-distilled

water (recently boiled and essentially CO^-free) until free of Bodium hydroxide. Washing with absolute ethanol, benzene, and absolute ethanol yielded an essentially anhydrous residue of barium carbonate. The thoroughly washed powder was dried at

50°C over a 24-hour period. The dry barium carbonate was stored under desiccation until counted.

Milligram quantities of barium carbonate were transferred sis an ethsuaol slurry to weighed steel planchets and dried with an infrared heat lamp. The planchet wan allowed to come to room temperature while in a petri dish said after cooling the planchet and dried sample were re-weighed. The difference between the weight of the planchet said sample and the planchet alone repre sents the quantity of barium carbonate counted.

(4) Detection of radioactive compounds on paper

Chromatograms containing radioactive compounds were cut into strips 1.5 inches wide and 21 inches in length. Radioactive areas were located by scanning the strips in the automatic strip counter. The strips and charts on which radioactivity was re corded moved at a rate of 0.75 inches per minute.

(H) Organic Preparations

(l) Synthesis of dihydroxymaleic acid

Tartaric acid was converted to dihydroxymaleic acid ac cording to the procedure of Hartree (91)• The product was twice recrystallized from an acetone solution which was mixed with an ice-water mixture. The thoroughly washed precipitate of dihy droxymaleic acid was made anhydrous by storage over phosphorus ^8 pentoxide In an evacuated desiccator* The dihydroxymaleate became beige-white approximately 2k hours after initiation of the desiccation* The partial characterization of the anhydrous product was accomplished through examination of its ultraviolet spectrum and its solubility in water, alcohol, and acetone, and its co-chromatography with authentic dihydroxymaleic acid* The ultraviolet absorption of the desiccated material suggested that

it was 90 to 92 per cent pure*

(2) Adenosine-5’-triphosphate-P^ labeled ■52 The synthesis of ATP labeled with P^ in the |9 and

Y-phosphate positions was accomplished through the procedure

described by Hems and Bartley (88).

(5) N-methylation of heterocyclic compounds

Pyridine and a number of its derivatives were converted

to the corresponding N-methyl compounds through reaction with

methyliodide in benzene*

The 2-picoline used was a commercial product of Matheson,

Coleman, and Bell, k-picoline and 3-picoline were obtained from

the Eastman Kodak Organic Division and pyridine was an anhydrous

product obtained through the Ohio State University laboratory

supply system* All the bases were twice redistilled before use*

The solvent employed in the methylation procedures was thiophene-

free benzene* The reaction vessel was a 500 ml widemouth Erlen

meyer flask fitted with a magnetic stirrer* The methylating

agent was methyliodide which was delivered through a graduated

burette fitted with a condenser jacket through which was if9 circulated water at approximately 0°C. The delivery rate for methyliodide was a 2*0 ml per minute* The general procedure for purification of the methiodides involved separation of the crystal line material by suction filtration, washing the precipitate while in the filter funnel with cold absolute methanol and two re crystallizations from 70 per cent aqueous methanol*

Description of the course of reaction*—

^f-Picoline

The reaction mixture became white to light green during

the latter part of the addition* The solution appeared to contain

finely dispersed crystals and upon standing at room temperature

for about 15 minutes a voluminous precipitate was formed* The

precipitate was collected by suction filtration and the filtrate

allowed to stand overnight at 2°C* No additional crystalline

material other than benzene was deposited*

3-Picoline

The reaction mixture became yellow upon addition of

methyl iodide* There was no precipitation at 1 hour after com

pletion of the addition but the reaction mixture had separated

into two liquid layers. The lower layer was a dark yellow and

highly viscous liquid while the second layer was light yellow and

quite mobile* The two components were separated by decantation

and during the transfer the more viscous component began to . 1 crystallize* The yellow precipitate was allowed to stand in con

tact with the mother liquor overnight* No attempt was made to

recover additional 4-Picoline methiodide from the less viscous

liquid layer of the original reaction mixture* 2-Picoline

The reaction mixture became light green as addition of methyiiodide was completed. Upon standing for about a half hour at room temperature noticeable precipitation had taken place.

Precipitation was complete after 45 minutes. The white crystals were allowed to remain in contact with the mother liquor over night.

Pyridine

Reaction of Pyridine with methyiiodide resulted in the concomitant production of a flocculent white precipitate. Upon standing at room temperature the reaction mixture, which appeared yellow, yielded a mixture of yellow and white crystals.

