/
THE DEGRADATION OP PHENYLALANINE, TYROSINE, AND
RELATED AROMATIC COMPOUNDS BY A MARINE
DIATOM AND A HAPTOPHYCEAN ALGA
by
ARTHUR FREDERICK LANDYMORE
B.Sc., University of British Columbia, 1968
M.Sc., University of British Columbia, 1972
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in the Department
of
Botany
We accept this thesis as conforming to the
required standard
THE UNTOHSITY OF BRITISH COLUMBIA
March, 1976" In presenting this thesis in partial fulfilment of the requirements
for an advanced degree at the University of British Columbia, I agree
that the Library shall make it freely available for reference and
Study. I further agree that permission for extensive copying of this
thesis for scholarly purposes may be granted by the Head of my
Department or by his representatives. It is understood that copying
or publication of this thesis for financial gain shall not be allowed
without my written permission. iii. ABSTRACT,,
The degradation of phenylalanine and tyrosine was ex•
amined in axenic cultures of Isochrysls galbana Parke and
Navlcula lncerta Hustedt. Both species were able to metabolize
L-phenylalanine and L-tyrosine as the sole nitrogen source, but severe growth Inhibition was observed for _I. galbana. No growth of I_. galbana was obtained on the D-isomers of these two amino acids, but N. lncerta was able to utilize both D- amino acids after an extended lag period.
Analysis of the growth medium and the algal cells from non-radioactive and radioactive experiments never revealed cinnamic or p-coumaric acids. This suggested that phenyl• alanine and tyrosine ammonia-lyases (PAL and TAL) were not involved in the initial degradative step of either these amino acids. This was confirmed as no enzymatic activity for PAL was detected in crude enzyme preparations.
Enzymatic activity for amino acid transaminase was ob• tained for both algal species. This suggested that phenylpyruvlc acid and p-hydroxyphenylpyruvic acid were the Initial respec• tive products from the metabolism of phenylalanine and tyrosine.
From the non-radioactive and radioactive experiments, a scheme for the degradation of L-phenylalanine and L-tyrosine was proposed for both algal species. The compounds in brackets were not identified but were expected. The pathways were:
L-phenylalanine —phenylpyruvlc acid >-[phenylacetaldehyde]
—y-phenylacetlc acid—^-mandelic acid—^-benzoylformlc acid—^>
[benzaldehyde]benzoic acid—*-p-hydroxybenzoic acid; and • ' _ • ( "
L-tyroslne —*-p-hydroxyphenylpyruvlc acid—^-J^p-hydroxyphenyl
acetaldehyde] —^-p-hydroxyphenylacetic acid—*-p-hydroxy-
mandelic acid —>-Jjj-hydroxybenzoylformic acid] —p-hydroxy-
benzaldehyde —»-p-hydroxybenzoic acid and p-hydroxybenzyl- alcohol. Benzoic acid was also hydroxylated in the ortho and
meta positions by both algal species. In both these schemes, the two C^-fragments removed from the side chain were iden• tified as CO^* Also, the carbon-3 of the side chain of both phenylalanine and tyrosine was removed and trapped as COg.
The relationship of these pathways to other algae is also discussed.
Evidence suggested that p-hydroxybenzoic acid by both
I. galbana and N. lncerta was (1) decarboxylated, probably resulting ln 1,4-dihydroxybenene, (2) bromlnated to 3-bromo- p-hydroxybenzoic acid, and (3) excreted into the medium. It was unknown if the 1,^-dihydroxybenene and/or 3-bromo-p-' hydroxybenzoic acid caused the browning observed mainly in cultures of both species grown ln the presence of tyrosine.
Saltcycllc acid (ortho-hydroxybenzolc acid) was also de• carboxylated, probably resulting in catechol (1,2-dihydroxy- benzene).
Ring cleavage observed for tyrosine and for phenylal• anine (Vose et al.. 1971) appeared to involve a C^-C^ com• pound, probably benzoic acid or one of its hydroxylated pro• ducts. Ring cleavage does not appear to be important in the degradation of either amino acid. No dihydroxyphenolic compounds were detected, but this does not eliminate the possibility of their formation. Evi• dence suggested that both species had problems hydroxylating not only the aromatic ring (eg. benzoic acid) but also the side chain of intermediates in the degradative pathway.
Both algal species degraded cinnamic and p-coumaric acids in a similar pathway to that reported in higher plants
The pathway involved 0-oxidation of the side chain to produc benzoic and p-hydroxybenzolc acids from cinnamic and p- coumaric acids respectively.
The uptake rates of both phenylalanine and tyrosine and the effect of other aromatic compounds on the growth constants and lag periods of both algal species are also presented. vi.
TABLE OF CONTENTS. Page ABSTRACT iii
TABLE OF CONTENTS vi
LIST OF TABLES ix
LIST OF FIGURES xii
ACKNOWLEDGEMENTS xviil
INTRODUCTION 1
LITERATURE REVIEW
MATERIALS [ AND METHODS 16
• 'I. Culturing. 16
A. The medium 16
B. Algal species utilized 16
C. Maintenance of stock cultures 18
De- Contamination testing 18
E. Cell enumeration 21 a,. Hemacytometer 21, b. Optical density 21
F. Optical density experiments and medium
preparation 23
G. One liter culture experiments 2k
H. Mass culturing 25 I.: Cell collection and storage 27 II. Chemical studies of aromatic compounds 29 • A. Sources 29 B, Chromatography 30 c.. Spectroscopy 31 vii.
Page D». Melting points 31
E. Chemical preparations of non-radioactive
compounds « 32
F, Chemical preparations of radioactive compounds 32
IIIo Isolation of products 32
IV, Radioactive feeding experiments 33
A. Source of ^C-isotopes 33 l4
B. Preparation and administration of C-compounds 34
Fission of the aromatic ring of tyrosine 3^
Side chain degradation of phenylalanine and tyrosine 35 14 Degradation of other C-labell.ed substrates 37 Uptake experiments with phenylalanine and tyrosine 38 14 C. Detection of C-products 39
Autoradiography 39
Scintillation counting 39
V, Enzyme assays 40
A» Phenylalanine ammonia-lyase 40
B. - Transaminase 4l
C. p-Hydroxybenzoate" hydroxylase 4l
D» Protein determination 42
RESULTS - ^3
I,. Culturing ky
A*. The effect of phenylalanine and tyrosine 43
B, Metabolism of phenylalanine and tyrosine 59 C. . The effect and metabolism of other aromatic compounds 62 - Page
De Results of radioactive tracers 62
Uptake of phenylalanine and tyrosine 62
The catabolic fission of the aromatic ring of
tyrosine 71
Side chain degradation of phenylalanine and
tyrosine 7k
Degradation of other C-labelled substrates 92
E.. Results of enzyme assays 96
Phenylalanine ammonia-lyase 96
Transaminase ^ 96
p-Hydroxybenzoic acid hydroxylase 101
Protein determinations ; 101
DISCUSSION 103
LITERATURE CITED 125
APPENDIX 134
Ai- ROUTINE STERILITY TESTS 134
B- SPRAY REAGENTS 135
C- CHEMICAL PREPARATIONS OF NON-RADIOACTIVE
COMPOUNDS 138
D- CHEMICAL PREPARATIONS OF RADIOACTIVE COMPOUNDS 146
E- THE EFFECT AND METABOLISM OF OTHER AROMATIC
COMPOUNDS 149
•ADDENDUM 159 ix.
LIST OF TABLES>
TABLE Page
1. The growth of algae on phenylalanine when
used as the sole N-source. 8
2. The growth of algae on tyrosine when used
as the sole N-source. 9
3. Total ^CC>2 measured as product of catabolism
from 2-weeks incubation of algae with ring
labelled ^C-phenylalanine. 10
Aromatic compounds Identified from algae. 13
5* Bromophenolic compounds identified from
algae. 14
6. Phytoplankton culture medium. 17
7. Initial source of axenic algal cultures. 20
8. Growth constants and cell yields from mass
.cultures. .44
9. The effect of aromatic compounds on the
growth constants and cell yields from one
liter cultures of Isochrysls galbana. 45
10. The effect of aromatic compounds on the
growth constants and cell yields from one
liter cultures of Navlcula lncerta. 45
1.1. A summary of the effect and metabolism of
other aromatic compounds on Isochrysls
galbana and Navlcula lncerta. 6y
12. Uptake rates of phenylalanine and tyrosine by
Isochrysls galbana and Navlcula lncerta at
"two substrate concentrations. 65 X.
TABLE Page
13. Total ^'C02 measured as a product of cata-
bolism from 2-weeks incubation of algae with
uniformly ring-labelled ^C-tyrosine, 72
14. Radioactivity in products Isolated from 14 uniformly ring labelled C-tyrosine products. 73 14
15* Total COg measured as a product of cata-
bolism from incubation of pre-adapted cells
of Isochrysls galbana with labelled phenyl•
alanine and tyrosine. 76
14
16. Total CO2 measured as a product of cata-
bollsm from incubation of pre-adapted cells
of Navlcula lncerta with labelled phenyl•
alanine and tyrosine. 77 14
17. Radioactivity ln products Isolated from x C-.
labelled feedings from incubation of pre-
adapted cells of Isochrysls galbana. 83
18. Radioactivity In products Isolated from -Re•
labelled feedings from incubation of pre-
adapted cells of Navlcula lncerta. 84 14
19« Total C02 measured as a product of cata-
bolism ln the dark from incubation of non-
adapted cells with labelled aromatic
compounds. 93
20. Total ^C02 measured as a product of cata-
bollsm from a 12-hours dark incubation of xi.
TABLE Page
non-adapted cells with labelled aromatic
compounds. 95
21. Phenylalanine ammonia-lyase activity ln
marine algae. 97
22.. Transaminase activity in cell extracts of
Navlcula lncerta and Isochrysls galbana. 99
23. Phenolic compounds detected from feeding non•
radioactive phenylalanine and tyrosine to
various algae and the relationship of the
phenylalanine feedings to the metabolism of
phenylalanine ring-l-^C. 121 xii.
. . LIST OF FIGURES
FIGURE . . '._ Page
1. The shlkimlc acid pathway for the bio•
synthesis of aromatic amino acids. 2
2. The pretyrosine pathway in blue-green algae. 6
3« Photomicrographs of the algal species
utilized in this investigation. 19
4. The linear range of OD in relation to cell
number for planktonic species. 22
5. Diagram of mass culture apparatus and trap
for volatile product(s). 26 14 6. Diagram of apparatus for COg regeneration
and retrapping, 36
7. Diagram of the effects of L-phenylalanine on
the growth-constant and lag period of
Isochrysls galbana and Navlcula lncerta. 46
8. Diagram of the effects of L-ty.rosl.ne on the
growth constant and lag period of Isochrysls
galbana and Navlcula lncerta. 46
9« Growth curves of Navlcula lncerta on nitrate
and L-phenylalanine with and without nitrate. 47
10. Growth curves of Navlcula lncerta on nitrate
/ and L-tyrosine with and without nitrate. 48
11. Diagram of the effects of L-phenylalanine as
the sole N-source on the growth constant and
lag period of Isochrysls galbana and Navlcula
lncerta. 50 xiii.
FIGURE Page
12. Diagram of the effects of L-tyrosine as the
sole N-source on the growth constant and lag
period of Isochrysls galbana and Navlcula
lncerta. 50
13. Growth curves of Isochrysls galbana on nitrate
and L-phenylalanine with and without nitrate. 51
Ik,. Growth curves of Isochrysls galbana on nitrate
and L-tyroslne with and without nitrate. 52
15. The effect of L-tyroslne with and without L-
phenylalanine on the growth yields of
Isochrysls galbana. 5k
16. Growth curves of Navlcula lncerta on nitrate
and D-phenylalanlne. 56
17. Growth curves of Navlcula lncerta on nitrate
and D-tyroslne., .57
18.. Diagram of the effects of D-phenylalanine
and D-tyrosine on the growth constant and
lag period of Navlcula lncerta. 58
19. Absorption spectrum in ethanol of the
phenolic acid tentatively identified as 3-
bromo-p-hydroxybenzolc acid Isolated from
Isochrysls galbana and Navlcula lncerta. 6l
20. - Time course of uptake of DL-phenylalanine-3-
C for Isochrysls galbana at an Initial,
phenylalanine concentration of 10 uM. 6k
21. Time course of uptake of DL-phenylalanine-3-
. C for Isochrysls galbana at an initial xiv.
FIGURE Page
phenylalanine concentration of 0.1 mM. 64
22. Time course of uptake of DL-phenylalanine-3-
•^C for Navlcula lncerta at an initial phenyl•
alanine concentration of 10 uM. 66
23. Time course of uptake of DL-phenylalanine-3- 14 C for Navlcula lncerta at an initial phenyl•
alanine concentration of 0.1 mM. 66
24. Time course of uptake of DL-tyrosine-3-1^C for
Isochrysls galbana at an initial tyrosine
concentration of 10 uM. 68
25. Time course of uptake of DL-tyrosine^-^C for
Isochrysls galbana at an initial tyrosine
concentration of 0.1 mM. 68
26. Time course of uptake of DL-tyrosine-3-^C for
Navlcula lncerta at an Initial tyrosine
.concentration of 10 uM. 69 14 27. Time course of uptake of DL-tyroslne-3- C for
Navlcula lncerta at an initial, tyrosine
concentration of 0.1 mM. 69 28. Autoradiography of the ether extracts from 14 'C-slde chain labelled phenylalanine and tyrosine fed to Isochrysls galbana., 80 29.. Autoradlographs of the ether extracts from 14
C-slde chain labelled phenylalanine and
tyrosine fed to Navlcula lncerta. 81 Autoradiographs of the ether extracts from
C-slde chain labelled phenylalanine and tyrosine to be fed to Isochrysls galbana and Navlcula lncerta.
Autoradiographs of the ether extracts from l4 1U cinnamic acid-2- C and p-coumaric acid-2- C fed to Isochrysls galbana and Navlcula lncerta.
Autoradiographs of the ether extracts from 1U l4
C-phenylalanine and C-tyrosine trans• aminase experiments with extracts of
Isochrysls galbana and Navlcula lncerta.
The degradative route of L-phenylalanine in
Isochrysls galbana and Navlcula lncerta.
The degradative route of L-tyrosine in
Isochrysls galbana and Navlcula lncerta.
The degradative route of cinnamic and p- coumarlc acids in Isochrysls galbana and
Navlcula lncerta.
Absorption spectrum of J,5-dlbromo-p- hydroxybenzolc acid and it*s degradation product.
Absorption spectrum of J,5-dibromo-p-
hydroxybenzaldehyde.
Absorption spectrum of p-hydroxyphenyl- .
acetaldehyde. Absorption spectrum of p-hydroxybenzoylformic acid and it's spontaneous degradation product p-hydroxybenzaldehyde.
Absorption spectrum of synthetic phenylhydra- crylic acid.
Diagram of the effects of phenylacetlc acid on the growth constant and lag period of
Isochrysis galbana and Navlcula lncerta.
Diagram of the effects of p-hydroxyphenyl- acetlc acid on the growth constant and lag period of Isochrysls galbana and Navlcula lncerta.
Diagram of the effects of DL-mandellc acid on the growth constant and lag period of
Isochrysls galbana and Navlcula lncerta.
Diagram of the effects of DL-p-hydroxy- mandelic acid on the growth constant and lag period of Isochrysls galbana and
Navlcula lncerta.
Diagram of the effects of benzoic acid on the growth constant and lag period of
Isochrysls galbana and Navlcula lncerta
Diagram of the effects of p-hydroxybenzoic acid on the growth constant and lag period of Isochrysls galbana and Navlcula lncerta.
Diagram of the effects of p-hydroxybenzalde• hyde on the growth constant and lag period of
Isochrysls galbana and Navlcula lncerta. Diagram of the effects of 3i5-dibromo-p-
hydroxybenzolc acid on the growth constant and lag period of Isochrysls galbana and
Navlcula lncerta.
Diagram of the effects of 3.5-dibromo-p- hydroxybenzaldehyde on the growth constant and lag period of Isochrysls galbana and
Navlcula lncerta.
Diagram of the effects of cinnamic acid on the growth constant and lag period of
Isochrysls galbana and Navlcula lncerta.
Diagram of the effects of p-coumaric acid on the growth constant and lag period of
Isochrysls galbana and Navlcula lncerta.
Absorption spectrum in ethanol of the phenolic tentatively identified as p-
.hydroxyphenylhydracrylic acid from Iso• chrysls galbana and Navlcula lncerta.
Diagram of the effects of m-hydroxybenzoic acid on the growth constant and lag period of Isochrysls galbana and Navlcula lncerta.
Diagram of the effects of o-hydroxybenzolc acid on the growth constant and lag period
°f Isochrysls galbana and Navlcula lncerta. xviii.
ACKNOWLEDGEMENTS.
I wish to express my sincere appreciation to Dr. G. H. N.
Towers and Dr. N. J. Antia under whose supervision this study
was conducted, and for their valuable advice and criticism
during the research and preparation of this manuscript.
To Dr. C. K. Wat, Mr. J. Y. Cheng, and my collegues for
their assistance and helpful discussions during this research.
To Dr. J, S:. Craigier N. R. C. Atlantic Regional Lab.,
Halifax, N.S, for his kindness in providing samples of synthetic
bromophenolic compounds.
To the members of my committee for their helpful comments
on this manuscript.
To the Fisheries Research Board of Canada for providing
financial assistance during this study.
Finally, but firstly, to my wife Marlene who typed this
manuscript and withstood the many problems I had during" the
research and preparation of this thesis. INTRODUCTION :
' Both phenylalanine and tyrosine are important pre• cursors not only for protein "biosynthesis, but also a host of other compounds found in higher plants, lower plants, fungi, bacteria, and. animals- (see* Luckner, 1972). The bio• synthesis of these two amino acids by bacteria, fungi, and plants through the shlkimlc acid pathway (Figure 1) is well known (see Towers and Subba. Rao, 1972); however most insects and mammals including man are unable to synthesize them and they must be obtained from either their diet or the.-micro-, flora within their alimentary canal. Axenlc cultures of algae do not require either of these two amino acids for autotrophic growth, suggesting that they possess the shikimic acid pathway*
The metabolism of phenylalanine and tyrosine is of special interest. For plants, fungi, bacteria, and animals the metabolic products and pathways are well described
(Luckner, 1972; Towers and Subba Rao, 1972), but little is known for algae. Both of these amino acids can serve as the sole N-source for growth of certain algal species, but the metabolic path of their utilization has not been investigated.
