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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 TOTAL SUGAR AS KLETT UNITS o> oo o DAYS ro o oo ro o ro ro pH $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 o> 00 o Fig. 12 Fig. *<co ro <n oo io ro 68 rate of carbohydrate release from the mycelium into the growth y.- . medium was but slightly less than the rate of carbohydrate up- take. 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.
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