(4) Preparation of lead tetraacetate

The procedure employed was a combination of the methods

described by McClenahan and Hockett (92) and by Oesper and Deasy

(93). The reaction vessel was a 2-liter, J-neck round bottom*

flask which was provided with a thermometer and an electrically-

driven teflon stirrer. All operations were performed rapidly in

a well-ventillated hood and in the presence of subdued light.

Acetic anhydride was redistilled before use with the aid of an

air condenser.

Eleven hundred grams of glacial acetic acid and 250 ml

of acetic anhydride were mixed and brought to 75°C through heating

of the reaction vessel with a thermostatically-controlled heating-

mantle. A glass tube through one sidearm provided for the 51 introduction of a continuous stream of dry chlorine gas (gener ated by the action of concentrated HC1 on manganese dioxide).

Red-lead (350 grams) was added in small quantities to the warmed mixture of acetic anhydride and acid. The solution was allowed to become colorless between additions of red lead. Precautions were taken to maintain the temperature of the system below 90°C.

Upon completion of the reaction the mixture was turbid and contained significant quantity of unreacted undissolved red lead. The solid and soluble components of the mixture were separated by decantation. The solid residue was returned to the reaction vessel to which fresh acetic anhydride and acetic acid had been added and the addition of red lead repeated. The filtrates which resulted were cooled to room temperature and then kept at 4-°C in the dark for 3 hours. Lead tetraacetate precipi tated as a mixture of plates and short grey-white needles. The precipitate was collected by rapid suction filtration and washed with small volumes of cold glacial acetic acid. The washed lead tetraacetate was stored in vacuo over Pg05 u^il used.

(5) Synthesis of l-methyl-3- carboxy-2-pyridone

A procedure for the chemical conversion of 1-methyl- nicptinic acid (trigonolline) to the corresponding 6-pyridone has been described by Huff (89). The procedure leads to the un equivocal and exclusive production of the 6-pyridone; none of the

2-pyridone is formed. A method for the synthesis and isolation CONTENTS--(Continued) Page

(G) Quantitation of Radioisotopic Compounds ..... 42

(1) General Methods ...... 42 (2).Preparation, Transfer, and Counting of Samples . 43 (3) The Conversion of Sodium br Potassium Carbonate-C^*- to Barium Carbonate-C^ ...» 46 (4) Detection of Radioactive Compounds on Paper . 4?

(H) Organic Preparations...... 47

(1) Synthesis of DihydroxymaleiCpAcid ...... 47 (2) Adenosine-5'-Triphosphate-P -Labeled .... 48 (3) N-Methylation of Heterocyclic Compounds . . . 48 (4) Preparation of Lead Tetraacetate ...... 50 (5) Synthesis on l-Methyl-3-Carboxy-2-Pyridone • 51

RESULTS ...... 55

(A) Growth of P. charlesii and Production of Oligo saccharides in the Presence of Various Di- carboxylic Acids ...... 55

(1) The Effect on Metabolism of Various Di- carboxylic Acids Substituted for Tartaric Acid in the Raulin-Thom Growth Medium .... 55 (2) The Effect on Metabolism of Varying Ammonium Salt Concentration of Several Dicarboxylic Acids...... 66 (3) The Effect on Metabolism of Varying Concen trations of Nitrogen of the Growth Medium .. • 93 (4) The Effect on Metabolism of the Enantiomorphs of Tartaric Acid ...... 117

(B) The Metabolism in vivo of Phosphorylated Compounds by P. charlesii...... 122

Studies on the Timer-Course1 of Utilization of Tartaric Acid-1,4-C by i. charlesii...... 16?

(E) Incorporation of Various Carbon-l4 Labeled Carboxylic Acids by Penicillium charlesii .... 208 14 (F) The Incorporation of Tartaric Acid-1,4-C by Penicillium charlesii Grown in the Presence of Tartronate ...... 211

vi 52 of the 2-pyridone which corresponds to trigonolline has also been reported (90)•

The former procedure involves the introduction of oxygen into the aromatic heterocyclic ring through the agency of ferricyanide. The methodology employed in the present case represents a modification of the procedure reported by Huff (89)*

Trigonolline hydrochloride ClO grams, O.O58I moles] was dissolved in 120 ml of 2*9 normal sodium hydroxide. To the resulting solution, maintained at 24° C was added 100 ml of 35 per cent potassium ferricyanide at a rate of 0*3 ml per minute. The reaction vessel was fitted with a magnetic stirrer and the con tents of the vessel were stirred for 90 minutes after completion of the addition of ferricyanide. The reaction system was initi ally pale green in color but became red-brown as the reaction proceeded.