Vose et al. (1971) reported that nine of twenty-two phyto• plankton species could metabolize the total carbon skeleton
of phenylalanine to C02y but they did not investigate the route of degradation. They also detected the presence of an unidentified volatile product or products. To date, there COOH Phosphocnolpyruvatd —<~— HO— C—H Pi OH HOT-V ^OH ©O T OH + H—C—OH OH OH OH OH Shikimic acid CHO I 5- Dehydroquinic acid 5-Dehydroshilcimic acid 5-Phospho I H—C—OH shikimic acid PEP H—,C—OH CHjO® 3-Deoxy-D- arabinoheptulo H—C—OH sonic acid 7-phospbate CHjO—® D-Er>1hrose-4-phosphate CH, O-t ceo COOH OH J-Enolpyruyyl shikimic acid 5- phosphate CH2CHNH2COOH HOOCV CH,CC00H o CH, O—C L-Phenylalanine OH ZOOM Prcphenic acid Chorismic acid OH OH L-Tryptophaii —— —- L-Tyrosine p-Hydroxyphenyl pyruvate Figure 1. The shikimic acid pathway for the biosynthesis of aromatic amino aclds0 are no further reports on the metabolism in algae of these two amino acids. An examination of their metabolism in algae was therefore considered to be worthy of study. Two species, Isochrysls galbana Parke and Navlcula lncerta Hustedt were selected for this investigation based on the results of Vose et al.(1971). Upon feeding ring- Ik labelled C-phenylalanine, they reported I. galbana produced an unidentified volatile compound(s) which contained a large percentage of the radioactivity while the radioactivity obta• in lned in C02 was low. In N. lncerta, on the other hand, the reverse situation was observed. Out of twenty-two species they examined, the greatest radioactive count collected in ^COg was from N. lncerta while the greatest radioactive count collected in the unidentified volatile C-compound was from I, galbana. Both these planktonlc algae were con• sidered the most promsing species to use in order to invest• igate the.degradative pathway of not only phenylalanine but also tyrosine. This dissertation will present evidence for the de• gradative routes of phenylalanine and tyrosine in both axenic planktonlc algal species. The metabolic routes were examined with specifically labelled radioactive tracers. Several enzymes, involved in the degradative pathways, were investi• gated in cell-free extracts from both, algal species. The effects on growth of various aromatic compounds and their metabolism by both algal species were also examined. 4. LITERATURE REVIEW. The biosynthetic pathway for aromatic amino acids begins with the condensation of phosphoenolpyruvic acid and erythrose-4-phosphate (Figure l)'r the first of seven enzym• atic reactions that lead to the formation of chorismic acid. Chorismate is converted to prephenlc acid or can be used for tryptophan synthesis. So far as is known, all organisms that are able to synthesize L-phenylalanine and L-tyrosine utilize prephenate as the last common intermediate of a branching pathway. This pathway is inferred to be the bio• synthetic route in algae. No investigation to confirm this has been conducted, although~several key enzymes have been extensively studied. The initial enzyme, 3-deoxy-D-arablno heptulosonic acid 7-phosphate synthetase has been studied in Agmenellum quadrupllcatum (Ingram .et a^,, 1973l Jensen et al.. 1974j Stenmark et al,, 197*0, Anacystls nidulans (Weber et al., 1968j Stenmark et al., 1974; Phares et al., 1975)» Anabaena variabilis. Osclllatorla tenuis. Nostoc sp., Meso-taenlum sp., Anklstrodesmus sp.and Euglena. gracilis. (Weber et al. , 1964). High enzymatic activity was obtained for this enzyme and its control by phenylalanine and tyrosine was examined. The branch point enzyme, chorismic acid mutase has also been Investigated in Anacystls nidulans, Zygnema sp,, Chlamydomonas relnhardtl and Anklstrodesmus sp,, (Weber et al., 1969). Euglena gracilis (Weber et al,.. 1964, 1970), Ac^enellura quadrupllcatum and Anacystls nidulans (Stenmark et al,, 1974). Again, control of this enzyme by phenylal• anine and tyrosine was studied. ; '"•y.;;£:'":jv.Htgh specific activity for. prephenate dehydratase and an aromatic transaminase'was detected for Agmenellum quadrupllcatum and Anacystls nidulans, however no activity /for prephenate dehydrogenase was obtained (Stenmark et al,. 1974). In blue-green algae (or bacteria) a new pathway from prephenic acid to tyrosine was reported (Stenmark et al,. 1974| Jensen and Plerson, 1975? Jensen and Stenmark, 1975). This pathway has been shown by enzymatic studies to be present In Agmenellum quadrupllcatum, Anacystls nidulans, Coccochloris elabens, Nostoc muscorum, Lyngbya lagerhelmll, and Oscllilatorla sp, and involves a transamination to give pretyroslne followed by dehydrogenation-decarboxylatiort to give L-tyroslne (Figure 2), In the analysis of various algae, both these amino acids were found not only in proteins (Fowden, 1962j Schmld, I969J Kempner et al.. 1974) but also in the free state (Fowden, 1951» Watanabe, 1951J Schmld, 1969? Kempner et al., 1974» Impelllzzerl, 1975). In one case, it. is reported, that phenyl• alanine is excreted by Calothrlx scupulorum Into the medium (Stewart, 1963K The presence of phenylalanine and tyrosine in algae and of enzymes involved in their synthesis as well as the .lack of either amino acid for growth, leads, one to conclude that algae as a.rule, can synthesize both amino acids, prob• ably by. the shikimic acid pathway. : Although the metabolism of phenylalanine and tyrosine- has been poorly investigated, their utilization as the sole source of nitrogen has been extensively studied. In 0 NH2 ^H2 CH -C-COOH HOOC CH2-CH-COOH CO- CH2-CH-COOH HOOD 2 prephenate pretyrosine, transaminase dehydrogenase NAD+ OH OH OH Prephenic Acid Pretyrosine L-tyrosine Figure 2. The pretyrosine pathway In blue-green algae. Table 1 a list is compiled of algal species examined for their ability to utilize the nitrogen of phenylalanine and a corresponding list for tyrosine is shown in Table 2. Approxim• ately one-third of the species were able to metabolize phenyl• alanine and tyrosine^ The growth in most cases was never comp• arable to cultures grown on nitrate. Several species could me• tabolize D-phenylalanine, although D-tyrosine never served as a nitrogen source. No algal species capable of heterotrophic growth on either phenylalanine or tyrosine are known. In all the nitrogen utilization studies, the fate of the carbon skeleton of the amino acid was disregarded. The only investigation of this problem was that by Vose et al. lit 14 (1971) who Incubated DL-phenylalanine- C (ring-1- C label• led) with 22 axenic phytoplankton species. In the light, nine species metabolized a fraction of the total carbon skeleton to COg whereas in the dark only four species retained this ca• pacity (Table 3). It must be noted that this study only indicated the species that have the capability to degrade the aromatic ring. No Indications as to which species can partially metabolize the carbon skeleton or which species are able to utilize phenylalanine as a N-source were obtained. The rate of uptake of phenylalanine by various algal species has been investigated. In Meloslra nummuloldes (Mel- 3), 0.1 mM phenylalanine was taken up at 2,7 x 10 umoles/ mln/cell for the first ten minutes; when averaged over six -9 hours however, the rate decreased to 0.14 i 10 jimoles/min/ cell (Hellebust et al.. 1967). Platymonas subcordlformis (Wille TABLE 1 The Growth of Algae on Phenylalanine When Used as the Sole N-source. Concentration Alga Isomer fed (mM) Growth Reference Chlorophyta Anltlstrodesmus amalloides L 21.1 Den Dooren (I969) » : a D 21.1 NG L 21.1 NG " angustus D 21.1 NG " falcatus var stlnltatus • » i» * if L 21.1 NG 0 21.1 NG " nanoselene L 21.1 ? » « D 21.1 NG Brachlinonas subaiarlna ? 0.24 NG Droop (1961) Chlorella sp_. (F. B. Wann's No. 11) ? 12.5 55* <2 ) Ludwlg (1938) pyrenoldosa DL ii.5 NG (2)'(hos h et al. (1950] vulgaris DL 6.1 Arnow"Tl953) (strain 11) L ^.o ? Ballard (1966) " "Delft" DL 12.1 Den Dooren (I967) « It L 12.1 +++ (1969) D 12.1 ++++ • . Strain 260 L 12.1 KG m m « D 12.1 NG " Strain Z6\ L 12.1 ++ D 12.1 ? m an L 12.1 NG Strain 262 D 12.1 ++ :L 12.1 NG. • n n D 12.1 ? Strain 263 DL 9.1 9.1 + Loefer (1932) « it n DL + n 1 ? NG n Chlorogonlun elona;atum • Droop (1955) " euchlorum 1 0.24 NG ? ? NG (1961) Eaematoooccus Pluylalls L (1955) " " (2 strains) 3.0 +++ La Hue et al. (1966) Kannochlorls oculata Strain No.66 D 3.0 +++ Prototheca elferrl 60-49 L 3.0 +++ 1. n n D 3.0 +++ ? 0.24 NG Droop (1-961) " morIformis 60-50 Stephanosphaera pluvlalls DL 12.1 ++++ DuDuss l (1931)(1933b) DL 12.1 NG n S'jglenophyta DL 12.1 ++++ (1933a) Saglena anabaer.a var minor DL 12.1 + (1933b) * deses DL 12.1 NG (1931)(1933b) " gracilis DL 12.1 NG " fclebsli DL 12.1 ++ " plsclformis Chrysophyt" 9tellata a Kor.ochr,y3l3 lutherl NG Droop (1955) Prysr.eslnm paryum +++ Syracosphaera elongata ? Ba^mgrloohyta T 0.1 KG Hellebust et al. (1967) Msloslra nujimjTold33 Clone Mel-3 Cryotpphyta L 0.5 NG Antla et al. (1968) P.'ndoohyta rihoden.a roaculata DL 0.42 NG Turner (1970) Xanthonhyta £iao.'3li2 aubterraneus DL 0.31* NG ,,, Killer et al. (1958) Trlbonema aequale DL 0.25 8iy2' Belcher ej al. (1958) Cyanophyta (2) Amtnellum quail ru pi lea turn 0.9 50* Kapp si al. (1975) ' 3traln P!l-6 (1) NG-no growthi ?-doubtfuli +-poon ++-falri +++-goorti ++++-eicellent. (2) of control. 9. TABLE 2 The Growth of Algae on Tyrosine When U3ed as the Sole N-Souree. Concentration Alga Isomer fed (mM) Growth Reference Chlororhyta (1) L 10.8 NG Den Dooren (I969O Anklatro'jesmus amallolde3 D 10.8 NG • ~ •* L 10.8 NG " angU3tu3 D 10.8 NG * m L 10.8 NG " falcatus var -s'tlpltatus D 10.8 NG m- mm m L 10.8 NG " nano3elene D 10.8 NG- ? 0.21 Droop (1961) (1938) • n ? (2) Ludwig Brachlesnaa submarlna L 6k% Arnow (1953) Chlorella sp. (F. B. Wann's Ko. 11). L 10.8 1 Den Dooren (1967)(1969) " vulgaris 0 10.8 ? (1969) • " "Delft" L 10.8 NG ...'••» » D 10.8 NG • " Strain 260 L 10.8 NG m m ' m m D 10.8 NG " " Strain 261 L 10.8 NG D 10.8 NG • • " Strain 262 L 10.8 NG m it m n D 10.8 NG • " Strain 263 ? 27.0' ++ Bond (1933) L <9.1 ++++ Loefer (1932) ' m m mm M I. L <9.1 '++ Chlamydomona3 pulvlr.ata ? 0.21 NG Droop (1961) " euchlorua ? 27.0 ++ . Bond (1933) Haeniato^occus oluvlall3 (2 strains) 1 27.0 NG Bond (1933) 'Lobonor^s sp. ? 27.0 ++++ N II- PolvtoTa uvella ? 0.21 Prototheca zopfIt Droop (1961) Stephar.olpfaaera pluvlalls Euglenophyta L 11.0 NG Euglena aniiaana var minor L U.O NG Dual (1931)(1933b) * ~~ deses L 11.0 ++ II N H " alaollls L 11.0 NG (1931a)' L 11.0 NG (1931b) • '• fclebsll L 11.0 NG (193D(1933b) " plsclformis L 11.0 NG " atellata Chrysonhyta Hymenoronas sp. 156 0.5^.^ ++ Printer et al. (1963) Baclllarlophvta 0.1 NG Hellebust et al. (1967) Melo3lra nummuloldes Clone Mel-3 Crypto^hyta Bemlselals vlre3cen3 L 0.5 NG Antla et al. (1968) Rhodor>h7ta Rhodella maculata L 0.38 NG Turner (1970) Xantho^hvta Monodu3 subterraneus L 0.17 + (2) Miller et al. (1958) Trlbongia aequale L 0.25 85# Beloher et al. (1958) Cyanop'-.yta Agmenellum quadrupllcatum Strain 0.1-»-2.1 NG Kapp et al. (1975) PR-6 (1) NG-no growth: 7-doubtfuli +-poori ++-faln +++-goodi ++++-excellent. , (2) of control. 10. TABLE 3 Total l*COz measured as product of catabolism from 2-weeks' incubation of algae with ring-labeled i4C-phenylalanine • -. Light Dark Alga "CO:, dpra X 10-3 Chlorophyta Dunaliella tertiolecta 0 0:-. Nannochloris oculata 0 Chrysophyta .'• Isochrysis galbana 1.08(0.05%)* 0 ' Prymnesium parvum 2.94 (0.13%) 2.61(0.12%) Monochrysis lutheri 0 •o - Coccolithus huxleyi o • 0 BacQIariophyta Pennate diatoms Amphiprora paludosa 0 0 Cylindrotheca fusiformis 8.22(0.37%) 0 Phaeodactylum tricornutum 2.56(0.12%) 2.62(0.12%) Navicula incerta 32.94(1.50%) 10.07(0.46%) Nitzschia angularis 13.25(0.60%) 0 Centric diatoms Cyclotella nana 0 • 0 Chaeloceros gracilis 0 0 Skeletonema costatum 0 o Thalassiosira fluvial His o . 0 :. Cryptophyta Chroomonas salina 0 0 Rhodomonas lens 18.92(0.86%) 7.66(0.35%) Hemiselmis virescens 0 .'• o Vyrrophyta 1.66(0.07%) Amphidinium carteri •' 0 Rhodophyta Porphyridium omentum 0 0 Cyanophyta : Anacystis marina 0 • o Agmenellum quadruplicatum 1.05(0.05%) 0 •Figures in parentheses give data as % of added "C-pheoylalanine. Promt '"• . ' J, H. Vose, J. Y. Cheng, N. J. Antia, and G. E. N. Towers. 1971. The catabolic fission of the aromatic ring of phenyl• alanine by marine planktonlc algae. Can. J. Bot., ^9:259- 261, • • ' • • ; \-- • • ii.;. Hazen assimilated 50% of the supplied phenylalanine over a concentration range of 0.2-1.0 uM/1 (North et al., 1967). and at a concentration of 0.7 ;u;M/l, an accumulation ratio of 670:1 was obtained (North et ai., 1969). In more detailed studies, Chlorella fusca showed an apparent KM of 5 MM, a V^y of 0.1 nmoles/min/10' cells and at a concentration of 2 juM. an accumulation ratio of 1000:1 (Pedersen et al., 197*01 whereas Chlorella vulgaris had a Kffl of 7.2 uM or 15.0 uM for L-phenylalanlne-l-^C or 10.0 uM for L-phenylalanlne-4-T (Dedonder et al., 1968; Van Sumere et al., 1971)• It was also observed that C_. fusca has the ability to assimilate. D-phenylalanine (Pedersen et al., 1974). The metabolic routes for the degradation of both phenylalanine and tyrosine are unknown. Phenylalanine and tyrosine ammonla-lyases could not be detected in Alarla esculenta (L.) Grevllle, Ulva lactuca. Rhodymenla pal-. mata (L.) Grevllle, and Polyslphonla lanosa (L*) Tandy, (Young et al., 1966). In several algae an aromatic amino, acid transaminase has been detected. In Chlorella vulgaris»» the amino group was transferred from L-phenylalahine to dloxovalerlc acid (an a-keto acid) (Gassman et al., 1968) while l>n Ulva lactuca pyruvate and a-ketoglutara.te were uti• lized as the a-keto acid (Jacobi, 1957). No transaminase activity .was detected ln U, lactuca for L-tyrosine with ei* ther of these a-keto acids. In Agmenellum quadrupllcatum and Anacystls nidulans, a transaminase was reported involving L- phenylalanine and activity with several a-keto acids (Stenmark et al,, 197*0* At present no other enzymes have been examined which could be Involved ln the degradation of phenylalanine and/or tyrosine. The fission of the aromatic ring by algae has also •.------ik . ' .. been studied by Craigle et al., 1965 • C-phlorogluclnoI •(•'1»3!i 5-trl hydroxybenzene ) was added to nine axenlc species of algae. Six were able to metabolize the ring carbons to C02« Fission of the aromatic ring was enhanced severalfold by preconditioning each species to phloroglucinol. No In• dication as to the pathway of ring fission was given* In the chemical analysis of algae, many aromatic •'. compounds have been identified. They range.from simple to large multi-ringed structures. In Table k % list is prese• nted of identified and non-Identified compounds along,with the algal species from which they were isolated. Only benzaldehyde can be hypothetically implicated In the degradation of aromatic amino acids, whereas the others based on phloroglucinol, are almost certainly derived from another1pathway • .An interesting group of phenolics contain^ bonded bromine have, been identified* They are found in the Hhodophyta, mainly in the Rhodomelaceae, although several have been reported in two other algal divisions (see Table 5 )•• In Table 5 are listed the alga and the bromophenollc compounds contained-in each along with the references and chemical .. , structures. The ethyl and methyl ethers are now considered, to be artifacts of isolation and only the corresponding aldehyde was present (Pedersen et al., 197^1 Glombitza et al., 197^1 13 : • TABLE 4 ';. Aromatic Compounds Identified Prom Algae. Arotnatlo Algal species compound Reference Benzaldehyde Ulva pertusa. Enteromorpha sp., Katayama (1962) Codlum fragile. Sargassum sp., CH Laminaria sp., P_arp.hy.ra temerar 0" ° Dlgenea simplex Cladostephus sponglosus. C. vertl- Glombltza et al. Phlorogluclnol cllUtus. DletTQta dlchototya, .Fucus U973b7 veslculosus. F. serratus, F, spir• alis . Hlmanthalla elongata. Cysto- selra tamarlsclfolla. C_. baecata. C. H0 Fucus veslculosus Glombltza £_£ al. (1975) • ; Bagan ejt iai^V<1975) Fucus veslculosus Glombltza e£ al. (1975) Bl-and polyphenols Fucus veslculosus Glombltza e£ al. (197H (2-lf phlorogluclnol units) Glombltza et al. (1975) Bl-and polyphenylethers Cystoselra tamarlsclfolla Glombltza al. C_. baecata (1975) (2 or 3 phlorogluclnol units)' Mixed polyphenyl-polyphenol Fucus veslculosus Glombltza e£ al. (1974*) ethers (Z-U- phlorogluclnol units Bagan et al. (1975) Polyphenol ether (2-7 units) Halldrvs slllguosa. Blfurcarla Glombltzafi£ al. (non galloyl hydroxylatlon rotunda (197*) pattern) Fucus veslculosus Bagan et al. (1975) Sargalin C20H2«v08Nl»K Sargassum confusum Salto et.al. (1951) Phenolic (unknown) Fucus veslculosus HcLachlan ej ,al..,(196'0 Alarla esculenta. Aacophyllum Cralgle et al. (1964) nodosum. Chorda f 1'1'lum. Chord- aria flagelllformls. Ectocarpus sp. Laminaria agardhll. dlgltata Sargassum natans Sleburth et al. (1965) TABLE 5 Bromophenollc Compounds Isolated Prom Algae. Bromophenollc oompound Algal species ln table (Reference 1) Cytnophy.ta Calothrlr brevlsslma ?(A) , 14(A)2. 15(A)2, 16(A) lEhweoohyta Fucus veslculosus 7(B), 13(B), 16(B) Rhodoohyta Antlthitsnlon piuaula 11(c), 13(c) CeraTtum .rubrum 11(c). 13(C) Coralllna officinalis 11(C) Cystoslonlua purpureum 10(C.J) Halopltys lncurvus ~ 1(D), 7(D). 16(K.P) Odonthalla dentata 2(C). 3(C.G) 4(C). 5(C). 6(C), 7(C). 10(C). ll(C.G) 12(C). 13(C) ." corymblfcra 11CQ). 12(S.Q). 9?(D. 16(Q.) PhycoArys rubens 13(C) Polyslphonla brodlacl 1(D), 3(C.D). 6(C), 7(C.D). 10(C)., ll(C.D), 12(C), 16(D,0) " elongata 6(D)..10(D), 11(D). l6(D.O) * frutlculosa 1(D), 10(D), 11(D), 16(D,0) " . lanosa 3(D), 6(D,K), 7(D,M), 10(D,H). ll(D.L) 15(D), 16(D,H.H,0). 17(D) " morrowl'l 6(F) " nigra 1(D), 10(D), 11(D).. 16(D,0) " nlgrescens 3(C,D), 6(C), 7(D), 10(D). U(C,D), 12(C), 16(D,0) " thuyolfles .. 10(D), 11(D), 16(D) " urceolata 1(D), 6(C,D), 7(C,D), 10(C)., 11(C),. 12(C) " violacea 10(D). 11(D), 16(D) Bhodonela confervoldes 3(C.G), 10(C). lKC.P.G), 12(C) larlx 10(E). 12(E) " subfusca 10(D,B), 11(D), 12(H), 15(D), 16(D,0.3) Vldalla volubllls 16(N) (1) letter refers to reference, while number refers to chemical structure. (2) .Isolated from medium. A. Pedersen, !K. arid E. J. Da Silva (1973), B Pedersen, K. and L. Fries (1975). C Pedersen, K., P. Saenger, and L. Fries (197*0. D Glombltza, K. H. Stoffelen, U. Murawskl, J. Blelaczek, and H. Egge (1974) E Katsul, K. , Y. Suzuki, S. Kltamura, and T. Irle (1967) P Salto, T., and Y. Ando (1955) G Cralgle, J S.. and D. E. Gruenlg (1967) H Hodgkln, J H., J. S. Cralgle, and A. G. Kclnnes (1966) I Katsumoto, T., and S. Kagawa (1964) J Pedersen, K. (1974) K Augler, J , and P. Kastagll (1956) L Kastagll, P., and J. Augler (1949) R' Stoffelen, E., K. -W. Glombltza, U. Murawskl. J. Blelaezek, and H. Egge (1972) K Augler, J. , and K. H. Henry (1950) 0 Glombltza, K. -W., and H. Stoffelen (1972) • P Peguy, M. (1964) Q Kurata, K. , T. Anlya, and-K. Yabe (1973) Kurata, K, , and T. Araiya (1975) © © © H20H CH2OCH3 CH2OCH3 OC H CHCH0 0 ^2 2 S Br-\^-Br B OH OH OH @LAN0S0L @ © © CH2OCH3 C^CX^Hg CH20H ^OCHj Br. B rS^OH B OH OH OH OH © © -or- © © + CH0 CH20H CH 0H Mi 2 CH20S0^K ChL^OCjHy B, Br Br,^ Br- Bt^s^-OH Br_\^0H ; + 6 Br'* ^0S03-K Br^s^-OH . Br\^OH OH BrS^-OH OH OSOj-K* OSO^K* OH OH REF.-H REF.-O and Weinsteln et al.,1975 - see Addendum p.159). The biosynthesis of these phenolics is thought to proceed through the shiklmic acid pathway with bromination occurr• ing in the presence of a suitable peroxidase (Craigie and Gruenlg, 1967). Two reports of a peroxidase in red algae are known. Pedersen (1974) studied a partially purified enzyme preparation from Cystoclonlum purpureum which cata• lyzed the formation of monobromoprotocatechualdehyde (structure 6 - Table 5) from protocatechualdehyde in the presence of H202 and NaBr. Murphy and 0 HEocha. (196-9)) also obtained a peroxidase from the same algal species and they partially characterized it but no details as to sub• strates were given. Many investigators have examined the effect of aromatic compounds on algae. The number of compounds tested are too numerous to present in a table. In the discussion, the effect of the aromatic compounds import• ant in relation to the degradation of phenylalanine and tyrosine will be introduced. ; * 16. MATERIALS AND METHODS I. Culturing. All methods were based on standard principles of aseptic techniques used in culturing phytoplankton. A. The medium The composition of the phytoplankton culture medium used In this investigation of aromatic metabolism is presented In Table 6. The medium is based on that used by Dr. N. J.. Antia for mass culturing planktonlc algal species (Antia and Kalmakoff, 1965). Open ocean seawater (see McAllister et al. i960) was always used. All the inorganic chemicals used for the culture medium were 'Baker Analyzed' Reagents obtained from J. T. Baker Chemical Co., Phlllipsburg, N.J. The pH was 7.9-8.1 after autoclaving or 7.7-7.9 after filter steriliza• tion. The salinity was calculated to be 27$°. B. Algal species utilized. Axenic cultures of Navlcula lncerta Hustedt and Isochr• ysls galbana Parke were obtained from Dr. N.J. Antia, Vancouver Laboratory of Fisheries and Marine Service of Environment Canada, Navlcula lncerta Hustedt was Initially obtained as strain Lewin #66-M from Dr. Joyce Lewln. Dr. Ralph Lewin isolated this strain (73-M) from a saline pool in San Francisco, California. Isochrysls galbana Parke was obtained from Woods Hole Oceanography Institute as clone "Iso" by Dr. J. D. H. Strickland. Dr. Mary Parke isolated this strain (Flagellate I) from a fish pond at the Marine Biological Station, Port Erin, Isle of Man, United Klngdon.- Light 17. TABLE 6 Phytoplankton Culture Medium KN05 126.0 mg ( 1.25 mmoles) NaH2PO^'H20 40.2 mg ( 0.40 mmoles) 1 Na2S103'9H20- - 268.8 mg ( 0.96 mmoles) Vltamins: Thiamine*HC1 0.96 mg ( 2.84 umoles) Biotln 1.92 wg ( 7.8 nmoles) B 12 (Cyanocobalamin) 3.84 ug ( 2.57 nmoles) Trace Metals (chelated): Na2*EDTA*2H20 8.1 mg (21.8 umoles) PeCl3'6H20 2.7 mg (10.0 umoles) MhS0^»4H20 1.125 mg ( 5.0 ;umoles) ZnSO^.'7H20 0.575 mg ( 2.0 umoles) Na2MoO^*2H20 0.243 mg ( 1.0 umole ) CuSO^'5H20 0.025 mg ( 0.1 umole ) CoSO^*7H20 0, 014 mg (50.0 nmoles) Buffer: Tris'HCl (pH 7.5-7.6)2 1.0 gm ( 8.3 mmoles) Sea Water: Open ocean, salinity 33&> -767.0 ml Distilled Water to 1 liter (1) one half this concentration was utilized ln mass cultures of Isochrysls galbana. (2) this gives a pH of 7.9-8.1 In the medium after autoclaving (15 minutes at 120°C). 18. microscope photographs of both species are presented in Figure 3. Other phytoplankton species utilized in various ex• periments were also obtained from Dr. Antia. Their initial source is listed in Table 7. Non-planktonic algal species were collected from Stanley Park, Vancouver, B.C. near Lumber• man^ Arch. C, Maintenance of stock cultures. N. lncerta and I± galbana were maintained in 125 ml screw cap Erlenemyer flasks containing kO ml of the culture medium in Table 69 The silicate concentration used was one half the value stated in the table for only stock cultures. These two species were subcultured every two months or when exponentially growing cells were required for an experiment. These cultures were maintained at 19-21°C and at approximately 100 foot-candles (Weston Footcandle Light Meter) under con• tinuous illumination from cool-white fluorescent lights (No, F1^T12/CW., Sylvania Electric Canada, LTD.). The other phytoplankton species were obtained in the exponential growth phase from Dr. Antia's collection. D. Contamination testing. A contamination test was performed whenever a stock culture was utilized to Initiate an experiment and at the end of the same experiment. One or two drops of the algal culture growth medium were added to a screw capped culture tube containing the sterility test medium (Appendix A). The A B Figure 3. Photomicrographs by interference contrast of the algal species utilized in this investigation, (A) Isochrysls galbana Parke and (B) Navlcula lncerta Hustedt. TABLE 7 Initial Source of Axenlc Algal Cultures Algal species Initial sourc Chlorophyta Brachlmonas aubmarlna (Bohlln) Droop Millport strain Dr. M. R. Droop var puisIfera 7/2a Droop Dunallella tertlolecta Butcher Woods Hole Clone "Dun", Dr. J. D. H. Strickland Nannochlorls oculata Droop Millport strain #66, Dr. M. R. Droop Haptophyta Emlllanla huxleyl (Lohm. ) Hay & Mohler (2) Woods Hole Clone "BT-6", Dr. T. R. Parsons Pavlova lutherl (Droop) Green(3) ' Millport strain #60, Dr. J. D. H. Strickland Baclllariophyta Aaphl'prora paludosa W. Smith Woods Hole "Lewin #73-K", Dr. J. Lewln var duplex Donk, Thalassiosira pseudonana (Hustedt) Woods Hole Clone "3H", Dr. R. R. L. Gulllard Hasle & Helmdal Skeletoneaa costatum (Grev. ) Cleve Woods Hole Clone "Skel", Dr. R. R. L. Gulllard Thalassiosira fluvlatllls Hustedt Woods Hole Clone "Actin", Dr. J. Lewln Cryptoohyta Chrooiconas sallna (Wislouch) Butcher Woods Hole Clone "3C", Dr. F. T. Haxo Cryptomonad str. :/ffll Hasklns WHl,Dr. L. Provasoli Rhodomonas lens Pasoher and Ruttner Hasklns Laboratories, Dr. L. Provasoli Pyrrophyta Woods Hole Clone "Amphl 1", Dr. L Provasoli Amphldlnium carterl Hulburt Rhodoohyta Vlscher's strain #107, Dr. F. T. Haxo Porphyrldluci cruentum Nag ell Millport strain #207, Dr. M. F. Turner Rhodella maculata Evans Cyanophyta Agtnenellum quadrupllcatum (Keneg. ) Van Baalen's strain "PR-6", Dr. C. Van Baalen Brebisson Van Baalen's strain "6", Dr. C. Van Baalen. Anacystls marina (Hansg.j Drouet & Dally Xanthophyta HeterothrlT sp. S. M. E. Marseille strain. Dr. S. Y. Maestrlnl Monallantus sallna Bourrelly S. M. E. Marseille strain. Dr. S. X. Maestrlnl (1) original Isolate and name of specimen-culture donor. (2) taxonomlcally revised from Cocsollthus huxleyl (Lohm.) Kamptner, (see Hay et al., 1967). (3) ta-xonomlcally revised from Konochrysls lutherl Droop, (see Green, 1975). (6) taxonomlcally revised from Cyclotella nana Hustedt, (see Hasle et al. , 1970). If tube was incubated about three weeks in the dark at room temperature. Contamination was extremely rare and when dis• covered the data therefrom was discarded. In Appendix A, the procedure undertaken when possible contamination was suspected is outlined, ? E, Cell enumeration, . Cell numbers were determined directly by counting the cells with a hemacytometer or indirectly by measuring the optical density of a cell suspension, a. Hemacytometer. A counting chamber (AO Spencer bright-line) was em• ployed when an estimate of cell number was required. Samples °f N, lncerta or I. galbana were not required to be preserved for counting (the motile stage of I. galbana was not encount• ered in exponentially growing cultures). However, when motile planktonlc species were to be counted, samples were treated with one drop of 5% formalin per ml for ca, 5 nin. before filling the hemacytometer chambers. For each sample, at least two replicate fields were counted to obtain an estimate of the cell number. For very small planktonlc species a Petroff-Hausser bacteria counter counting chamber (C, A. Hausser and Son) was used, b. Optical density. Optical density (O.D.) was used as an estimate of cell number when growth experiments were conducted ln 8 ml capacity .optically^ clear,, screw capped ....culture tubes,. The contents of —I—( i lilj 1 1—1 1 Mil 4.0x107-h i i i i-111 1 t •—•Isochrysis galbana Navicula incerta 1.0x# 4.0x1cPf CELL NUMBER PER ml 1.0x1064r 40x105f loxicr 4.0x10n-b—H-i Q004 0.010 0.040 0.100 0.400 1.000 OPTICAL DENSITY at 600 nm. Figure k> , The linear range of OD in relation to cell number for planktonlc species. 23. each culture tube was first thoroughly mixed with a Vortex Jr0 mixer. The tube.was then Inserted into a Spectronlc 20 Colorimeter (Bausch and Lamb, Inc., Rochester, N.Y.) and the OD read at 600 nm. . The range within which OD was linearly related to cell number was determined by using concentrated cultures of N. lncerta and Xi galbana obtained by centrifugation. A dilu• tion series was prepared and the OD of each dilution was measured. In Figure 4, the relationship of OD to cell number is presented. Additional experiments Indicated that the re• lationship between cell number and OD was almost constant throughout the exponential phase. F. Optical density experiments and medium preparation. When the simultaneous use of many compounds over a large concentration range was required for an experiment, the use of large culture volumes was impractical and small Kimax screw capped test tubes (Kimble No 45042, 15x125 mm) were used instead. These tubes were selected for ease of measure• ment without sampling every tube because they fit directly into the Spectronlc 20. Therefore there was little chance of cont• amination ruining the experiment. By using the algal culture medium, 2.1 mM stock solu- tions of each aromatic compound were prepared^then filter sterilized (0.2-um pore-size Gelman Metricel filter in • sterile disposable plastic units from Nalge Co., Rochester N.Y.). A dilution series (2.1, 1.05, 0.525, 0.262, 0.105, 0.0525, and 0.0262 mM) was prepared by aseptically pipetting . Into pre-sterillzed tubes the required volume of stock solu• tion and sterile culture medium to give a final volume of 4 ml per tube. These tubes, along with a control (no aromatic compound), were inoculated with 0.2 ml of an exponentially growing stock culture (7-14 days old with no previous ex• posure to any .aromatic compound). The dilution in each tube, caused by the Inoculum, decreased the initial aromatic con• centration and thus the actual concentration range examined was 2.0, 1.0, 0.5. 0.25, 0.1, 0.05, and 0.025 mM. The inoculated tubes were incubated stationary at an angle of 20-25° from the horizontal plane'under continuous illumination from cool-white fluorescent light placed hori- .zoritaily above them. ' The temperature was maintained^ between 19-21° C and the light intensity was estimated • at ca. 100-120 foot-candles. Growth measurements were made at 1-2 day. intervals, depending on the rate of OD change. Exponential growth constants (AOD/day) were calculated from the plots of 0D versus incubation time. The adaptation period, the number of days from inoculation to the*major'significant Increase in OD (expon• ential growth phase) was also calculated from the same plot. At the termination of the experiment, each tube was acidified to pH~2 then extracted with peroxide free diethylether for analysis of metabolic products. G. One liter culture experiments. In 2,8 liter capacity Erlenmeyer flasks, 1 liter growth experiments were conducted at 19-21°C under continuous [ ' 25. illumination from cool-white fluorescent lamps. The inten• sity of illumination was estimated at ca. 280-310 foot-candles. Each culture was incubated stationary and every day was thoroughly mixed by vigorous swirling of the flask. The growth of each culture was followed by taking cell counts, then plotting 1°S10 E^/EQ against growth period (t hr), where NQ and Nj. denote cell concentrations at time of inoculation and t hr_ respectively. NQ was calculated directly from the cell count of the inoculum used. The growth constant (K(io) hr and mean generation time (tg hr) were calculated from the expressions (Antia and Kalmakoff, 1965)1 log10 Nt/N0 = K(1Q) • t and log10.2 = K(l0) • tg The slope of the straight-line portion of the growth curve was used to derive K(io)» H. Mass culturing. In 6 liter capacity Erlenmeyer flasks, modified for mass culturing phytoplankton, 5 liter growth experiments were conducted. In Figure 5# the design of the mass culture vessel which is similar to that used by Antia and Kalmakoff (I965) is outlined. The temperature was maintained at 19- o 20 C by means of an incubator. Cool-white fluorescent lamps provided continuous illumination at an intensity estimated at ca. 1200-1500 foot-candles.. Each culture was kept in constant motion by means of a magnetic stirring bar and was aerated daily for 3-6 hours with a source of compressed air (95$ air and 5% C02) through the fritted-glass outlet. Growth parameters for each culture were determined as Diagram of mass culture apparatus and trap for volatile product(s). A, stream of gas containing' 95% air and 5% CO 2» Slass holder packed with cotton wool. C, autoclavable rubber tubing. D, ground glass fitting for aeration tube. E, 6-liter Erlenmeyer culture vessel. F, fritted-glass gas outlet. G, magnetic stirring bar. H, magnetic stirring block. I, cotton wool (non-absorbent) plug wrapped in gauze*. J, cotton wool plug wrapped in gauze, r K, autoclaved rubber stopper fitted with glass 27. Figure 5 (cont.). tubing (to replace the cotton wool plug (J) in order to connect the gas bubbler to the culture vessel). L, gas bubbler containing reactant for volatile product(s). * replaced by an autoclaved rubber stopper when gas bubbler was connected. described in Part G for one liter culture experiments. After filling with 5 liters of the algal culture medium the culture vessel was fitted with the aeration apparatus then autoclaved as a unit. After cooling, the gas-inlet line was connected to the source of COg-enriched air and the system was allowed to stabilize 1-2 days before inoculating with gentle aeration and mixing at 19-21° C. Just prior to inoculation, sterile vitamins and nitrate-phosphate solutions were added aseptically to the culture vessel. To trap the volatile material reported by Vose e_t al. (1971) from I. galbana, when grown on L-phenylalanine, the gas bubbler was connected to the culture vessel and the cotton wool plug was replaced by an autoclaved rubber stopper. The rubber stop• per with the glass tube and the connecting rubber tubing were autoclaved before connecting the culture vessel to the gas bubbler. Two trapping solutions were used: 2,k-dinitrophenyl- hydrazine (100 mg/100 ml in 2N HC1, see Dawson et al. 1969) and 5N KOH. I. Cell collection and storage. When required, cultures were harvested towards the end of the exponential phase of growth. All one.liter Cultures^ were haves ted aseptically by centrifugation (Servall RC-2 model centrifuge) in autoclaved 250 ml centrifuge bottles at ca.. 6,000 rpm for 20 min. Then the cells were washed with the sterile algal culture medium to remove any residual aro• matic compound