The pH of the solution was adjusted to 3*5 through the cautious addition of normal hydrochloric acid. The course of the pH adjustment was followed with a Beckman zeromatic pH meter.

The acidified solution was cooled to 0°C and the crystalline mass which formed was filtered by suction and washed several times with cold absolute ethanol, the washings being added to the main filtrate. The precipitate was designated PQ .

The combined filtrate and washings waB concentrated under reduced pressure to approximately 120 ml and the concentrate refrigerated at 2°C for ^5 minutes. The precipitate which formed was added to PQ. The combined precipitates were designated P^. 53

Precipitate Pg wa6 dissolved in 150 ml of warm water and the solution treated while hot with 600 mg of charcoal (Darco-G-60)•

Filtration of the hot mixture yielded a dark green solution from which a precipitate formed upon cooling overnight. The dark green mass contained traces of a blue-green component which might have been an oxidation product of the desired pyridone.

The precipitate was washed with cold 85 per cent ethanol and the

residue treated with 3 liters of absolute ethanol in 200 ml

volumes each. It was observed that the green precipitate was not

soluble in absolute ethanol or benzene. The initial reactant,

Trigonolline, was found to be slightly soluble in absolute ethanol } and this serial "extraction" served to separate the reactant and

the product. The alcohol extraction was accomplished by main

taining the mixture at the boiling point of ethanol for 10 minutes.

Evaporation of the alcohol yielded a mixture of light grey plates

and needles. Re.crystallization of this latter mixture yielded

needles which were identical in ultraviolet absorption spectrum

and melting point to authentic trigonolline.

The green precipitate described above, which was not

soluble under the conditions of extraction with absolute ethanol,

was recrystallized from 50 per cent aqueous ethanol. The material

which resulted upon washing the crystals with absolute ethanol had

the appearance of light green-yellow plates. The melting point of

these plates was 196°C. The melting point obtained for the thrice

recrystallized light-green plates was 203°C. When the solubilized (once crystallized) green-yellow plates were treated with Norite and the resulting aqueous

solution cooled to -5°C white needles were obtained* RESULTS

(A) Growth of P. charlesii and Production of Oligosaccharides in the Presence of Various Dicarboxylic Acids

Cl) The effect on metabolism of various dicarboxylic acids substituted for tar taric acid in the Baulin-Thom growth medium

■ In view of the sparcity of recorded information which relates to actual function(s) of tartaric acid in the Raulin-

Thom growth medium it was of interest to seek information which might argue for or against a unique role for this acid in the production of oligosaccharides by P. charlesii. Although it is i | 1^ known that carbon-14 from tartaric acid -1,4-C is incorporated into galactocarolose (5) it is not known that production of this oligosaccharide by P. charlesii growing on the Baulin-Thom medium does or does not specifically require tartaric acid.

An absolute requirement for tartaric acid in production of the oligosaccharides of P. charlesii was tested qualitatively by substituting various dicarboxylic acids for tartaric acid in the Baulin-Thom growth medium.

The Baulin-Thom growth medium was prepared as described under 'EXPERIMENTAL' except for the substitution of equimolar quantities of the indicated acids for DL-tartaric acid: dihydroxymaleic, maleic, fumaric, succinic, malic, or malonic acid. Ad-

55 56

ditionally, the growth media contained 0.667 mg/ml of diammonium

carbonate.

Because it was destroyed by high temperatures dihydroxymaleic acid was added aseptically to the Baulin-Thom salts

solution which had been autoclaved for 15 minutes at 15 psi of

pressure and a temperature of 120°C and cooled to room temperature.

Aliquots of 1.0 ml were removed at 24—hour intervals from

one of duplicate flasks and these aliquots tested for hydrogen

ion concentration (pH) and total-sugar concentration of each

system. Aliquots removed at 72-hour intervals were chromato

graphed in several solvent systems as a means of following the

course of change in concentration of the carboxylic acids and

carbohydrate of the medium.

iHt At the end of the growth period of 26 days the residual

growth medium and mycelium were separated by decantation and

filtration. The operations which followed this separation are

described under 'EXPERIMENTAL.'

The concentrated growth medium was fractionated and

hydrolyzed according to alternative procedure (A) under 'EX

PERIMENTAL.' When co-chromatographed with various standards in

three different solvent systems only galactose was detected in

the monosaccharide area of the chromatogram of the neutralyzed

medium hydrolyzate.