initially added to the medium. The medium was analyzed for aromatic products, and the pre-adapted cells were used for tracer experiments. The mass cultures were harvested by using the continuous flow system (Szent-Gyorgl and Blum) for the Servall RC-2 centrifuge. A speed of 10,000 rpm was maintained throughout the collection. The continuous flow system was rinsed at the end of the harvest with 3.0$ NaCl to remove excess seawater, then the cells were packed for another 20 min. To prevent cell lysis, the in- ternal temperature of the centrifuge was held between 17-21 C for harvesting all cultures. The collected cells from the mass cultures or from the C-tracer experiments were dried with P2O5 at room temp• erature in a desiccator which was continuously evacuated; by a high vacuum pump. After twelve hours, the P2O5 was changed and the desiccator was evacuated to 100-150 torr then stored at 0-Vc for several days during which time the P2O5 was changed until the cells were totally dry. The dried cell pellets were ground to a powder in a mortar and pestle, 0 weighed, and stored at -30 C in vials within a desiccator evacuated to 100-150 torr. These cell powders can be stored indefinitely under these conditions and were used for all 29. enzyme assays. The four mass cultures of .1* galbana grown on L-tyrosine were collected with the continuous flow system, but the steel tubes containing the cell pellets were stored frozen at -30°C, These cells were maintained at this temperature until extract• ed for the bromophenollc compound. II.. Chemical studies of aromatic compounds. A, Sources. The aromatic compounds used in this study were re• agent grade and were used without further purification. L- phenylalanlne and L-tyroslne were "gold label* products from Calbiochem, Los Angeles, California. p-Hydroxyphenylacetic acid was also obtained from Calbiochem. D-Phenylalanine and D-tyroslne were obtained from Aldrich Chemical Co., St. Louis, Missouri.. Phenylacetic acid was supplied by Nutritional Bio• chemical Corp., Cleveland, Ohio, whereas BDH Laboratories, Plalnview, N.Y. supplied DL-p-hydroxymandelic acid and Fisher- chemical Co., Fairlawn, N.J., supplied o-hydroxybenzoic acid (salicylic acid), Coumarin, DL-mandelic acid and p-hydroxy- benzoic acid were obtain from J. T. Baker Chemical Co., Phllllpsburg, N. J. The source of m-hydroxybenzolc acid was unknown, but It was a chromatographically homogeneous compound as were all the aromatics for metabolic studies. Phenol, obtained from Fisher Chemical Co., was re• distilled before use and p-hydroxybenzaldehyde, obtained from J,- T. Baker Chemical Co., was recrystalized. Both these 30. purified compounds were chromatographically homogeneous. All other aromatic compounds, used for as chroma• tographic standards, were obtained from various chemical com• panies or were synthesized whenever possible. The following bromophenol standards were supplied by Dr. J. S. Cralgle, N. R» C. Atlantic Regional Lab., Halifax, N.S, : 3,5-dibromo-^- hydroxybenzoic acid, 5-bromovanillin, 6-bromovanilTin, 3,5- dibromo-^-hydroxybenzyl methyl ether, 3-bromo-^,5-dihydroxy- benzaldehyde, 3.5-dibromo-^-hydroxybenzylalcohbl, and" 2,3- dibromo-^,5-dlhydroxybenzylalcohol.. B. Chromatography.. Paper chromatography employing Whatman No. 3MM paper, was used for two directional separation of phenolic acids In Ik the C-ring labelled L-tyrosine experiments. These chromato- grams were developed by descending solvent flow at room temp• erature. The .first direction (with the paper machining) was developed by using solvent system AiZ% aqueous formic acid (V/V) and the second direction by using solvent system Bi the organic upper layer of benzene: acetic acid: water (10:7*3r V/V/V). Thin layer chromatography (TLC), employing "0.5 mm Avicel TG-101 cellulose layers and solvent systems A and B, was used for most separations. Prepared chromatographic sheets (#1325^ cellulose with fluorescent indicator #6065) from Eastman Kodak Co., Rochester, N.Y. were used for the separation of Cr-C, aromatic compounds in solvent system C: lsopropanolt 6 1 Cone. NR4OH1 water (8:1:1, V/V/V). _ ' _ : Phenolic compounds were detected by spraying with diazotized p-nitroaniline reagent.or ferric chloride reagent • . . '.. '31. '• (see Appendix B), Aldehydes were detected with 2,4-dinltro- phenylhydrazine reagent (Appendix B) andacids by methyl red- bromothymol blue reagent (Appendix B). To aid ln identification of the various degradative products, spots were excised from the chromatograms, a UV-spectrum was obtained, and the compound was co-chromatographed with auth• entic standards. This was undertaken for both radioactive and non-radloactlve experiments. >. Gas liquid chromatography of aromatic acids was ac• complished by a Tracor 550 Gas Chromatograph with a column containing ]$% OV-1 on 80/100 mesh Chromosorb W-HP. Trimethyl- silyl derivatives of the acids were prepared with Tri-sil from Pierce Chemical Co., Rockford, 111. Temperature programming, increasing at 2°/minute from 90°C to 200°C, was used to separate the- :Siiylated-products' which • were detected by flame 1 ohization. C. Spectroscopy. Ultraviolet spectra were obtained using a Unlearn model SP .800 recording spectrophotometer. Nuclear magnetic resonance (NMR) spectra were obtained on a Varlan HA 100-100 •MHz spectrophotometer. Mass spectra (MS) were .obtained on a 1290G Nuclelde 90° Sector Instrument. Only the major character• istic MS peaks are presented with the relative peak Intensity ln brackets 1.. e. ( ) given as a percentage of the greatest peak. D. Melting points. Melting points were taken ln a Thomas Hoover Uni- melt Capillary melting point apparatus. The values obtained are uncorrected. 32. E. Chemical preparation of non-radloactlve compounds. In Appendix C, the methods for the synthesis of various aromatic compounds used in this investigation are presented. The methods used were based on previously reported synthetic routes. The aromatic compounds synthesised were,* 3• '5-dTbromo-p-hydroxybenzoic acid 3»5-dibromo-p-hydroxybenzaldehyde 3t5-dibromo-p-hydroxybenzylalcohol 3-bromo-p-hydroxybenzoic acid .p-hydroxyphenylacetaldehyde p-hydroxybenzoylformic acid phenylhydracry11c acid p-hydroxyphenylhydracrylic acid (unsuccessful) sulfate esters of phenolic compounds F-, Chemical preparations of radioactive compounds. In Appendix D, the methods for the synthesis of 14 C-labelled compounds, not commercially available, are presented. The compounds synthesised werei L-.t yr.os 1 ne-(U)-ri ng-llfC 14 p-hydroxyphenylacetic acld-1- C 14 p-.hydroxyphenylacetic acid-2- C 14 p-coumaric acld-2- C III. Isolation of products. In all cases the medium, with or without cells, was acid• ified to pH 2 with cone. HClf then extracted with redistilled peroxide free dlethylether. The extracts were washed with 0/5 N HC1 then allowed to evaporate to dryness at room temp• erature in a fume hood. The residue was redissolved in eth- .anol or ether for chromatographic spotting. In the extraction of I_. galbana cells for the bromo- phenolic compound B.bl gmwet cells were suspended in 100 ml 1.5 N HC1 then disrupted in a French press. This suspension -was extracted 'With ether and the resulting emulsion .was .centri• fuged to separate the phases. The phenolic acids ,ln the ether were extracted into 5% NaHCOy which was acidified and re- extracted with ether. This extract was evaporated to a small volume, banded on Eastman prepared cellulose plates with flu• orescent Indicator, and chromatographed in solvent system C. The band was scraped off and eluted with ethanol for UV and MS analysis. IV. Radioactive feeding experiments. Ik ' A. Source of C-lsotopes. From New England Nuclear, Boston, Mass. the -following lk lk C-compounds were purchased* L-tyrosine-1- C (specific ac- lk tivlty (SA), 58.2 uCi/mmole), DL-tyrosine-3- C (SA, 15.5 pCl/ lk , mmole), L-phenylalanlne-1- C (SA, 52.6 uCi/mmole)., DL-phenyl- \k lb alanine-3- C (SA, 1^.5 uCi/mmole), phenylacetic acld-2- mmole), and salicylic acid-7- C (SA, 2.3 uCi/mmole). The Radiochemical Center, Amersham, England supplied the following DL-tyrosine-2-l2|'C (SA, 50.0 uCi/mmole), L-tyrosine-U-^C (SA, (1) side chain. 10 juCi/mmole), DL-phenylalanine-2-iqC (SA, 32 juCl/mmole), L- phenylalanlne-U-^C (SA, ^92 juCi/mmole), and phenylacetic acid- l-^C (SA, 59 yuCi/mmole). p-Hydroxybenzoic acid-7-li*'C1(SA, 55#000 uCi/mmole) was obtained from Schwarz/Mann and cinnamie acld-2-1^C (SA, 1.4 juCi/mmole) from ICN Chemical and Radio• isotope Division, • lb ••: B. Preparation and administration of C-compounds. lb All the C-compounds used for metabolic studies were dissolved In distilled water, filter sterilized, and stored o frozen at -20 C until required. Fission of the aromatic ring of tyrosine. To a sterile Erlenmeyer flask fitted with a, center well, 1 uCl (20 pg) of L-tyrosine-uniformly ring-^C (SA, 9.3 uCi/ mmole) was-added aseptically outside the center well and 0.5 ml 10 N KOH with a filter paper wick (Whatman #1) for trapping C02 Inside the well. A 15 ml aliquot of a 2 week old culture was transferred aseptically outside the center well. The 0 flask was incubated stationary for 2 weeks at 19-21 C, under constant illumination (200-250 foot-candles). A second flask with the same contents was Incubated for the same period in continuous darkness. The contents of the center well were transferred to 15 ml scintillation fluid, and a fresh paper wick Impregnated with two drops of 10 N KOH was placed In the center well. The medium was acidified with two drops of $ N lb HC1 to release any remaining COg. After 2 hours of shaking, the paper wick was removed and placed in scintillation fluid along with all washings of the center well. The medium was (1) side chain. extract ed;.an^ for autoradiographic analysis. ' 14 . After the initial counting, the CO2 was regenerated and retrapped in cc-phenylethylamlne (PEA) using the apparatus in Figure 6. The scintillation fluid and paper wicks were placed ln the regeneration chamber (B) and nitrogen was pass• ed through the system such that the PEAtB^O ('3il, V/V) (G) rises Into the exchange column (H), and the bubbling effect was moderate but constant. Then 20 ml of 4 N R^SO^ was slovr- T4 ly injected Into the scintillation fluid releasing the CO2 which was carried across and into the PEA. Nitrogen was • passed through the system for 15 minutes, after which the PEA was drained from the column into the 20 ml test tube. The column was washed with 4 ml ethanol which was combined with the former PEA, mixed, and a 1 ml aliquot was removed for scintillation counting. Side chain degradation of phenylalanine and tyrosine. To sterile Erlenmeyer flasks with center wells, 2-uCi of the ^Crlabelled precursor (L-Phe-l-^C, DL-Phe-2-lIfC, DL- Phe-3-ll,'C, L-Tyr-l-^C, DL-Tyr^-^C, or DL-Tyr^-^C) was added aseptlcally along with enough sterile cold E-lsomer to give a final concentration of 0,05 mM. Each substrate was in• cubated in duplicate, under continuous illumination (280-210 foot-candles) or in continuous darkness for 6 and 12 hour periods at 19-21 C. The pre-adapted cells from one liter cultures (preadapted: on 0.1 mM of either L-phenylalanine or L-tyroslne - collection and washing of the cells was described under ."•Cell collection and storage*?. 27) were suspended in enou sterile algal culture medium to add 10 ml aliquots, of known 36. Figure 6. Diagram of apparatus for ^00% regeneration and retrapping. A, stream of nitrogen. B, regeneration 14 " chamber. Ct. C02-K0H-scintillation fluid. D, 30 ml syringe with needle. E, 4N H2S02j.. F, rubber stoppers. G, a-phenylethylamine and water (3*1) in a 20 ml test tube. H, 25 ml volumetric plpet containing glass beads. ceil number, aseptically to each flask. At the end of the incubation period, 2 drops of 5 N 14 HC1 were added to the medium to release any remaining CO^. After 6 hours of Intermittent shaking, the paper wick was re• moved and, along with all washings of the center well, was placed in scintillation fluid. After the initial counting, l4 •'' the CO2 was regenerated and retrapped for re-counting (method described above). The medium was extracted and chroma- tographed for autoradiographic analysis, 14 Degradation of other C-labelled substrates. The Erlenmeyer flasks fitted with the center well were used for experiments with the following radioactive compounds} phenylacetic acid-l-^C, phenylacetic acld^-^C, p- 14 hydroxyphenylacetlc acld-1- C , p-hydroxyphenylacetlc acid- 14 14 14 2- C, benzoic acid-uniformly ring- C, salicylic acld-1- C, Tit 14 and p-hydroxybenzolc acid-1- C. All these C-compounds, along with enough sterile cold compound to give in each case a final concentration of 0.05 mM, were added aseptical• ly to the pre-sterilized flasks. 10 ml aliquots containing a known number of non-adapted cells, concentrated from one liter cultures (see side chain degradation of Phe and Tyr) 14 were added aseptically to each flask. The C- phenylacetic . acids and ^C-p-hydroxyacetlc acids were Incubated for 6 and 12 hour periods in continuous darkness at 19-21° C while the . other acids were incubated for only a 12 hour period. The 14 14 termination, collection of C02, regeneration of the C02. (l) side chain labelled and extraction of the medium for chromatography was the same as described above ln the side chain degradation of phenyl• alanine and tyrosine. i h The degradation of cinnamic acid-2-• C and p- coumaric acid-2-^C was studied with pre-adapted cells. The cells were collected from a liter culture, washed (as described under 'cell collection and storage' p. 27), and resuspended in 5 ml algal culture medium. These cells were incubated for Zk hours under continuous illumination (280-310 foot-candles) o lh at 19-21 C with 2 uCi of either C-substrate ln a 50 ml Erlenmeyer screw capped flask. The incubation was terminated by centrifuging down the cells and decanting the supernatant. The cells were washed with fresh algal culture medium, cen• trifuged down, and the supernatent decanted. The cells were treated as described under 'cell collection and storage' p. 27 and the medium with the respective washing was acidified to pH 2 and extracted for chromatographic analysis. Uptake experiments with phenylalanine and tyrosine. Into pre-sterllized 50 ml Erlenmeyer flasks 2 ;uCi of Ik lh DL-phenylalanine-3- C or DL-tyrosine-3- C was added asep- tically along with enough L-lsomer of the respective amino acid to give a final concentration of 0.1 or 0.01 mM. With each concentration, 10 ml aliquots containing a known number of non-adapted cells were placed in two flasks, one to be in• cubated under continuous illumination (280-310 foot-candles) and the other in continuous darkness. Likewise, with another two flasks at the same concentration, 10 ml aliquots of pre- adapted cells were added aseptically. The pre-adapted cells 39. were collected from a-known volume of media containing 0,1 mM of-either L-phenylalanine" or L-tyrosine,washed, and re- suspended in the same volume of fresh algal culture medium for use in these uptake studies. From each flask, 1 ml samples were removed at 0, 10, 30, 60, 120, 240, 360, 5^0, and 720 minute intervals. Each sample was filtered using a pre-moistened (with algal culture medium) R Millipore HA (0.45-micron pore size) filter, gently sucked • dry, then washed with 5 nil algal culture medium, and again sucked dry. The filter was then placed in 15 ml scintillation fluid along with 1 drop 0.1 N HC1 to destroy the pigments. The samples were left 24 hours before being counted,, C. Detection of -^'C-products. Autoradiography. The developed chromatograms, dried overnight, were placed in light tight x-ray film exposure holders with a sheet of Kodak Blue Brand Medical X-ray film (Estar Base) for two to three weeks depending on the number of dpm(s) applied. At the end of the exposure period, the x-ray film was removed and de• veloped under a red-yellow safelight. The ^C-spots located by the autoradiograph were scraped from the TLC plates and placed directly into 15 ml scintillation fluid. Scintillation counting. The scintillation fluid utilized in all these studies consisted of 6 gm of PPO, 0,4 gm of P0P0P dissolved in 412 ml of toluene and 688 ml of ethanol. The samples were counted in a Nuclear-Chicago 720 series or Unllux II liquid • 40. scintillation spectrometer. Dual channel counting per• mitted calculation of efficiency from a quench curve prepared for each Instrument employing a series of variably quenched samples. The dpm values obtained for samples containing a g a known number of cells were expressed as dpm per 10 cells. V. Enzyme assays. A. Phenylalanine ammonla-lyase. (Young et al., 1966). To each tube, 10 mg cell powder (see 'cell collection and storage' for planktonlc species p. 27), 0.5 ml of K-Tricine buffer (0.2 M), and 0.4 ml distilled water were added. Each o tube was covered with Parafilm, then sonicated at 0-4 C for 5 minutes in a Raytheon 10-kcycle magnetostrictive oscillator at a maximum current output of 1.1 A. These sonicates were used directly for enzyme assays, without further treatment. Each reaction was initiated by adding 10 umoles of L-phenylalanine 14 containing 1 >uCi of L-phenylalanine-U- C (The Radiochemical Center, Amersham). At the end of the incubation period (2 hours at 30°C), the reaction was stopped with 1 ml 0.5 N HC1 then centrifuged at 5.000 rpm for 30 minutes. The supernatant was decanted and the pellet was washed with 0.5 N HC1. The supernatants were combined and extracted twice with 4 ml ether, then the aqueous phase was discarded. The ether was washed with 10 ml of 0.5 N HC1 and the ether was decanted Into a scintillation vial and evaporated to dryness at room temperature under a jet of nitrogen. The contents of the vial, after adding 15 ml scintillation fluid, was then counted,, 41,, B. Transaminase. A modification of the assay described by Dlamondstone (1966) was used. This was a fixed-time assay based on the alkali-catalyzed oxidation of p-hydroxyphenylpyruvlc acid or phenylpyruvic acid to respectively p-hydroxybenzaldehyde or benzaldehyde. The reaction mixture contained 5 umole of tyrosine or phenylalanine, 5 jumole of a keto acid (pyruvic, oxaloacetic, or a-ketoglutaric acid) and 0.2 umole of pyri- doxal phosphate in a final volume of 0.9 ml of 0.1 M-sodium phosphate buffer, pH 7.6. The enzyme was prepared as described under phenyl• alanine ammonla-lyase then centrifuged at 20,000 g for 20 minutes. The supernatent was passed through a 2 cm x 20 cm column of Sepadex G-25 (Pharmacia, Uppsala, Sweden),prepared with the 0.1 M-sodium phosphate buffer, pH 7.6, for removal of the low molecular weight molecules. 12,5 ml of eluate was collected and 0.3 ml aliquots were used in the enzyme assay. All procedures were performed at 0-5°C until the assay was begun. o After a 2 hour Incubation at 30 C, the reaction was stopped by addition of 0.1 ml of 10 N KOH with continuous agitation. The reaction mixtures were left at room temp• erature for 30 minutes and then the absorbance was read against a blank at 331 nm. The amount of product formed was calculated from a molar extinction coefficient of 19,900 M 1 -1 cm •• C. p-Hydroxybenzoate hydroxylase. A crude enzyme preparation (0.2 ml of sonicated cells) + was Incubated with: NADP f 0.5 jumolej- glucose-6-phosphate, 5.0 umolej glucose-6-phosphate dehydrogenase, 0.1 ml of a stock diluted 25 fold; p-hydroxybenzolc acid, 0.3 umole; in a total volume of 1.0 ml at pH 7.2, Including 0.17 ml of 0.1 M-sodlum o phosphate buffer. The reaction mixture was incubated at 30 C for one hour and terminated by the addition of 1 ml of 0.5 N HC1, The dihydroxy-product was measured by_ the method described by Arnow (1937) for 3,4-dlhydroxyphenylalanine (D0PA). To the 2 ml solution (1 ml reaction mixture +1 ml 0.5 N HC1), 1 ml of nitrite-molybdate reagent (10 gm sodium nitrite and 10 gm sodium molybdate in 100 ml), and 1 ml 1 N sodium hydroxide solution was added, mixing well after each addition. The samples were read at 510 nm. D. Protein determination. The method of Lowry et al. (1951) was used for the determination of protein. The supernatant from sonicated cells grown on nitrate or phenylalanine was utilized after removal of cell debris by centrifugation.. Bovine serum albumin (frac• tion V, Sigma Chemical Co., St. Louis, Mo., U.S.A.) was used as the standard.. •*3. RESULTS I. Culturing.. A.. The effect of phenylalanine and tyrosine. Under optimal growth conditions (mass culturing), the addition of either of these amino acids at 1.0 mM had no effect on the growth of, N. lncerta (Table 8),. while the small differences observed for Ij. galbana were probably not significant. Data from one liter cultures Indicated that these amino acids reduced the cell yield for 1^ galbana (Table 9) with only a slight decrease in the growth constants. Phenylalanine not only reduced the growth constant but also the cell yield for i, lncerta (Table 10), while tyrosine was stimulatory, Increasing both the cell yield and the growth constant. The feeding of L-phenylalanlne or L-tyroslne over the concentration range of 0.025 to 2.0 mM had no effect on the growth constant or lag period of N. lncerta (Figures 7 and 8 ). Similarily L-phenylalanine had no effect on either of these growth parameters of Ij galbana (Figure 7 ) while L-tyrosine was stimulatory at lower concentrations and in• hibitory at higher concentrations (Figure 8 ). Generally, ln the presence of nitrate, neither amino acid exhibited . any consistent influence over the growth of either algal species. When nitrate was. omitted from the culture medium, TABLE § Growth Constants and Cell Yields from Mass Cultures .Growth Mean gener- Cell count Dry cell Size of Algal species constant ; atIon time at harvest yield culture and (tg) (£(io)> Aromatic addition 6 -i hr xlO /ml mg/llter liters hr culture Isochrysls galbana (1) (1) (1) Nitrate 0.012 (i) 24.5 13.3 ' 178 2 (3 +• L-tyroslne^ ^ 0.009 33.4 15.2 NB > 4x5 (?) + L-phenylalanlnev ': 0.015 20.1 ND ND 2x5 Navlcula lncerta Nitrate 0.040 7.53 9.0 185 2x5 (2) + L-tyroslnev ' 0.039 7.72 8.6 l6l 2x5 (?) + L-phenylalanlneVA 0.039 7.72 11.5 196 2x5 (1) value from Antia and Kalmakoff, 1965. (2) concentration of 1.0 mM. (3) ND-not determined. TABLE 9 .The Effect of Aromatic Compounds on the Growth Constants and Cell Yields from One Liter Cultures of Isochrysls galbana •Growth Mean gener• Cell count Dry cell constant ation time at harvest yield Aromatic..« (tg) Percentage i; yield . • addition* Nitrate 0.016? 18.0 3.38 117 0.0 + L-tyroslne 0.0166 18.1 3.32 99 15.5 + L-phenylalahtne 0.0160 18.8 3.01 89 24.0 + t-clnnamate 0.0159 18.9 2.56 71 39.5 + phenylacetate .0.0167 18.0 2.80 87 25.5 + benzoate O.OI70 17.7 3.01 94 19.5 + ,p-hydroxybenzoate 0.0162 18.6 2.91 97 17.0 + t-p-obumarate4^ 0.0141 21.4 1.50 68 42.0 (1) concentration .0.1.0 mM. (2) based on dry cell yield and using nitrate yield as reference. (3) concentration 0.05 mM. TABLE 10 The Effect of Aromatic Compounds on the Growth Constants•and Cell Yields from One Liter Cultures of Navlcula lncerta Growth Mean gener• Cell count Dry cell constant ation time at harvest yield Aromatic, . (tg) Percentage 11 yield ... addition ' 6 OL(io)> hr xl0 /al mg/llter reduction*" culture hr"1 Kltrate 0.0200 15.1 2.03 191 0.0 + L-tyroslne 0.0202 14.9 2.47 219 +15.0<3> + L-phenylalanlne 0.0186 16.2 1.61 129 32.5 + t-elnnamate 0.0173 17.4 0.78 59 69.I + phenylacetate 0.0194 15.5 1.71 166 13.0 + benzoate 0.0199 15.1 2.13 191 0.0 + p-hydroxybenzoate 0.0193 15.6 1.13 88 5^.0 + t-p-ooumarateC^ 0.0195 15.* 2.26 188 1.5 (1) concentration 0.10 mM. (2) based on dry cell yield and using nitrate yield as reference. (3) percentage stimulation. (4) concentration 0.05 nd. •. 4 ( o o 1 cn -r~ 1: PERIOD PERIOD (days) (days) LAG LAG —r (• ) (• ) I-l \ (x—x) (x—x) I / cn p / 13- ,0 p /I .A o ) (AOD/day incerta (AOD/day) incerta 3 \ T GROWTH CONSTAN GROWTH CONSTANT o o CP o o Navicula Navicula Isochrysis galbana Isochrysis galbana o o I / I ! x / \ • t constan constant INHIBITON of T—1—1—r o cn o cn N of INHIBITO O D O O O - Vo V.. growth growth £3 o. Oi In fc> SS x, *. ro "cn cn cn % 0 % ^ >0 1—1 o O O ° 1—' 6% m o O Oi 1 » So CI• S- CD CD 0> 1 a o tr a cr ca !~» -•> o 1 P 3- Ct 3 P o >-» • 3 r-> a! 3 3 CD •1 cr cr CD cr 3" O O CD 3 3- CO 3 E cr CD (X) 0 cr 1 0 r* 3" O VJ ca f •o O cr 0 CD CD 3" CD •"-*> CN cr P ff a •-» CR •I P 0 h* Figur e 00 • Figur e « 3 p. 0 SO CO >i o> M a o 3 01 cr P 3 cr io H o o 3" 01 O o "> o P (-» 0 8" *! P P P- 3 P M 3 p O- P 3 3 P. H* CO n H" 1-1 01 9) T3 h-i 01 O O P. "1 •t •* O O P CD P^ 3: cr p. ca ct- 3: 0 0 3 x> 3 P X~ x ^ ~~ ~~ — - * 0.3 N. lncerta G c NO3 (control) o--o L-Phe (0.50 mK) + NO3 X--X L-Phe (0.50 mM) - NO3 0.2 h • • L-Phe (0.05 mM) - NO3 OPTICAL DENSITY 0.1 h 0.0 Figure 9. Growth curves of Navlcula lncerta on nitrate and L-phenylalanine with and without nitrate. / G 0.3 N. lncerta e—e NO-J (control) o—o L-Tyr (0.50 mM) + NO3 0.2 X x L-Tyr (0.50 mM) - NO3 OPTICAL —• L-Tyr (0.05 mM) - MO 3 DENSITY 0.1 0.0 J 1 1 I l_ -I I • ' 0 8 12 16 20 24 DAYS Figure 10. Growth curves of Navlcula lncerta on nitrate and L-tyroslne with and without nitrate. 49. several distinct patterns were observed. N* lncerta could utilize both amino acids as the sole N-source with no change in the lag period from nitrate grown cells (Figures 9 and 10). The growth constants were approximately one half of the control value (Figures 11 and 12), indicating that both amino acids were not as efficient as a nitrogen source as was nitrate. The lag period was the same on nitrate as it was: on both amino acids.- When cells were removed from the 0.5 mM tube and inoculated into another tube with the same concentration of amino acid, very little difference was measured in the growth parameters. Growth constant Lag period (hr"1) (days) L-phenylalanine 1st transfer 0.085 3.6 2nd transfer 0.080 3.8 L-tyrosine 1st transfer 0.067 3.6 2nd transfer 0.05? 3.5 This confirmed that the enzymes required for the amino nitrogen utilization were present and no enzymes were in• duced. is galbana. on the other hand, utilized the higher concentrations of phenylalanine as efficiently as nitrate (Figure 11), although as the concentration decreased, the growth constant also decreased. When the optical density data was plotted (Figure 13). it became evident that there was a four day lag period beyond that observed for cells grown on nitrate. An inoculum from the 0.5. mM .tube (only L-phenylalanine) when used to initiate a similar tube gave no change in growth constant but the lag period was extended 3.0 a control ao O o 16 -Ji t—X: control i—i -X K ' *- 1—4 3A UJ «n 2.5 rr a- m 2.0 control < control UJ 2.0 < 0.022 _ 0.021 Q017 ] 0.020 1/5 control 1/1 — roc 0.01 9 8^ 0.15 8^ •v con_t_rql_ Q 0.016 o 0.125 £3 0.018 -# x x— v w *- * I O au I o t\J t<3 control cr control Ul 0.017 fr or o 0.015 o &0.01 6 u o 0.015 o ~ o - + 20 ~Z- 3 2: fi o*— c„ + 5 o ,o + 10. 'K- V -V. < / til o 0 •—»- 0 • X x- z £ 5 2 5 10 10 20 5 5 3 3 2.5X10 5x10 10"'* 2.5x# 5x10*' 10 2.0x1a 2.5 xlO5 5x105 10"'' 2.5X104 ExTO*1 10"3 2OX103 CONCENTRATION (M) CONCENTRATION (M) Figure 7. Diagram of the effects of L-phenylalanlne on the growth Figure 8. Diagram of the effects of L-tyroslne on the growth constant and lag-period of Isochrysls galbana and constant and lag period of Isochrysls galbana and Navlcula lncerta. O Navlcula lncerta. I. galbana C G NO3 (control) o o L-Phe (0.50 mM) + NO3 X—-X L-Phe (0.50 mM) - NO3 • • L-Phe (0.05 mM) - NO3 0.3 0.2 OPTICAL DENSITY 0.1 0.0 Figure 13. Growth curves of Isochrysls galbana on nitrate and L-phenylalanine with and without nitrate. 52. I. galbana G C NO3 (control) o—o L-Tyr (0.50 QM) + NO3 X—-X L-Tyr (0.50 mM) - NO3 • L-Tyr (0.05 mM) - NO> a—-a L-Tyr (2.0 mM) - N0- 0.3 0.2 OPTICAL DENSITY 0.1 0.0 Figure 1 ¥. Growth curves of Isochrysls galbana on nitrate and L-tyroslne with and without nitrate. 53 to about 12 days. A similar observation was reported by Antia et al. (1975) for growth on glycine. Growth of Xi galbana on L-tyrosine as the sole U- source was very poor. The growth constants were always less than the nitrate control (Figure 12) and a very definite re• duction in growth yield was obtained (Figure 14). -..-As the concentration of L-tyrosine increased the cell yield or op• tical density maximum also decreased. Also from the optical density data (Figure 14), it became evident that there was a four day lag period beyond that observed for cells grown on nitrate. When an inoculum was removed from the 0.5 mM tube and used to initiate growth ln a fresh tube containing the same concentration of tyrosine no growth was observed sugr'. g|s|^ng^^ In an attempt to reverse the Inhibition of I. galbana by L-tyrosine, two different concentrations of L-tyrosine with L-phenylalanlne (at one half the tyrosine concentration) were used. When cells with no prior exposure to either amino acid were used, a lag period of four days beyond that ob• served for cells grown -on nitrate was observed but no change in the growth yield was- obtained (Figure 15)» That suggested that L-tyrosine or one of it's metabolic products was: toxic* Growth of Isochrysls on nitrate in the presence of L-tyrosine^. (Figure 14) was normal which Indicated that one of L-tyrosine*s metabolic products caused the growth inhibition, (l) L-tyrosine was assimilated in the presence of nitrate. 54. oris G—G NO^ (control) X---X L-Tyr (0.50 mM)'- NO^ • ' L-Tyr (0.50 mM) + L-Phe 0.10 (0.25 mM) - NO, OPTICAL DENSITY 0.05 -XL. 0.00 \%~%^X-X-X< _i t 0 12 16 20 2U 0.15 1 C G NO^ (control) X—-X L-Tyr (1.0 mM) - NO^ • • L-Tyr (1.0 mM) + L-Phe 0.10 (0.5 mM) - NO3 OPTICAL DENSITY 0.05 / J L QjQjfr-X-x-x aoo 1 I- • I 1 1— 0 8 12 16 20 24 DAYS Figure 15, The effect of L-tyroslne with and without L-phenyl• alanlne on the growth yields of Isochrysls galbana. Neither D-phenylalanlne nor D—tyrosine were utilized "by I. galbana as the sole N-source. No growth was detected over the concentration range of 0„025-2,0 mM for a period :of at least forty days. IJ. lncerta. however was able to utilize both D-amino acids, but only after a long lag period"(Figures l6 and 17). After bO days, growth on 0.5 mM D-phenylalanlne was 78% of that observed on L-phenylalanine (Figure 9)* whereas on 0.5 mM D-tyrosine,. growth was bQ% of that observed on L-tyroslne (Figure 10) and was still increasing (Figure 17). The lag period appeared to be dependent on the concen• tration of the D-isomer, the greater the concentration the shorter the lag period (Figures 16, 17f and 18). The growth constants were also reduced from that observed for nitrate •and as the concentration decreased so did the:growth constant. In all cases, the cells grown on the D-isomers were light brown in pigmentation unlike cells grown on nitrate or the L-isomers which were dark brown. The difference In pigmentation and decreased growth constants indicated the D-amlno acids were a poor source of nitrogen for N. lncerta. Both species when grown In the presence of L-tyrosine released an ether insoluble brown coloured material Into the medium. This occurred not only with mass cultures and one liter cultures but also In the OD experiments. This appear• ance of browning occurred at the end of the exponential phase, approximately after twenty-two days of growth for I. galbana (Figure lb) and seven days for N. lncerta (Figure 10}:. No Figure 16". Growth curves of Navlcula lncerta on nitrate and.D-phenylalanine. £ £. control (nitrate) • —• 2©0 mM D-phenylalanlne 0.2 + + 0.5 mM D-phenylalanlne X x 0.25 mM D-phenylalanine A A 0.1 mM D-phenylalanlne OPTICAL o- o .0.025 mM D-phenylalanlne / DENSITY / 0.1 •0.0 :-«4r-f-t>-4-+-^-x-'-o-<>-+-«rr:— 0 12 18 24 30 36 DAYS Figure 17. Growth curves of Navlcula lncerta on nitrate and D-tyroslne. G £ control (nitrate) .— _. 2.0 mM D-tyroslne + • + 0.5 mM D-tyroslne 0.2 X- x 0,25 mM D-tyroslne A A 0.1 mM D-tyroslne o o 0.025 mM D-tyroslne OPTICAL DENSITY 0.1 0.0 DAYS 26.0 2U.0 o o 22.0 20.0 1—4 CC — UJ 16.0 TO 18.0 < o . U.0 12.0 A— control (NCh) 3.2 3.2 control (N03) 0.091 0.091 0.009 ^ 0.017 o 0.008 I 0016 to — y >~ 0.007 c 8^ o I o £ 0.006 ^ 0.006 e o.oo5 g 0.005 or -C o S 0.00/, A 0.00/, 0.003 0.003 0.002 Q002 20 o o ^- o IT) K 15 10 10 I o 5 5 or 0 0 o 2.5X105 5x105 104 2.5x# 5x10'* 10"3 2Dx1(? CONCENTRATION (M) Figure 18. Diagram of the effects of D-phenylalanlne and D-tyroslne on the growth constant and lag period of Navlcula lncerta.. - 59. colouration occurred with growth of either species on L- phenylalanine. The identity of this coloured substance is unknown. The volatile product(s) reported by Vose et al.(1971) were not detected in in vivo experiments with I, galbana mass cultures. No aromatic compounds were trapped in the KOH and no aldehydes or ketones were detected in the 2,4-dinitro- phenylhydrazine. This suggests that possibly the volatile product(s) was only released upon acidification of the medium, thus it would not. be released under the conditions of these two trapping experiments. B. Metabolism of phenylalanine and tyrosine. When the medium, totally devoid of cells, was ex• tracted with ether and the ether chromatographed, very few aromatlcs were detectedi For both species the only phenolic :compound observed:.from both the phenylalanine and tyrosine media was p-hydroxybenzoic acids An acid, positive area for benzolc-phenylacetlc acids was also observed from the phenylalanine medium. No other compounds were detected. Extraction of the cells from growth on both amino acids revealed p-hydroxybenzolc acid. From the tyrosine grown cells, p-hydroxyphenylacetlc acid and traces of both p-hydroxybenzylalcohol and 3-bromo-p-hydroxybenzoic acid were also detected for both species. Acid positive areas from phenylalanine grown cells were observed not only for the benzoic-phenylacetic acid area, but also for phenyllactic- phenylpyruvic acid area. For both species, the major com• pound present from growth on tyrosine was p-hydroxybenzoic acid, and for phenylalanine, benzoic and/or phenylacetic acid. At no time was p-coumaric acid or indications of the presence of cinnamic acid ever observed. The aromatics produced from phenylalanine and tyro• sine metabolism were identified mainly by Rf positions and co-chromatography with authentic samples. When possible, UV spectra were obtained for comparison with known spectra. The colour reaction of the phenolic acids with diazotized p-nltroanlline (PNA) aided in identification as did gas chromatographic analysis of the non-phenolic acids. In al• most all cases, so little product was obtained that further chemical characterization was impossible. The bromophenollc compound from I_. galbana was tentatively identified as 3-bromo-p-hydroxybenzoic acid. In solvent system C, p-hydroxybenzolc acid had an R^ of 0,36 while 3-bromo-p-hydroxybenzoic acid had an Rf of 0o.l5« Spraying with PNA, 3-bromo-p-hydroxybenzoic acid turned salmon-red in colour while both 3,5-dibromo-p-hydroxybenzolc acid (Rf of 0.18) and p-hydroxybenzolc acid reacted to give red coloured spots. The major peak in the UV spectrum (Figure 19) of the isolated 3-bromo-p-hydroxybenzoic acid corresponded to that of p-hydroxybenzoic acid while the fine structure, also in the spectrum, was very similar to. 3,5-dlbromo-p-hydroxybenzoic acid (Figure 36J and to the im• pure synthetic sample of 3-bromo-p-hydroxybenzoic acid. The great Instability of the Isolated 3-bromo-p-hydroxyben- zoic acid resulted in failure at further attempts to fully Figure 19. Absorption spectrum in ethanol of the phenolic acid tentatively identified as 3-bro.mo-p-hydroxybenzoic acid 1.6 isolated from Isochrysis galbana and Navicula incerta. 