The material which remained at the starting line, when

the neutralyzed medium hydrolyzate was chromatographed in the

n-Butanol:Pyridine:Water (6:4-:3) solvent system, was eluted, concentrated and the concentrate hydrolyzed with 3*0 normal sulfuric acid. The time of hydrolysis was 90 minutes and the temperature was 95°C« The cooled hydrolyzate was neutralyzed with barium carbonate and the neutralyzate treated with mixed resin as described under 'EXPERIMENTAL,' The resin-treated neutralyzate was chromatographed in the n-ButanolsPyridine:Water

(6:4-:3) solvent system, Mannose was the only monosaccharide observed. In the latter case, faint carbohydrate positive sub stances were present in areas which migrated less rapidly than disaccharide standards.

Observations on the metabolic patterns developed,— With

the exception of flasks containing P, charlesii grown in the . presence of tartaric acid growth of the organism was quite slow in all of the cases tested. The removal of glucose from the

growth medium was quite rapid in the tartaric acid system as

shown in Figure 3 but noticeably slower under other conditions.

In the maleic, malic, nuLLonic, and succinic acid systems the rate

of glucose uptake from the medium and changes in pH were very

similar to the patterns representing the.fumarate series.

Changes in total sugar and pH of the medium for the fumarate

series are shown in Figure 4, Metabolism of glucose in the

presence of dihydroxymaleic acid was quite unusual. After 23

days of growth in this latter system less than 50 per cent of

the sugar initially present had disappeared. The mycelium did

not form a visible green matting of spores in the dihydroxymaleate

system and the initial appearance of the medium was unaltered Fig. 3.--Variation in hydrogen.,ion and carbohydrate concentration with time of growth of P. charlesii on the Raulin-Thom medium. ~

The curve representing pH changes is that which remains positive in slope after 4- days of growth

The second curve reflects changes in carbohydrate concentration of the growth medium

The abscissa reflects days of growth of the culture of P. charlesii

58 CD O 01 0 1 pH TOTAL SUGAR AS KLETT UNITS ro

DAYS 46 3 vm 01 Fig* If.—-Variation with time of growth of P. charlesii of the hydrogen ion and carbohydrate concentration of the fumarate-modified Raulin-Thom medium*

The curve defined by closed triangles reflects changes in carbohydrate concentration of the growth medium

The curve defined by closed circles reflects changes in the hydrogen ion concentration of the growth medium

The abscissa reflects days of growth of the culture of P* charlesii

60 90

5 0 X CL

3 0

■ * ...... 2 4 6 8 10 12 14 16 18 20 22 24

DAYS

Fig. k CONTENTS— (Continued) Page

(G) Incorporation of Glucose-u-C by Penicillium charlesii in the Presence of Tartronic Acid . . . 229 l^t (H) The Incorporation of Glucose-u-C■ by Penicillium charlesii Grown in the Presence of Tartaric Acid 236 l^f (I) The Incorporation of Glucose-u-C by Penicillium charlesii Grown in the Presence of Dihydroxymaleic Acid ...... 249

(J) Studies on the Dihydroxymaleic Acid Dependent Oxidation of NADH_ Catalyzed by Extracts of P. charlesii ...... 292

DISCUSSION...... ■...... 320

SUMMARY ...... 357

LITERATURE CITED ...... 359

AUTOBIOGRAPHY ...... 368

vii Fig. 5«— Variation with, time of growth of the hydrogen ion and carbohydrate concentration of the dihydroxymaleate- modified Baulin-Thom medium.

The curve defined by closed triangles represents changes in the carbohydrate concentration of the growth medium

The curve defined by closed circles reflects changes in the hydrogen ion concentration of the growth medium

The abscissa records days of growth of the culture of P. charlesii

62 TOTAL SUGAR AS KLETT UNITS 0 0 9 0 0 5 300 700 100 6 8 10 Fig.5 AS a, DAYS 12 14 16 18 20 22 24 0 3 0 7 5-0 0 9 after 26 days of growth of P. charlesii* The plots of changes in medium sugar concentration and pH are recorded in Figure 5•

The appearance of carbohydrate material other than

glucose, in the medium (as revealed by paper chromatography) after about 7 days was observed in all the cases studied. The

formation of mycelium was depressed in the cases of malonic, malic, maleic, dihydroxymaleic, fumaric, or succinic acid sub

stitutions for tartaric acid. Highest yields of oligosaccharides

were obtained in the presence of malonic and tartaric acids, as

shown in Table 1. The ratio of oligosaccharide to mycelium pro

duced was highest when dihydroxymaleic acid substituted for tar

taric acid.