12 OPTICAL DENSITY 0.8 OA 0.0 200 225 250 275 300 325 350 WAVELENGTH (nm) characterize and confirm it's structure. C. The effect and metabolism of other aromatic compounds. In Table 11, a summary of the effect of the various aromatic compounds on the growth of I. galbana and N. lncerta is presented. The detailed effects of each aromatic compound on the growth constant and lag period are presented for refer• ence in Appendix E, The aromatic products detected from the metabolism of each aromatic substrate are also presented in Table 11. A general chromatographic map of the locations of the various aromatic compounds is presented for reference in Appendi B. For all but p-hydroxybenzaldehyde and p-hydroxymandelic acid, the aromatic compounds which were added to cell cultures caused no reduction in cell yield. Where growth inhibition had occurred, the maximum cell numbers were always obtained after an extended incubation period. D. Results of radioactive tracers. - Uptake of phenylalanine and tyrosine. In Figures 20 to 27 the time course uptake of DL-tyro- sine-3--^C and DL-phenylalanlne-3-^C at an initial concentra• tion of 10 and 100 juM are presented. Both algal species were capable of uptake of both amino acids, but great variations existed in their ability to do so. The uptake rates for both these amino acids were calculated (Table 12) and were based o on 10 cells per ml. In these calculations, the D-isomer com• ponent of either amino acid was disregarded. At the concen• tration levels utilized ln these studies, neither species was able to achieve more than a thirty percent uptake of the TABLE 11 A summary of the Effect and Metabolism of Aromatic Effect on Details compound growth ln constant1 2 Phenylacetic H.C. inhibitory 41 acid L.C. stimulatory Tables 9 and 10 p-Hydroxyphenyl- H.C. Inhibitory acetlc acid L.C. no effect 42 DL-Mandelic H.C. mildly Inhibitory 43 acid L.C. no effect DL-p-Hydroxy- H.C. > L.C. 44 mandellc acid Inhibition decreased Benzoic acid H.C. Inhibitory 45 L.C. stimulatory Tables 9 and 10 p-Hydroxybenzolc H.C. inhibitory k6 acid L.C. no effect Tables 9 and 10 p-Hydroxy- H.C. Inhibitory 47 benzaldehyde L.C. no effect 3,5-Dlbromo-p- H.C. Inhibitory US hydroxybenzolc L.C. no effect acid 3,5-Dlbromo-p- H.C. very inhibitory 49 hydroxybenz- L.C. no effect aldehyde Cinnamic acid H.C. Inhibitory 50 L.C. stimulatory p-Coumarlc acid H.C. no effect 51 L.C. stimulatory m-Hyd roxybonzolc H.C. mildly inhibitory 53 acid L.C. no effect o-Hydroxybenzoic H.C. Inhibitory 54 acid L.C. stimulatory (1) H.C. = high concentration 0.25-2.0 mM, L.C. = low (2) In Appendix E. o (3) TLC = thin layer chromatography j GLC = gas liquid benzoic acldj m-OHBA = m-hydroxybenzolc acid; benzaldehyde? p-OHPAA = p-hydroxyphenylacetic acid) PHCA = phenylhydracryllo acid. TABLE 11 (oont.) 63. Other Aromatic Compounds on Isochrysls galbana and Navlcula lncerta Chromotographlc results - Other comments (compounds detected)"^ TLC.GLC -presence of PAA suggested that It was poorly PAA,BA,p-OHBA metabolized. TLC -presence of p-OHPAA suggested that it was poorly p-OHPAA,p-OHBA,p-OHBAlc metabolized. TLC.GLC -no DL-mandelic acid was detected which suggested BA,p-OHBA that both isomers were metabolized. TLC -reduced cell yield for X» galbana at 1 & 2 mM. • -no DL-p-hydroxymandelic acid was detected-'which - suggested that both isomers were metabolized. p-OHBA,p-OHBAlc -presence of BA suggested It was poorly metabolized, -no o-OHBA or any dlhydroxy phenolic acids were TLC detected for either species. p-OHBA,m-OHBA 3-Br-p-OHBA, BA -UV-spectrum of 3-Br-p-OHBA identical to that in TLC Figure 19. p-OHBA.3-Br-p-OHBA -no dlhydroxy phenolic acids were detected, -medium turned brown (similar to growth on tyrosine) (colour was ether insoluble). -extremely toxic for I_i galbana at H.C. p-OH BA,3-Br-p-OHBA -no adaptation to this aldehyde when I. galbana was p-OHBAlc subcultured.' -medium turned brown (including a control with no cells) therefore this aldehyde was photochemically unstable. -brown colour was not extractable into ether. TLC -medium turned brown therefore photochemically unstable, nothing detected -colour was not extractable into ether. TLC -medium turned brown therefore photochemically -unstable, nothing detected -colour not extractable into ether. TLC.GLC -spectrum of PHCA identical to spectrum of synthetic PHCA.BA,p-OHBA sample (Figure 1*0). TLC -purple spot tentatively identified as p-hydroxyphenyl- Cis & trans-p-CA, hydracrylic acid, (see Appendix E). p-OHBAld,p-OHBA, purple spot not chromatographed • .«——• not chromatographed concentration 0.025-0.10 mM. chromatography! BA = benzoic acldj PAA = phenylacetlc acidi p-hydroxy- o-OHBA = o-hydroxybenzolc acldj p-OHBALo = p-hydroxybenzylalcoholj p-OHBAld = p-hydroxy 3-Br-p-0HBA = 3-bromo-p-hydroxybenzoic acldj p-CA = p-coumarlc acldt 3000 o—o non-adapted cells, Illuminated o--o non-adapted cells, Illuminated •—• non-adapted cells, no Illumination non-adapted cells, no Illumination A—A pre-adapted cells, Illuminated A—A pre-adapted cells. Illuminated A—A 2.500 pre-adapted cells, no Illumination 1)500 pre-adapted cells, no Illumination 1400 2,000 1,200 DPM PER / o 107 CELLS 1,000 1,500 DPM PER 107 CELLS 800 1J00O 600 400 200 6 8 10 12 HOURS Figure 20. Time course of uptake of DL-phenylalanlne-3- Figure 21, Time course of uptake of DL-phenylalanlne-3-A^C for Isochry3l3 galbana. The initial phenylalanine for Isochrysls a;albana. The Initial phenylalanine concentration Has 10 ;iM, concentration, was O.JQ mH. ON TABLE 12 Uptake Hates of Phenylalanine and Tyrosine by Isochrysls galbana and Navlcula lncerta at Two Substrate Concentrations Uptake Hates I*. galbana Ex lncerta (2) L-phenylalanlne 10 uM 100 uM ' 10 uM 100 uM Non-adapted cells. Illuminated 0.020 0.07 10.8 Non-adapted cells, no Illumination 0.010 0.05(^ 2.6 9.1 Pre-adapted cells, illuminated 0.003 4.4 10.0 Pre-adapted cells, no Illumination 0.003 0.05 4.6 8.9 . (2) L-tyroslne 10 uM 100 uM 10 uM 100 uM Non-adapted cells, Illuminated 0.014 0.10 34.0 Non-adapted cells, no Illumination 0.004 0.06 5.1 31.5 Pre-adapted cells. Illuminated 0.005 0.12 13.5 Pre-adapted cells, no Illumination 0.004 0.03 9.>> 8.1 (1) jumoles/hour/10'' cells. (2) based on the assumption that the L-isomer was selectively assimilated prior to the D-lsomer (see text). „ (3) averaged over 12 hours the rate was 0.11 umoles/hour/10' cells. (4) if averaged over 12 hours, the value was comparable to the others at this concentration* _ ON 100000 8QQ00 60J300 DPM PER 107 CELLS 40000 0--0 non-adapted cells, Illuminated 20,000 •—• non-adapted cells, no illumination A—A pre-adapted oells, Illuminated pre-adapted cells, no Illumination _i i i_ 10 12 (HOURS Figure 22. Time course of uptake of DL-phenylalanlne-3-tTC for Navlcula lncerta. The Initial phenylalanine concentration was 10 uJii 1 available L-amlno acid. Phenylalanine was assimilated by JE. galbana at both 10 and 100 uM concentrations (Figures 20 and 21). At both these concentrations, illumination enhanced the uptake rates (Figures 20 and 21, Table 12) regardless whether or not cells had been pre-exposed to phenylalanine. At 10 uM substrate level, non-adapted cells appeared to accumulate phenylalanine more rapidly than pre-adapted cells, but. this did not appear to be true at 100 uM. This observation may have been the result of pre-adapting the cells with 100 uM of L-phenylalanine which resulted in an internal pool of phenylalanine. This suggested that I± galbana had the cap• acity to accumulate phenylalanine against a concentration gradient. The time course uptake of phenylalanine by N. lncerta Is presented in Figures 22 and 23. At least a 100- fold increase ln uptake rates was evident when compared to Isochrysls- (Table 12).. At both concentration levels, an initial rapid uptake occurred followed by a decrease in rate This was especially evident at the 10 uM concentration where the "levelling off" occurred with about 7-10% of the avail• able L-phenylalanine assimilated. The variation between 2-12 hours may have been the result of N. lncerta* s ability to adhere to the walls of the flask which resulted in a lower cell number than expected (thus a lower number of DPM) (only in these uptake experiments were the adhering cells of Navlcula not scraped from the walls of the experimental 67. vessel). At both concentrations, except for pre-adapted cells in 10 uM phenylalanine- C, light appeared to enhance the uptake rates (Figures 22 and 23, Table 12). The uptake of tyrosine by I_. galbana was comparable to that observed for phenylalanine (Table 12). In Figures 2k and 25 the time course uptake of tyrosine is presented. Similar to the phenylalanine results, light also enhanced the uptake of tyrosine at both concentration levels. Un• like the results with phenylalanine, non-adapted cells with no illumination appeared to have difficulty assimilating tyrosine at the 10 ;uM level (Figure 2k, Table 12). For both tyrosine and phenylalanine pre-adapted cells of Isochrysls appeared to be able to assimilate both these amino acids more efficiently at 100 ;uM whereas non-adapted cells were more efficient at 10 uM, The assimilation of tyrosine by N_, lncerta is presented "in Figures 26 and 27. The uptake rates were comparable to phenylalanine (Table 12), but non-adapted cells at 100 uM had a 3-fold greater rate. A 3-fold dif• ference in rate was also observed between the pre-adapted and non-adapted cells on L-tyroslne at 100 uM. This was probably the result of pre-adapted cells containing a large internal pool of tyrosine. At 100 uM light appeared to stimulate uptake (Figure 27), but this effect was only ob• served over the first eight hours of assimilation. Light also o—o non-adapted cells, Illuminated •—• non-adapted cells, no Illumination o—o non-adapted cells, Illuminated 1,800 A-—zV pre-adapted cells, illuminated /a •—• non-adapted cells, no illumination s • A-—A pre-adapted cells, Illuminated A—A pre-adapted cells, no Illumination / / 1£00 1,400 1200 1000 DPM PER 107 CELLS 800 600 400 200 HOURS it Figure 25. time cctirse of uptake of DL-tyrdslne-3-A C for Figure Zl*. time course of uptake of DL-tyroslne-S-^C for Isochrysls galbana; The initial tyrosine concen• isochryais galbana. The Initial tyrosine concen• tration »a3 0;10 mM; tration was 10 uM. o—o non-adapted cells, Illuminated non-adapted cells, no illumination 180,000 A—A pre-adapted cells, illuminated ? A—A pre-adapted cells, no Illumination 160,000 uqooo 120,000 100,000 DPM PER 107 CELLS 80,000 .60,000 40,000 20,000 6 HOURS HOURS 1 Figure 26. Time course of uptake of DL-tyroslne-3-^C for Figure 27. Time course of uptake of DL-tyroslne-3- C for Navlcula lncerta. The Initial tyrosine concen• Navlcula lncerta. The Initial tyrosine concen• tration was 10 JJH. tration was 0.10 mK. stimulated tyrosine uptake at 10 uM but only for non-adapted cells (Figure 26). The pre-adapted cells at 10 uM for tyro• sine (Figure 26) and for phenylalanine (Figure 22) were not stimulated by illumination. Why the uptake rates in con• tinuous darkness were greater than under continuous illum• ination was not resolved. The "levelling off" observed in the uptake of tyrosine at 10 uM (Figure 26) occurred when 10-13# of the available L-tyrosine had been assimilated. The variation in curves was probably the result of cells --• . adhering to the walls of the flask. In all these uptake studies no corrections were made lk for the metabolism of either amino acid. The loss of C02 and possible excretion of products into the medium were dis• regarded. Both were possibly significant after twelve hours lk of amino acid uptake. The error due to the loss of C02 was minimized under illumination due to photosynthetic fixation of COg. It is unknown if the greater uptake rates In the light were related to this fixation of QQ^ or to the greater provision of energy for amino acid transport. In the uptake experiments, no account of the Re• label going into an internal pool or into proteins was made. After uptake of both amino acids for one hour, the total Internal radioactivity'1*was generally larger for non-adapted cells than fqr pre-adapted cells of both species when cell volume and number were taken into account. The Internal radioactivity was also greater when the cells of both species were Illuminated. (l) in the soluble and insoluble pools. 71. The ability of both algal species to concentrate both amino acids and the stimulatory effect of light, le, increased uptake rates and generally larger concentration ratios, was very suggestive of active transport. No in• hibitors of photophosphorylation and, mainly, oxidative phosphorylation were examined in relation to amino acid uptake to confirm this suggestion. No other parameters of uptake were examined. The catabollc fission of the aromatic ring of tyrosine. When axenic cultures of nine planktonlc species, from three divisions, were Incubated 2-weeks with uniformly C-ring-labelled L-tyrosine, all species were capable of ring cleavage to produce "^CC^ (Table 13);. Whether in con• tinuous darkness or under constant illumination each species retained this capability. Unlike the results reported for the degradation of phenylalanine (Table 3)» U» lutherl. C, huxleyl-. and S. costatum were able to degrade tyrosine to COg. The presence of a p-hydroxyl group on the aromatic ring appeared to facilitate ring cleavage. Analysis of the medium from these experiments sug• gested a possible route for degradation of the carbon skel• eton of tyrosine.. For each species, both p-hydroxyphenyl- acetic and p-hydroxybenzoic acids were detected (Table 1^), p-Coumaric acid was not detected in any species examined. The presence of p-hydroxyphenylacetlc acid suggested a C^- fragment was initially removed, but if a C2-fragment was 72. TABLE 13 Total ^C02 Measured as Product of Catabolism from 2-weeks1 14 Incubation of Algae with uniformly Ring-labelled C-tyrosine Light Dark 14 _3 Algal species C02, dpm x 10 Haptophyta, Isochrysl-s-galbana- 11.1 (0.50$)* 3.1 (0.14$) Konochrysis lutherl 4.0 (0.18$) 8.7. (0.3.9$) Coccollthus huxleyi 56.0 (2.52$) 76.8 (3.46$) Bac1llarlophyta Penr.ate diatom Navlcula Incerta 82.1 (3.70$) 12.4 (0*56$) Centric diatom Skeletonema costatum 2.5 (0.11$) 5.6 (0.25$) Cryptophyta Rhodomonas lens 49.8 (2.24$) 71.1 (3.20$) •Figures in parentheses give data as $ of added C-tyrosine. TABLE 14 14 Radioactivity In Products Isolated from Uniformly Ring-labelled C-tyroslne Feedings (1) p-OHB P-OH0AO p-OHBA p-OH0CH2OH Algal species dpm x 10"3 Haotophyta Isochrysis galbana Dark 180.2 ( 8.11)(2) 7.77 (0.35) 7.3^ Light 137.8 ( 6.21) 8.00 (0.36) 7.09 Monochrysls lutherl Dark 1.11 ( 0.05) 1.11 (0.05) Light 3.33 (0.15) 2.89 (0.13) 0.91 Coocollthu3 huxleyl Dark 0.89 ( 0.04) 1.11 (0.05) Light 3.12 ( 0.14) .1.31 (0.06) Bacill^rlophyta Pennate diatom Navlcula lncerta Dark 151.2 ( 6.81) 14.2 (0.64) 2.10 (0.09) 8.46 (0.38) Light 358.0 (16.1 ) 22.9 (1.03) 2.06 (0.09) 16.0 (0.72) Centric Diatom Skeletonema costatum Dark 8.22 ( 0.37) 1.11 (0.05) Light 2.4? ( 0.12) 2.00 (0.09) Cryptophyta Rhodomonas lens Dark 19.8 ( 0.89) 3.78 (0.17) Light 19.1 ( 0.86) 0.89 (0*04) (1) abbreviations aret p-OHB = p-hydroxybenzolc acid, p-OH0Ac = p-hydroxyphenylacetio abld, p-OHBA = p-hydroxybenzaldehyde, p-OH0CH2OH = p-hvdroxybehzylalcdtiol. (2) figures in parentheses give data as % of - added l*C-tyrosine. i removed this, acid would not be detected. Similarly, if a unit was removed as in the case of tyrosine-phenol lyase (see preparation of uniformly ring labelled tyrosine) neither of these acids would be detected. If a.C'^ followed by a C2- fragment was- removed, p-hydroxybenzoic acid would not be de• tected. Therefore these results suggested two C-^-f ragments, probably as CQ2, were removed to produce p-hydroxybenzalde- hyde. (detected in some instances-Table 14). p-Hydroxybenz- aldehyde would be expected to be oxidized to p-hydroxybenzoic acid and this was detected in the extracts of each species examined. For both I. galbana and JJ. lncerta the reduction product, p-hydroxybenzylalcohol was also detected (Table 14) but most of the radioactivity was observed in the p-hydxoxy- benzoic acid,. Side chain degradation of phenylalanine and tyrosine. To confirm the loss of C02 units from the side chain of both aromatic amino acids, specifically side chain label• led phenylalanine and tyrosine were fed to both I, galbana and ,Ni lncerta. Two uCl were fed, but the side chain-2-^C and side chain-3-^C were mixtures of the DL-isomers. • 'iPre- vious results suggested that Isochrysls did not metabolize the D-isomer of phenylalanine and tyrosine while Navlcula metabolized both D-isomers but only after a fourteen day lag period (Figures 16 and 17). Immediate growth of Navlcula on the L-isomers (Figures 9 and.10), and the short time length (12 hours) for these experiments allowed 75. the D-isomer content of both amino acids to be disregarded. Therefore, all calculations for both species were based only on the content of L-lsomer which was assumed to be 50# of the added DL-mixture. The cells utilized in these stud• ies were pre-adapted on 0,1 mM L-phenylalanine or L-tyrosine because 1^ galbana had a lag period before either amino acid was utilized as a nitrogen source (Figures 13 and 1*0. l/4- In Tables 15 and 16 the results of the C02 trap• pings for both algal species and both amino acids are pre• sented. To enable a comparison between species, the values in both tables were based on 10 cells. For lncerta (Table 16) this was the actual radioactive counts trapped, but twice this number of radioactive counts were trapped for I. galbana. The percentage values should be used to compare the radioactivity trapped for each series of amino acids (le. ll4 -l-^C, -Z^C. and -3- 'c). lk The values obtained for the CO,, in the KOH trap 14 were confirmed by retrapping the CO2 in a a-phenylethyl- amine. Any difference was associated with a volatile pro• duct or products. In the feedings of I. galbana with phenyl• alanine (Table 15)» a volatile product was detected at the Cg-Cg level which suggested the volatile product of Vose et al. (1971) was a Cg-C^ compound.