These results illustrate that the replacement of tartaric

acid by a variety of dicarboxylic acids in the Baulin-Thom

medium does not result in complete cessation of oligosaccharide

formation by Penicillium charlesii.

Whereas growth was slow in the presence of all dicar

boxylic acids tested, other than tartaric acid, the behavior of

P. charlesii in the presence of dihydroxymaleic acid was most

unusual. Although there was little change in the concentration

of carbohydrate of the medium and very little mycelial material

was present after 26 days of growth, the ratio of oligosaccharide

to mycelium was higher when P. charlesii was grown in the presence

of dihydroxymaleate than when the mould was grown in the presence

of tartrate. 65

TABLE 1 PRODUCTION OF MYCELIUM AND OLIGOSACCHARIDES BY P. CHARLESII GROWN 26 DAYS IN THE PRESENCE OF VARIOUS DICARBOXYLIC ACIDS

Units of Oligosac Total Dicarboxylic Mycelium charide Per Unit Galactose Acid in Formed of Mycelium pH of Medium Isolated Me ditun GramB Initial Final Moles Moles/Gram

Tartaric 5.7 *U5 6.3 186.5 3.25 Malonic 2.6 3.6 ^.•3 ^9.5 1.90 Fumaric 2.3 ?A ^ A 31.5 1.37 Succinic 2.1 *f.O 3.7 2lA 1.02 Maleic 1.8 3.2 3.6 9.0 0.50 Malic 1.6 3.3 3.7 11.5 0.78 Dihydroxy 1.1 3.0 2.3 36.7 3.33 maleic

The limited mycelial synthesis observed in these experi ments may reflect the low level of nitrogen available for the synthesis of important metabolites. Under conditions of such limited formation of mycelium the absolute amounts of glucose and dicarboxylic acid available for oligosaccharide formation would be high.

That such low amounts of oligosaccharide were formed in the present experiments may be a reflection of a negative in fluence on oligosaccharide production of the low pH which pre vailed in all cases studied. It is also possible that the di carboxylic acids employed actually inhibited oligosaccharide formation. 6*

These three possible explanations of the present obser vations were subjected to the experimental tests described in

succeeding sections*

(2) The effect on metabolism of varying ammonium salt concentrations of several dicarboxylic acids

These experiments were designed to extend 'and clarify

the results described in the previous section* It was observed

that substitution of various dicarboxylic acids for tartaric acid

in the Raulin-Thom medium, while omitting the corresponding am

monium salts, exerted an appreciable effect on mycelium and oligo

saccharide production* A possible effect of pH was discussed and

it was inferred that the ammonium ion concentration may have been

a determining factor in influencing the pH of the medium and

limiting the nitrogen available for protein, nucleic acid, and

lipid synthesis from other metabolites during growth of Penicil

lium charlesii*

In the previous section the studies described were results

of experiments in .which the acids tested (for capacity to sub

stitute for tartaric acid) were added in the free-acid form and

the sole sources of nitrogen were the ammonium salts of the in

organic acids specified. Thus, the possibility exists that growth

was limited by the amount of nitrogen available. This would ex

plain the limited utilization of glucose*

If on the other hand, glucose metabolism in the presence

of the substituted acids were regulated and diminished because

the mould could not metabolize these "new" acids, then growth, as 67 measured by mycelium weight increase and glucose disappearance, might not be altered by increasing or decreasing the absolute quantity of ammonium salt of the dicarboxylic acids under test*

The possibility is not to be overlooked that the "second

ary11 organic carbon source (dicarboxylic acid of the growth medium) is important in the metabolism of P. charlesii* Should mycelium and oligosaccharide production be related by direct or

indirect proportion and total' nitrogen of the medium be related

to mycelium formation, experiments performed in which the total

ammonium ion concentration is varied might yield useful infor

mation.

For these studies the concentration of the free dicar

boxylic acid was kept constant and the concentration of the cor

responding ammonium salt was varied. With the exception of di

carboxylic acid (and/or salt) concentrations the Raulin-Thom

medium was constructed in the usual fashion. That some oligosac

charide synthesis occurs in the presence of malonic, fumaric,

and dihydroxymaleic acids was shown in the preceding experimental-

results section. These three dicarboxylic acids and tartaric

acid were tested in the present case.