- A volatile product also was detected for N. lncerta (Table 16) but it appeared to lk be a C.-C compound. In the feedings of tyrosine- C, a o 1 volatile product at the Cg-Cg level was detected for Isochrysls (Table 15) while no volatiles were observed for 76. TABLE 15 Total ^COj Measured a3 a Product of Catabollsm from Incubation of Pre-adapted Cells of Isochrysls galbana with Labelled Phenylalanine and Tyrosine Incubation juCl Total conc• Trapped Re-trapped In Volatile pro- Labelled conditions added entration of ln KOH precursor of L-lsomer 14' (hours) L -Isomer (uM) 3 8 3 8 COg. dpm x 10~ /10 cells dpm x 10" /10 L-Phe-l-lUC Light- 6 2.0 50.0 5.2 0.122)* 5.4 (0.122) 0 Llght-12 2.0 50.0 10.3 0.232) 10.3 (0.232) 0 Dark - 6 2.0 50.0 ' 21.2 0.482) 21.3 (0.482) 0 Dark -12 2.0 50.0 50.0 1.132) 50.3 (1.132) 0 14 .0 0.29 L-Phe-2- C Light- 6 1 1*6.9 4.3 0.202) (0.022) 4.04 (0.182) Llght-12 1.0 46.9 10.2 0.462) 2.2 (o.io2) 7.96 (0.362) Dark - 6 1.0 U-6.9 17.0 0.772) 5.32 (0.242) 11.7 (0.532) .0 16.1 Dark -12 1 •*6.9 0.732) 15.0 (0.682) 1.10 (0.052) L-Phe-S-^C Light- 6 1.0 46.7 0.66 0.032) 0.15 (o.oi2) 0.51 (0.022) 4.4 Llght-12 1.0 • 46.7 0.202) 0.75 (0.032) 3.61 (0.162) Dark - 6 1.0 1*6.7 9.2 0.422) 1.3 (0.062) 7.9 (0.362) Dark -12 1.0 06.7 14.1 0.632) 7.6 (0.342) 6.5 (0.292) L-Tyr-l-lUC Light- 6 2.0 50.0 24.1 (0.542) 24.5 (0.552) 0 Llght-12 2.0 50.0 52.4 (1.182) 52.6 (1.182) 0 . Dark - 6 2.0 50.0 79-3 (1.792) 79.5 (1.792) 0 Dark -12 2.0 50.0 169.0 (3.762) 167.0 (3.762) 0 L-Tyr-2-lUC Light- 6 1.0 ^7.0 9.0 (0.412) 9.0 (0.412) 0 Llght-12 1.0 47.0 18.3 (0.822) 15.4 (0.702) 2.9 (0.132) Dark - 6 1.0 47.0 • 11.8 (0.532) 6.4 (0.292) 5.3 (0.242) Dark -12 1.0 47.0 22.2 (i.oo2) 18.8 (0.852) 10.0 (0.452) L-Tyr-3-1,fC Light- 6 1.0 43.4 6.9 (0.312) 2.1 (o.io2) 4.8 (0.212) 43.4 Llght-12 1.0 6.5 (0.292) 1.5 (0.072) 5.0 (0.222) Dark - 6 1.0 43.4 6.1 (0.282) 2.0 (0.092) 4.2 (0.192) Dark -12 1.0 43.4 12.4 (0.562) 10.0 (0.452) 2.4 (0.112) 14„ 'figures ln parenthesis give data as 2 of C-precursor fed. 77. 4 TABLE 16 Total l^COj Measured as a Product of Catabollsm from Incubation of Pre-adapted Cells of Mavlcula lncerta with Labelled Phenylalanine and Tyrosine Incubation pCl Total cone- Trapped . He-trapped In Volatile- pro- Labelled conditions added entratlon of In KOH g-phenyleChvlamlne ducts In- KOH: precursor of L-lsomer 1 4 3 3 8 (hours) L-lsomer (uM) ' 'C02, dpm x 10" /10°oells dpm; x: 10~' /10 cell3 L-Phe-l-^C Light- 6 2.0 50.0 147.0 ( 3.32**149. 0 ( 3.35*) 0: Llght-12 2.0 50.0 195.0 ( ^.39* 195.0 I 4.40*) 0: Dark - 6 2.0 50.0 '+48.0 (11.1 * 450.0 (10.1 *) 0 Dark -12 2.0 50.0 656.0 (14.8 * 659.0 (14.8 *) 0: L-Phe-2-l!*C Light- 6 1.0 46.9 25.3 ( 1.14* 25.4 ( 1.14*) 0 Llght-12 1.0 46.9 14.8 ( 0.67* 15.6 ( 0.70*) 0 Dark - 6 1.0 46.9 59.*+ ( 2.68* 5^.2 ( 2.44*) (0.23*). Dark -12 1.0 46.9 83.3 ( 3.75* 83.6 . ( 3.77*) 0. L-Phe-3-lUC Light- 6 1.0 46.7 10.9 ( 0.49* 0.64 ( O.03*) 10.2 (0.46*) Llght-12 1.0 19. ^ ( 0.87* 0.43 ( 0.02*) 19.0 (0.85*) Dark - 6 1.0 1*6.7 13.6 ( 0.61* 1.9 ( 0.09*) 11.7 (0.53*) Dark -12 1.0 1*6.7 21.7 ( 0.98* 3.4 ( 0.15*) 18 ..4- (0.83*) L-Tyr-l-lIfC Light- 6 2.0 50.0 301.0 ( 6.78* ) 304.0 ( 6.84*), 0 Llght-12 2.0 50.0 233.0 ( 5,25*) 234.0 ( 5.26*) 0 Dark - 6 2.0 50.0 648.0 (14.6 * ) 648.0 (14.6 *) 0 Dark -12 2.0 50.0 605.O (13.6 * ) 606.0 (13.7 *) 0 L-Tyr-2-1)+C Light- 6 1.0 47.0 32.1 ( 1.46* 32.5 ( 1.46*) 0 Llght-12 1.0 47.0 41.1 ( 1.85* 42.9 ( 1.92/0 0: Dark - 6 1.0 47.0 '71.0 ( 3.20* 71.4 ( 3.22*) 0 173 Dark -12 1.0 ' 47.0.. .0 ( 7.80* 174.0 ( 7.84*) 0 L-Tyr-3-l2tC Light- 6 1.0 t*3.i* • 1.84 ( 0.08* 1.85 ( 0.08*) 0 Llght-12 1.0 43.4 3.04 ( 0.14* 3.12 ( 0.14*) 0 Dark - 6 1.0 i*3.i* 30.5 ( 1.38* 31.0 ( 1.39*) 0 Dark -12 1.0 i*3.i* 77.0 ( 3.52* 77.7 ( 3,55*) 0 •figures In parenthesis glye data as * of1 C-precursor fed. 78 Navlcula (Table l6). The results ln Tables 15 and 16 confirm that C02 units were removed from the side chain of both amino acids.. l4 1 k The detection of C02 when phenlalanlne or tyroslne-3- C were fed to both species indicated that carbon-3, next to 14 the aromatic ring, was removed as C02. When specifically side chain labelled phenylalanine and tyrosine was fed to both algal species the radioactivities decreased in the or- der C-l>C-2>C-3., The uptake rates of both phenylalanine .and tyrosine for pre-adapted cells of each species were compar• able (Table 12), but tyrosine appeared to be metabolized 14 more rapidly than phenylalanine based on the .amounts of C02 produced. The cells 'Utilized for these studies were actively photosyntheslzlng (le. still growing anabollcally);. This was confirmed for both species by comparing the radioactivev lty for cells under continuous illumination with those in continuous darkness. The values in darkness were always^" greater than in light. In almost all instances, the radio• activity obtained for a 6 hour incubation was less than .ob• tained for the 12 hour incubation.; The outstanding example where this was not the case was for N. Incerta tyroslne-1- 14 C light and dark incubations (Table 16). This was possi• bly related to the "levelling off observed in the uptake of tyrosine (Figure 26), but the cause was unknown. The chromatographic analysis,of the medium from the (1) except in Table 15 L-Tyr-3-1^C-Light-6 vs Dark-6. 79. above experiments with phenylalanine and tyrosine specifically ^C-labelled in the side chain revealed similar patterns for both species. Photographs of the autoradiographs are presented in Figures 28 and 29. The controls, Figure 30, were in• cluded to show the contaminants that were present In the 14 C-amino acids to be fed. The DPM values were deducted for any contaminant that corresponded to a metabolic pro- 14 duct from the feedings. Both phenylalanine-2- C and -3- 14 C feedings contained faint hot areas that could possibly be interpreted as cinnamic acid, but these were also ob• served In the respective controls and were unidentified contaminants. Before any chromatograph was developed, cold p-hydroxybenzoic acid was spotted for reference with the 14 C-ether extract. The position of this acid, spot 1, was outlined with a dotted line. In Tables 17 and 18 the DPM values obtained in the various spots observed in the autoradiographs of I. galbana (Figure 28) and N. lncerta (Figure 29) are presented. All 8 the radioactive values In both tables were based on 10 cells. Spot #9. detected for both species, was an un• identified Cg-C^ compound. It was considered to be pos- ' slbly a N-malonyl or N-acetyl derivative of phenylalanine., but neither of these derivatives have been reported in algae for any amino acid (see Pbkorny et al.. 1970), Phenylpyruvic-phenyllactlc acids as one spot were detected In the extracts of both species when phenylalan• ine was fed. The percentage values for phenylpyruvic- phenyllactic acids were greater when phenylalanine-2- C\ gure 28. Autoradiographs of chromatograms prepared from the ether extracts of C-side chain labelled phenylalanine and tyrosine fed to Isochrysls galbana. Dotted line in• dicates the chromatographic position of authentic p- hydroxybenzoic acid. A. 2% aqueous formic acid. B. Benzene : acetic acid i water (10:7«3, V/V/V). 1. p.-Hydroxy benzoic acid. 2. p-Hydroxybenzaldehyde. 3. p-Hydroxyphenylacetic acid. h, p-Hydroxyphenyllactlc acid. 5. p-Hydroxyphenylpyruvic acid. 6. Benzoic acid. 7. Phenylacetic acid. 8. Phenyllactic and phenylpyruvic acids. 9. Unknown Cg-C-j compound (Unknown #1). 10. 3-hromo-p-hydroxybenzoic acid. 11. Unknown Cg-C2 compound (Unknown #2). Figure 29. Autoradiographs of chromatograms prepared from the ether lk extracts of C-side chain labelled phenylalanine and tyrosine fed to Navlcula lncerta. Dotted line Indicates the chromatographic position of authentic p-hydroxy• benzoic acid. A. 2% aqueous formic acid. B. Benzene » acetic acid 1 water (10i7:3, V/V/V). 1. p-Hydroxybenzoic acid. * 2. p-Hydroxybenzaldehyde. 3. p-Hydroxyphenylacetlc acid. k, p-Hydroxyphenyllactic acid. 5. p-Hydroxyphenylpyruvic acid. 6. Benzoic acid. 7. Phenylacetic acid, 8. Phenyllactic and phenylpyruvic acids, 9. Unknown C,-C! compound (Unknown #1), 0 3 10. 3-cromo-p-hydroxybenzoic acid. 11. Unknown C£-C2 comPound (Unknown #2). 'lgure 30. Autoradiographs of chromatograms prepared from the ether lk extracts of C^slde chain labelled phenylalanine and tyrosine to be fed to Isochrysls galbana and Navlcula lncerta. Dotted line indicates the chromatographic position of authentic p-hydroxybenzoic acid. A. 2% aqueous formic acid. B. Benzene : acetic acid 1 water (10i7»3. V/V/V). 1, p-Hydroxybenzoic acid. 2, p-Hydroxybenzaldehyde. 3, p-Hydroxyphenylacetic acid. k, p-Hydroxyphenyllactic acid. 5. p-Hydroxyphenylpyruvic acid. 6. Benzoic acid. 7. Phenylacetic acid. 8. Phenyllactic and phenylpyruvlc acids. 9. Unknown cg-c^ compound (Unknown #1). 10. 3-Drom°-P-nydroxyDenzoic acid. 11, Unknown Cg-C2 compound (Unknown #2). Control m Control Tr-3- C Li9ht-12hr. LiM-IZ hr. TABLE 17 Radioactivity in Products Isolated from ^C-labelled Feedings from Incubation of Pre-adapted Cells of Isochrysis galbana. 0Ao Benz p-OHB p-OHBBr Total Incubation juCl Unknown #1(1) 0Pyr-0Lact Labelled conditions £tdd ed precursor of dpm x 10~3 per 10 cells (hours) L-lsomer 1.55 (0.032) L-Phe-l-^C Light- 6 2.0 1.07 (0.022) 0.48 (0.012) 0.81 (0.022) Llght-12 2.0 o.48 (o.oi2) 0.33 (0.012) 2.0 o.4o (o.oi;?) 0.09 (o.o 2) 0.48 (o.oi2) Dark - 6 0.3} (o.oi2) Dark -12 2.0 0.27 (0.012) 0.05 (o.o 2) 4.27 (0.192) 1 1.0 1.98 (0.092) 1.52 (0.072) 0.77 (0.032) L-Phe-2-'*C Light- 6 3.96 (0.182) 8.63 (0.392) Llght-12 1.0 2.15 (0.102) 2.53 (o. n2) 1.06 . Dark - 6 1.0 0.43 (0.022) o.io (o.o 2) 0.53 (0.02*) (0.052) Dark -12 1.0 0.83 (0.042) 0.65 (0.032) 0.38 (0.022) I.85 (0.082) lll 0.08 (0.0 %) 0.38 (0.022) 0.20 (0.012) 6.97 L-Phe-3- C Light- 6 * 1.0 5.12 ( 0 . 232) 1.19 (0.052) (0.312) Llght-12 1.0 1. 58 (0.072) 1.81 (0.082) 0.59 (0.032) 0.23 (o.oi2) 0.14 (0.012) 4.20 (0.192) Dark - 6 1.0 0.84 (0.OU2) 0.70 (0.032) 2.31 (o.io2) 0.08 (0.0 2) 0.05 (0.0 2) 3.80 (0. 172) 1 0.48 (0.022) O.07 (0.0 2) 2.20 Dark -12 .0 . l.io (0.052) 0.48 (0.022) 0.15 (0.012) (o.io2) (3) p-OHBBr Total p-OH0Pyr p-OH0Lact 1p-OH^A o Unknown #2 p-OHBA P-0H3 dpn x 10"' per 108 cells L-Tyr-l-^C Light- 6 2.0 0.45 (0.012) 0.51 (0.012) 0.97 (0.022) 1.88 (0.042) Llght-12 2.0 0.96 (0.022) 0.92 (0.02^) Dark - 6 2.0 0.13 (o-o 2) 0.07 (0.0 2) 0.21 (0.0 %) 0.20 (0.0 Dark -12 2.0 0.15 (o.o 2) 2) 0.35 (O.012) L-Tyr-2-^C Light- 6 1.0 0 5.62 (0.252) 1.3? (o.o62) (o.oi2) 7.14 (0.322) Llght-12 1.0 0 2.86 (0.132) 0.3' (0.022) 0 3.20 (o.i42) Dark - 6 1.0 0 5.62 (0. 252) 1.39 (0.062) 0-32 (o.oi2) 7.34 (0.332) 0 (0. Dark -12 1.0 9.52 432) 0.91 (O.OW 0.48 (0.022) 10.9 (0.492) L-Tyr-S-^C Light- 6 1.0 0 1.25 (0.062) . 2.04 (0.092) 1.37 (0.062) 3.84 (0.172) 5.99 (0.272) 14.5 (0.652) Llght-12 1.0 0 3.51 (0. 162) 1.82 (0.052 ) 0.71 (0.032 ) 3.30 ( 0.152) 4.62 (0.212) 14.0 (0.632) Dark - 6 1.0 0 3.54 (0. 162) 4.10 (0.192) 2.77 (0.132) e.46 (0.382) 5.55 (0.252) 24.4 (i.io2) Dark -12 1.0 0 5.70 (0. Z6h) 5.19 (0.232) 3.34 (0.172) 8.53 (0. 382) 11.0 (0. 502) 34.3 d.552) (1) abbreviations arei 0Pyr = phenylpyruvlo acid, 0Lact = phcnyllactlc acid, tfAc => phenylacetic acid, Benz = benzolcacld, p-OI!B = p-hydroxybenzolc acid, p-OHBBr = 3- bromo-p-hydroxyhenzolc nold, p-OH0Pyr « p-hydrozyphenylpyruvic acid, . p-OH0Laot = p-hydrozyphenyllactlc acid, p-OH0Ac » p-hydroxyphenylacetlc acid, p-OHBA =• p-hydroxybenzal-lehyde. OO (2) figures ln parenthe3e3 elve data as % of adder! 1 C-precur3or. V^j (iS unknown hao same Rf valuos as p-hydroxybcnzaldehydo. • TABLE 18 - Radioactivity In Products Isolated from li+C-iabelled Feedings from Incubation of Pre-adapted of Navlcula lncerta. Incubation JJCI (1) Unknown #1 0Pyr-0Lact 0Ao Benz Labelled conditions add ed p-OHB p-OHBBr Total precursor of (hours) L-lsomer 8 dpn x 10"3 per 10 cells L-Phe-l-^C Light- 6 2.0 5.52 (0.12*) 3.74 (0.08*) Llght-12 2 9.26 (0.21*) .0 2.33 (0.05*) 2.40 (0.05*) 4.73 (0.11*) Dark - 6 2.0 1.33 (0.03*) 0.96 (0.02*) Dark -12 2.0 1.23 (0.03*) 1.62 (0.04*) 2.29 (0.05*) 2.85 (0.06*) L-Phe-2-ltlC Light- 6 10.7 (0.48*) 40.2 1.0 (1.9 *) 21.5 (O.97*) Llght-12 .0 8.94 (0.40*) 1 50.5 (2.3 *) 11.7 (0.53*) 73.5 (3.3 *) 77.0 (3.5 *) Dark - 6 1.0 (0 8.92 .40*) 38.6 (1.7 *) 33.2 (1.5 *) Dark -12 1.0 (0.33*) 8O.5 (3.6 *) 8.53 48.1 (2.2 *) 37.2 (1.7 *) 14 93.8 (4.2 *) Light- 6 1.0 5 (0.26*) .72 54.5 (2.5 *) 36 (1.6 *) 16.5 L-Phe-3- C Llght-12 1.0 5.64 (0.30*) .5 (0.75*) 1.23 (0.06*) 0.73 (0.03*) 115.0 (5.2 *) 45.3 (2.0 *) 23.8 (1.1 *) 17.7 (0.80*) .06*) 1.49 (O 0.77 (0.03*) •95.8 (4.3 2) Dark - 6 1.0 8.93 (0.40*) 45.7 (2.1 *) 61.3 (2.8 *) 9.40 (0.42*) Dark -12 1.0 9.67 (0.44*) 0.97 (0.04*) 0.60 (0.03*) 127.0 (5.7 *) 46.7 (2.1 *) .3 (2.6 *) 14.1 (O.63*) 2.13 58 (0.10*) 0.89 (0.04*) 132.0 (5.9 *) p-OH0Pyr p-OH0Lact p-0Hf$Ac Unknown #2^ c-OHBA „ r,tm p-UHdA p-OHB p-OHBBr Total dpm x 10" 3 per 108 cells L-Tyr-l-^C Light- 6 2.0 0 2.24 (0.05*) ? ,L ln Llght-12 2.0 0 1.03 (0.02*) 2.24 (0.05*) Dark - 6 2.0 0 1.99 (0.05*) Dark -12 2.0 0 2.78 (0.06*) 1$ jo.ogl 1 L-Tyr-2- "^ Light- 6 1.0 0 5.20 (0. 23*1 12.u (0.56*) 2.94(0.13*) ,n, ,fto9i, Llght-12 1.0 0 3.19 (0. 1^*5 2.63 (0.12*) ^.33 (0.19*) ^0.5 (0.9g) r k 0 ^ , 'A H 9.90 (0. 