A series of duplicate flasks wa6 prepared to contain the

Raulin-Thom medium with the acids and acid salts present, in the

amount stipulated in Table 2.

It was observed that autoclaving a solution of the Raulin-

Thom salts in the presence of "free11 dihydroxymaleic acid resulted

in the formation of a dark-red suspensoid which did not revert

4 6-3

TABLE 2 CONCENTRATION OF CARBOXYLIC ACID AND AMMONIUM SALTS PRESENT IN THE RAULIN-THOM GROWTH MEDIUM

Molar Concentration Dicarboxylic Acid Species

Flask Diammonium Salt of "Free" Series Designation Dicarboxylic Acid Dicarboxylic Acid

Tartrate RTj 0.04368 0.01772 r t 2 0.01456 0.01772 . RT^ 0.00485 0.01772 RTq 0 .01^56

Dihydroxy DHM_ 0.04368 maleic acid pgfj 0.01456 DHM1 0.00485

Fumarate FUM^ 0.04368 0.01772 f u m 2 0.01456 0.01772 FUM^ 0.00485 0.01772

Malonate m a l 3 0.04368 0.01772 m a l 2 0.01456 0,01772 MALX 0.00485 0.01772

upon cooling the mixture to 24°C. This phenomenon was suggested

due to the complexing of the dienol grouping of dihydroxymaleic

acid with ferrous ion of the medium, a process observed to be

facilitated by heating or low ammonium ion concentration. The

diammonium salt of dihydroxymaleic acid, when isolated, was ap

parently stable to this interaction and it was found feasible to

use this salt exclusively with the omission of the free-acid in

these studies. For this reason glucose metabolism, pH changes* 60 « total-sugar changes and. oligosaccharide formation in the presence of diammonium-dihydroxymaleate were compared to the HT^ flasks in which only the diammonium salt of tartaric acid was present*

At 24-hour intervals 1*0 ml was withdrawn from each flask for pH and total-sugar determinations. Ten microliters of each aliquot was assayed for total sugar according to the method of

Dubois and co-workers (75)•

Chromatograms were prepared by spotting 20 microliters of the medium on Whatman number 3 KM chromatography paper. This process was conducted at 3-da.y intervals. Ascending chromatography in the n-ButanolxPyridinesWater (6:4:3) solvent system was employed.

At the end of the 26-day growth period the media and mycelia from duplicate flasks were combined. The combined medium was concentrated to a small volume which was stripped upon Whatman number 3 MM chromatography paper. The dried paper was developed through ascending chromatography in the Methanol:Formic acid:Water

(80:13:5) solvent system. After drying, the chromatogram was developed in the same direction in the n-Butanol:Pyridine:Water

(6:4:3) system. The oligosaccharides remained at the origin throughout these two operations and were eluted, after the second development of the chromatogram, with cold distilled water at 4°C.

Qualitative analyses, performed to show that mannocarolose and galactocarolose were the only oligosaccharides synthesized by u P. charlesii under the experimental conditions, were carried out as described under 'EXPERIMENTAL.1 The experimental results sug

gested that homogeneous galactocarolose and homogeneous mannocaro- 70 lose were the only oligosaccharides present in the 26-day old growth medium of P. charlesii. Three solvent systems were used to establish that the monosaccharides released through hydrolysis, as described under 'EXPERIMENTAL,' were chromatographically identical to galactose and mannose respectively. When portions of the monosaccharide areas of the chromatograms were eluted and

concentrated the resulting solutions gave a positive response in

the Fisher assay (86) for galactose and mannose areas respectively.

The chromogen which resulted in the latter assay was examined for its capacity to absorb light at various wavelengths. A plot of

optical density against wavelength yielded an absorption spectrum

which was symmetrical with respect to galactose and mannose

standards respectively.

Observations on the metabolic patterns— Tartrate series.—

Significant differences in the morphology of the mycelium and

appearance of the medium were noted in this series. In the case

of the RTq flasks the initial inflection in pH was much greater

than at other levels of grow-medium-nitrogen. A green covering

layer of spores formed on the top side of the mycelium after 5

days. After 12 to 14 days of incubation the medium began to

assume a dark brown-red color.

Spore formation in RT^ occurred after 6 to 8 days of

growth of the mould. The fully developed mycelium was not compact

and the growth medium had not assumed the characteristic dark

brown color after 24 days. The mycelium of RT^ was compact and the typical mat of spores began to form during the 3rd to 4th days; thereafter, sporulation was rapid. The medium became dark-brown after 14- days of incubation.

In the presence of high concentrations of ammonium tar trate (4-3«7 moles/ml) spore production was not observed although the medium began to darken after 6 days. The pad was quite compact and rigid in appearance after 24- days of growth.

The curves which relate total sugar remaining in the medium and pH as a function of time for RT^, shown in Figure 6 revealed that carbohydrate was rapidly removed from the medium.

The total carbohydrate in the medium was reduced to 35 per cent of the initial value after 7 days of growth of P. charlesii. A noticeable lag period of about 11 days followed and during this period the change in the carbohydrates concentration of the medium was only slight. The pH curve revealed that only a very small change occurred in the hydrogen ion concentration of the medium over the first 21 days of growth. Both these unexplained effects may result from the low quantity of ammonium ion avail able for utilization in intracellular metabolism of glucose and other metabolites.

Figure 6 also summarizes pH and carbohydrate changes in the media which contained approximately 44 and 13 )Xmoles per ml

(RTj and RT^ respectively) of.diammonium tartrate. The 'curves for RT^ and RT^ are nearly superimposable although they differ in absolute magnitude at most points along the abscissa. The very ILLUSTRATIONS Figure Page

1 Biochemical processes involving tartrate . • . • 2k

2 "Metabolic train" used in studies of P. charlesii ...... 7 ...... 37

3 Variation with time of growth of P. charlesii of the hydrogen ion and carbohydrate concentration of the Raulin-Thom medium ...... 58

k Variation with time of growth of P. charlesii of the hydrogen ion and carbohydrate concentration of the fumarate-modified Raulin-Thom medium • . 60

5 Variation with time of growth of the hydrogen ion and carbohydrate concentration of the dihydroxymaleate-modified Raulin-Thom medium . . 62

6 Variation with time of growth of the hydrogen ion and carbohydrate concentration of systems containing various concentrations of diammonium tartrate ...... ?2

7 Variation with time of growth of the hydrogen ion and carbohydrate concentration of systems containing various concentrations of diammonium-malonate ...... 75

8 Variation with time of hydrogen ion concentration of systems containing various concentrations of diammonium malonate ...... 78

9 Variation with time of hydrogen ion concentration of systems containing various concentrations of diammonium fumarate...... 82

10 Variation with time of carbohydrate concentration of systems containing various concentrations of diammonium fumarate ...... 8*f

11 Variation with time of hydrogen ion concentration of systems containing various concentrations of diammonium dihydroxymaleate ...... 86

viii Fig, 6,~Variation with time of hydrogen ion and carbo hydrate concentration of systems containing various concentra tions of diammonium tartrate.

The curves defined by X represent changes in hydrogen ion and carbohydrate concentration of the RT^ system

The curves defined by the closed triangles represent changes in hydrogen ion and carbohydrate concentration of the RT2 system

The curves defined by closed circles represent the changes in hydrogen ion and carbohydrate concentration of the RT^ system

For each of the three systems defined above, the curve representing changes in hydrogen ion concentration is that which demonstrates a positive slope after 10 days' growth of P, charlesii

72 TOTAL SUGAR AS KLETT UNITS Q1

H- at O ro o\ §

OJ o

£ 1 ?zf rapid rise in pH of RT, after 11 days was quite unusual. It is noteworthy that the mycelium isolated from this system was quite compact and rigid despite the final pH of the medium. There was no evidence of lysis of the mycelium at 26 days.

Malonate series.— When grown on medium MAL^ P. charlesii assumed a crusty, well defined form which was covered by a light- green mat of spores. After days the growth medium had assumed

a dark color but definitely lacked the dark-brown hue which is

characteristic of the growth media under normal conditions.

The MALg medium began to assume a rust-red type appear

ance after about 10 days. The mycelium was compact and covered by a dark green matting of spores. After about 14 to 15 days a

portion of the upper mycelium had begun to submerge. Significant

submersion continued through the 26th day.

The medium from MAI, began to assume a red-brown color

after about 8 days. No perceptible green spore covering was

present even at 26 days. At the end of the growth period the

mycelium was compact and gray-white in appearance; the medium

was dark red-brown.

Figure 7 shows that definite lags in carbohydrate uptake

occurred over the first days of growth in all three members of

this series. In the case of the highest concentration of diam

monium malonate (MAL,) the carbohydrate concentration of the

medium was reduced by about 50 per cent during the 5th through

8th day interval. At 11 days the carbohydrate concentration in Fig* 7*— Variation with time of growth of the carbo hydrate concentration of systems containing various concentra tions of diammonium malonate*

The curve defined by X represents the MAL^ system

The curve defined by closed triangles represents the MAI<2 system

The curve defined by closed circles represents the MAL^ system

75 01 pH TOTAL SUGAR AS KLETT UNITS o

DAYS U 77 this system was less than 5 per cent of the Initial value* The pH of this system gradually decreased to a minimum of about k ,3 at 9 days and increased, slowly at first, then rapidly to a final value of about 8*1. No visible autolysis was observed at this high pH range*

The pH and carbohydrate curves for MAL. and MAL, are also represented in Figures 7 and 8* The rapid decrease in the carbohydrate concentration of the growth medium was followed by a lag period over which the total level of carbohydrate in the

growth medium did not change significantly* This lag was more pronounced in the system containing the lowest diammonium malo nate concentration (MAI^) than the growth medium of intermediate

concentration (MAI^)* The pH changes of these systems, shown in

Figure 8, were quite gradual*

Fumarate series*— The medium of FUtt^ retained a deep

yellow-green color throughout the 26-day growth period* Spore

formation begem at 5 to 6 days after inoculation and at 2k days

the top of the compact mycelium was almost completely green

while the reverse side (exposed directly to the medium) was a

pale yellow-orange color*

The FOM2 medium was red-brown in appearance after about

10 days of growth of P. charlesii. The change of the. medium to

a dark red-brown color was gradual after 12 days, A visible green

matting was present at about 6 days of growth and after 2k days

the compact and rigid mycelium was green on its top surface with

a pink-orange reverse* Fig. 8.— Variation with time of hydrogen ion concen tration of systems containing various concentrations of diam monium malonate.

The curves are defined according to the notations of Figure 7

78 7 0

20 In the case of the FUM^ system the medium began to assume the characteristic brown color on or about the 5th day of incu bation; the growth medium darkened gradually but more rapidly

than in either FUM^ or FUM^. No visible green matting was present and after 2b days the mycelium which covered the entire surface of the flask was compact and gray-white in color.

The pH of the FUM^ system remained essentially constant

throughout the first 22 days of incubation of P. charlesii. As

in the previous instances of growth of P. charlesii on low levels

of ammonium ion (RT^ and MAl^) the carbohydrate content of the

FUM^ medium dropped sharply to 50 per cent of the initial value

and after 7 dayd a definite lag in carbohydrate disappearance

occurred.

When P. charlesii was grown in the presence of the inter

mediate concentration of diammonium fumarate (FUM2) there was ah

initial lag in carbohydrate utilization and this lag was followed,

after 7 days of growth, by a rapid decline in the amount of sugar

residual in the medium. The pH of this system changed but little

during the first 15 days. A very sharp inflection in pH occurred

on the 16th day and this corresponded to the point at which the

relative carbohydrate concentration of the medium was less than

5 per cent of the original.

In the presence of the highest tested concentration of

diammonium fumarate (FTJM^) the initial lag in carbohydrate uptake

was brief though significant. Although the carbohydrate level

dropped after 4 days, there was little change in pH during the 81

first 13 days of growth* The pH of the medium began to rise at a point (12 days) when the carbohydrate had been almost completely

removed from the growth medium*

Figures 9 and 10 represent changes in the hydrogen ion

and carbohydrate concentrations, respectively for the fumarate

systems*

Dihydroxymaleate series*— Irregular alterations in pH

were noted in the dihydroxymaleate series* The.most unusual

pattern observed was that which represents the DHM^ system as

shown in Figure 11. In this case the pH gradually decreased

followed by an increase at 7 to 8 days and experienced a second ,

decrease which was followed by a slow increase from a value of

2*5 on the 13th day to 6*7 on the 2*fth day*

On the other hand, the pH of the DHM^ system gradually

decreased and remained relatively constant throughout the 6th to

i b t h day.interval; thereafter, the pH of this system increased

again at 19 days, decreased at 21 days and increased to a value

of 3*1 at 2b days* Changes in carbohydrate concentration in the

medium of this system also occurred in an unusual fashion* The

carbohydrate concentration was reduced 30 per cent between the

*fth and 7th days of growth; an additional 11 days were required

to reduce the level of carbohydrate at 17 days to 50 per cent of

the value which obtained for the first 7 days of growth* This

lag and reduced carbohydrate uptake ere represented in Figure 12*

Alternatively, it. might be argued that during this lag period the