45*) 4.64(0.21*) 2^.0 (1.1*) -R ,. / -. n LU 10 Llght-12 1.0 0 22.1 (1.0 *) 18.9 (0.85*) 10.1 (0.46*) 4l.O (1.9 *) 27.6 (1.2*) Jg^ (5*.4*) Dark - 6 1.0 0 11.0 (0.49*) 53^ (2.4 *) 38.9 ( 1.8 *) 52.9 (2.4 *) 39.7 (1.8*) wn /„ „ *, rk "12 1'° 0 1*.5 (0.66*) 32.5 (1.5*) 27.O (1.2*) 78.5 (3.5*) 33.3 (1-5*) Ht'.o (0.4 *j (1) abbreviations aroi 0Pyr = phenylpyruvlc acid, 0Lact » phenyllaotlc acid, 0Ao =• phenylacetic acid, Benz = benzoic acid. p-OHB = p-hyd roxyb-^nzolc acid, p-OHBBr - 3- brono-hydroxytsnzolc acid. p-Ol!0I'yr = p-hydroryphenylpyruvle acid, p-OHLoet = p-hF-lroxyphenyllactlc acid, p-OII0Ac = p-hydroxyphenylacetlc acid, p-OHBA = p-hydroxybenzaldchyde. ^ (2) figures ln parentheses give data as * of added C-precursor, 00 (3) unknown haa sao Br values as p-hydroicybenzaldehyde. _- 85. i 4 and -3— C were fed, which suggested that Gome C^-Cg com• pounds chromatographed with these acids.. It was known that both mandelic and benzoylformic acids chromatographed in this region (see Appendix B) and probably account for the increase in radioactivity. The results for Isochrysls sug• gested that mandelic and benzoylformic acids were present in very low amounts (Table 17), where with Navlcula these • two aromatic acids comprised the major portion of this 14 14 area when phenylalanlne-2— C or -3— C were fed. Phenylacetic acid was the only other spot detected 14 when phenylalanine-2-- C was fed to both species. The variations in the percentage values for this acid when fed to Isochrysls could be related to the Cg-C^ v°latlle pro• duct trapped In the KOH (Table 15). In CO trapping ex- 14 2 perlments, when C-phenylacetic acid was fed to both species, 14 a very large percentage of C-phenylacetic acid from the control was trapped in the KOH. This suggested that X* galbana may excrete phenylacetic acid into the medium and upon acidification this acid could be volatilized. The percentage values for N. lncerta were relatively constant and essentially no volatile product was detected. This suggested that phenylacetic acid was not excreted into the medium by this species. Two distinct spots were detected in the benzoic- phenylacetic acid region for r^. lncerta when phenylalanlne- 14 3- C was fed (Figure 29). Only one spot was detected for 86. I. galbana (Figure 28) which corresponded to phenylacetic acid Indicating little to no benzoic acid was present, p—' Hydroxybenzoic acid and 3-bromo-p-hydroxybenzoic acid were detected from both species (Tables 17 and 18) while no p- hydroxyphenylacetlc acid was detected. This suggested that benzoic acid was hydroxylated and not phenylacetic acid. The DPM values ln phenylacetic acid for N. lncerta 14 were greater when phenylalanlne-3- C was fed than when phenylalanine^-1^ was fed. This indicated that possibly a Cg-C-^ aromatic compound chromatographed with phenylacetic acid. The only Cg-C^ compound known to chromatograph In this region was o-hydroxybenzolc acid (salicylic acid)* If salicylic acid was present, Navlcula also possesses the capability to hydroxylate benzoic acid in the ortho-position. No indication of salicylic acid was obtained for I. galbana. The C^-C^ volatile product detected in the KOH for N. Incerta may possibly be benzoic acid (Table 16). Similar to phenylacetic acid, benzoic acid was observed to result ln 14 a very high number of DPM ln the KOH trap when ring- C- benzolc acid was utilized. Benzoic acid is relativef' 9: ly volatile and great care must be taken when it is - used in feeding experiments (B. Ellis, personal communica^ tion).. This suggested that benzoic acid may be excreted in• to the medium and volatilized upon acidification. In Figures 28 and 29, along the B axis and near the solvent front, areas of radioactivity were observed when "l It T L phenylalanine-2-x-C and -3- C were fed. These may have 87. corresponded to phenylacetaldehyde when phenylalanine^-1^! and phenylacetaldehyde-benzaldehyde when phenylalanine^-1*^ were fed. _Both these aldehydes are known to chromatography in this region, but many other compounds, especially lipids, chromatograph with these two aldehydes. Very similar patterns were obtained in the degrada• tion of tyrosine, but the degradation products all contain• ed a para-hydroxyl group. p-Hydroxyphenylpyruvic acid was not detected for N. incerta (Figure 29, Table 18), but a very faint spot (#5-Figure 28, Table 17) was observed for I. galbana when tyrosine-1- C was fed. This acid was not 14 14 observed for I. galbana when tyrosine-2- C and -3- C were fed. This being possibly due to only'.ljiCi of these two amino acids being utilized as opposed to 2 uCi for •• tyrosine-' l-^C and the tailing of other spots over this region. The 14 only other spot detected when tyrosine-1- C was fed cor• responded to p-hydroxyphenyllactic acid. When the percent• age values obtained for this acid were compared to those obtained when tyrosine^-1^ was fed to both species (Tables 17 and 18), a distinct Increase was observed. This sug• gested that a Cg-C2 compound, p-hydroxymandelic acid, chromatographed with the p-hydroxyphenyllactic acid (see Appendix B). A similar increase was noticed when the values of p-hydroxyphenyllactic acid were compared for the 14 14 tyroslne-2- C and -3- C feedings of N. lncerta (Table 18). This Indicated a Cg-C^ compound, probably p-hydroxybenzyl- alcohol (see Appendix B) also chromatographed in this . 88. position. No indication of a Cg-C^ compound was obtained for I. galbana. The only other phenolic.compounds detected for both 14 species when tyroslne-2- C was fed were p-hydroxyphenyl• acetlc acid and an unknown compound. This unknown compound (#2) chromatographed in the same region as p-hydroxybenzaldehyde. It's identity was unknown, but the only Cg-Cg compound in the degradative sequence of tyrosine not accounted for was p-tiydroxybenzoylformic acid. The chromatographic location of this phenolic acid was not determined (see Materials and Methods) and the unknown compound may be p-hydroxybenzoyl- formic acid. The Cg-Cg volatile compound detected for galbana in the ^COg studies (Table 15) was possibly p-hydroxyphenyl• acetlc acid. This acid was found to be quite volatile under acidic conditions and it would be expected to be trapped ln the KOH. - Chromatographic analysis of both algal species when 14 tyroslne-3- C was fed also revealed p-hydroxybenzaldehyde, p-hydroxybenzolc acid, and 3-bromo-p-hydroxybenzolc acid. When the radioactivity for unknown #2 (Tables 17 and 18) was subtracted from the combined values for p-hydroxybenz• aldehyde, values were obtained for p-hydroxybenzaldehyde. Generally the greatest radioactivity observed in any product 14 from the metabolism of tyrosine-3- C by both species was ln p-hydroxybenzoic acid and/or 3-bromo-p-hydroxybenzoic acid. This suggested that these two compounds appeared to be the 89, end product ln the degradation of tyrosine by both algal species. The radioactivity observed ln the light for these two acids appeared to be less than ln dark which suggested that light promoted their degradation. At no time were any dlhydroxyphenollc compounds detected for either algal species l4 when tyroslne-3- C was fed. For both I±. galbana and N. lncerta the percentages for the total radioactivity in the isolated products for phenylalanine and tyrosine (Table 17 and 18) increased as 14 the C-carbon moved closer to the aromatic ring. In the same experiments (Tables 15 and 16), the percentage activity 14 / in the COg decreased. When the total radioactivity (in the KOH and total ether extract) obtained in the dark for 14 ^ 14 14 phenylalanine-1- C, -2- C, and -3- C were compared, the values were within reasonable error of each other. The l4 14 l4 same was true for tyroslne-1- C, -2- C, and -3- C. A comparison of the degradative rates' for both species Indi• cated that N± lncerta degraded phenylalanine and tyrosine ten-fold faster than £. galbana. This ten-fold difference cannot be explained by uptake rates as a 100-fold difference was obtained, but suggested that Navlcula and possibly Isochrysls assimilated both amino acids into a storage pool. These amino acids were then metabolized at governed rates, 14 When the chromatograms from the tyrosine-1- C and 14 phenylalanlne-1- C feedings were sprayed with PNA or the spray for acids, only p-hydroxybenzoic acid was detected,. i 90. This was because of the addition of unlabelled p-hydroxy- benzoic acid. The other aromatic and phenolic acids were ln such low concentrations that they were not detectable. At no time in any autoradlograph from the phenyl- 14 14 alanine- C or tyrosine- C studies was cinnamic or p-cou- maric acids detected. When these two acids were fed to both algal species, they were metabolized. The metabolic patterns (Figure 31) were totally different from those ob• tained in the metabolism of phenylalanine and tyrosine. This suggested that these two acids were not involved in the degradation of either amino acid. The metabolism of cinnamic acid revealed mainly one other hot spot (spot #9) which corresponded to phenylhydracrylic acid. The identity of the spot to the upper left of spot #9 in XJ galbana and N„: lncerta (very faint) was unknown, It's location suggest• ed that it was benzoylacetlc acid, an oxidation product of phenylhydracrylic acid. (A similar chromatographic rela• tionship was observed for phenyllactic acid and phenyl- pyruvic acid, see Appendix B). The identity of the spot to the upper right of spot #8 in N, Incerta was unknown. 14 It's location and the position of the C-label in cin• namic acid suggested that it was either a cg"c-j or Cg-C2 compound. No attempt was made to identify this spot, but possibly it could have been phenylpropionic acid. No evi• dence was obtained from either species that the aromatic nucleus of cinnamic acid was hydroxylated. Figure 31. Autoradiographs of chromatograms prepared from the ether extracts of cinnamic acld-2- C and p-coumarlc acld-2- C fed to Isochrysls galbana and Navlcula lncerta. Dotted lines Indicate the chromatographic positions of authentic standard compounds. A. 2% aqueous formic acid B. Benzene : acetic acid : water (10*7:3, V/V/V). 1. p-Hydroxybenzoic acid. 2. p-Hydroxybenzaldehyde. 3. p-Hydroxyphenylacetic acid. b. Trans-p-coumaric acid. 5. Cis-p-coumaric acid. 6. p-Hydroxyphenylhydracrylic acid (?). 7. Trans-cinnamlc acid. 8. Cls-clnnamic acid. 9. Phenylhydracrylic acid. Autoradiographs of the ether soluble metabolic 14 products from p-coumarlc acid- C revealed only one spot for both species. This corresponded ln R^, to the tenta• tively .Identified p-hydroxyphenylhydracrylic acid. As was observed ln the unlabelled feedings a portion of p- hydroxyphenylhydraorylic acid remained at the origin. This was also apparent in the autoradiographs. 14 Degradation of other C-labelled substrates. 14 14 The CO,, released when phenylacetic acid-1- C 14 14 and -2- C and p-hydroxyphenylacetic acid-1- C and ^2- 14 C were fed to X« • galbana and N_. lncerta are presented ft ln Table 19. The DPM values were based on 10 cells,, Twice the listed number of DPMs were trapped for 1^ galbana but only 0.2 times the listed number for N_. lncerta. The low values were possibly the result of (1) the cells were not pre-adapted and (2) the cells were not excessively per• meable to either of these aromatic acids, .For both species when the percentage values were compared, a greater percent• age was obtained ln the CO^ when both acids were labelled in the C-l position rather than in the C-2, Similar to the re• sults obtained ln the degradation of both phenylalanine and tyrosine, the carbon next to the aromatic ring was removed as C02. Analyses of the autoradiographs, when phenylacetic 14 14 acid-1- C was fed to both species showed mandelic acid-. C 14 and benzoylformic acid- C. These two acids and p-hydroxy- 14 benzoic acid were also observed when phenylacetic acid-2- C TABLE 19 Total ^C02 Measured as a Product of Catabollsm In the Dark from Incubation of Non-adapted Cells with Labelled Aromatic Compounds Algal species Total 6-hour Incubation 12-hour Incubation and uCl concentration _^ -3 3 Labelled precursor added fed (mM)(D C02, dpm. x 10 per, 10 cells Isochrysls galbana phenylacetic acld-l-^C 1.00 0.05 1.5 (0.07%)^ 3.5 (0.l6#) phenylacetic acld^-^C 0.33 0.05 0.10 (0.01#) 0.30 (0.04#) p-hydroxyphenylacetlc acld-l-llfC 0.054 0.05 0..05 (0.04#) 0.11 (0.09#) p-hydroxyphenylaeetlo acld^-^C 0.24 0.05 0.04 (0.0 %) 0.12 (0.02#) Navlcula lncerta phenylacetic acld-l-^C 1.00 0.05 2.1 (0.10#) 2.9 (0.13#) phenylacetic acld-2-l(*C 0.33 0.05 0.31 (0.04#) 0.60 (0.08#) p-hydroxyphenylacetlc acld-l-l4C 0.054 0.05 0.31 (0.26%) 1.8 (l.50#) p-hydroxyphenylacetlc acld-2-l^C 0.242 0.05 0.12 (0.02#) 0.30 (0.05#) (1) labelled plus non-labelled aromatic precursor. (2) figures ln parenthesis give data as % of total concentration. 94. was fed. p-Hydroxymandellc acid was detected as the only product from the metabolism of p-hydroxyphenylacetic. acid, while p-hydroxybenzaldehyde and p-hydroxybenzoic acid were 14 also detected when p-hydroxyphenylacetic acld-2- C was fed to both species. These results are in agreement with the data obtained in the degradation of the side chain of phenylalanine and tyrosine by both algal species. In Table 20 are presented the results of feeding 14 various C-labelled Cg-C^ aromatic acids. Both species were able to degrade the aromatic ring of benzoicacid-(U)- 14 14 C-ring labelled to C02. In examining the autoradio• graphs, p-hydroxybenzoic acid, m-hydroxybenzoic acid, and o-hydroxybenzoic acid were observed as hydroxylation pro• ducts' for N. lncerta. Only o-hydroxybenzoic acid was not detected for ix galbana and this may have been the result of a very rapid decarboxylation of this acid as observed in 14 Table 20 (jje. a very high number of counts In the CO? 14 from salicylic acld-7— C). The presence of o-hydroxy• benzoic acid was speculated upon ln the N. lncerta phenyl- " 14 alanlne-3- C feeding experiment. Its presence was con- . firmed in this experiment. 3-Bromo-p-hydroxybenzolc acid was also detected in the autoradiographs from benzoic acid- 14 C for both species. One other compound was detected ln the autoradlo- 14 graphs from salicylic acld-7- C, but it was not identified. Salicylic acid was decarboxylated -by both species (Table 20). 95. TABLE 20 Total ^002 Measured-as a Product of Catabolism from a 12-hour Dark Incubation of Non-adapted Cells with Labelled Aromatic Compounds Algal species Total ^COg, dpm x 10"3 and >uCi concentration Labelled precursor added fed (mM.)(l) per 108 cells Isochrysls galbana Benzoic acid-(U)- ^C-rlng labelled 1.0 0.05 0.23 (0.01#)(2) salicylic acid -7-^C 1.0 0.05 20.5 (0.9W p-hydroxybenzoic lk 1.0 0.05 2.k (0.11$) acld-7- C Navlcula lncerta benzoic acid-(U)- ^C-ring labelled * 1.0 0.05 0.61 (0.03$) salicylic acid -7-^C 1.0 0.05 0.12 (0.01$) p-hydroxybenzolc acld-?^1^ 1.0 0.05 2.5 (0.11$) (1) labelled plus non-labelled aromatic precursor. (2) figures ln parenthesis give data as % of total concentration fed. p-Hydroxybenzoic acid was also decarboxylated to 14 yield CO (Table 20) or metabolized to produce 3-bromo- 14 p-hydroxybenzoic acid. The production of CO from p- 14 hydroxybenzoic acid-1- C was in agreement with the pro- 14 -\L duction of CO from tyrosine-3- C and p-hydroxyphenyl- 14 acetic acid-2- C. At no time was any dihydroxyphenolic compound observed in the autoradiographs from the metabo• lism of phenylacetic acid, p-hydroxyphenylacetic acid, benzoic acid, p-hydroxybenzoic acid, or o-hydroxybenzoic acid by either algal species. In the KOH center well from the experiments with these aromatic acids, a very high background count was ob• tained. This high background was eliminated when the C02 was retrapped ln the a-phenylethylamlne. The values in Tables 19 and 20 were results obtained after retrapping the CO . 2 E. Results of enzyme assays. Phenylalanine ammonia lyase. From the results presented in Table 21, this enzyme did not appear to be present ln algae. When positive values were obtained in this assay procedure, chromatography showed that the radioactivity resided in compounds other than cinnamic acid. Transaminase Transaminase activity was observed with both N„ lncerta and I. galbana. but X. galbana showed this activity only after.prior exposure to either phenylalanine or tyrosine TABLE 21 Phenylalanine Aramonla-lyase Activity In Marine Algae Algal species . Activity . Algal species ...... Activity Chlorophyta Pyrrophyta Dunallella tertiolecta nil Amphldlnium carteri nil Nannochloris oculata •0 nil Enteromorpha sp. nil, Rhodophyta Spongomorpha sp. nil Porphyridium cruentum nil Ulva so. nil Rhodella maculata nil Antithaminion sp. nil Haptophyta : ) Irldaea sp. nil Isochrysls galbana nil Porphyra sp. nil Monochrysis lutherl nil Cyanophyta Baclllariophyta Agmenellum quadrupllcatum nil Cyclotella nana " nil Navlcula lncerta nil Phaeophyta Fucus sp. nil Cryptophyta Lamlnaria sp. (blade only) nil Chroomonas sallna nil Nereooystls sp. (blade only) nil (Table 22). This difference may be related to the four day lag period shown by Isochrysis before growth on L-phenyl• alanine or L-tyrosine as the sole nitrogen source unlike N. lncerta0 When C-phenylalanine or tyrosine was incubated with an enzyme preparation from either species, the major part of the radioactivity was not located in the phenyl- pyruvic or p-hydroxyphenylpyruvic acids (Figure 32). There• fore, no true estimation of the enzymatic activity (based on the alkali-catalyzed oxidation of these two aromatic acids to their respective Cg-C-^ aldehydes) could be obtained and only the optical density readings from the enzyme assays are presented in Table 22. Only If the enzyme preparations were further purified could a true estimation of the trans• aminase activity be obtained. N. lncerta showed approxi• mately a ten-fold greater transaminase activity than I. galhana at comparable total protein concentrations (Table 22);. This suggested that the ten-fold difference in the degradative routes of both phenylalanine and tyrosine may be related to the transaminase activity. Incubation of non-adapted cells of I_. galbana with lk C-phenylalanine or tyrosine confirmed that no transamin• ase activity was present. Some radioactive products were extracted into ether (Figure 32), but they did not corre• spond to the aromatic compounds expected from the degrada• tion of phenylalanine or tyrosine. When pre-adapted cells of I. galbana were used, autoradiographic patterns TABLE 22 Transaminase Activity in Cell Extracts of Navicula incerta and Isochrysls galbana Transaminase actlvity^^ Amino acid and N. incerta I. galbana a-keto acid substrates Non-adapted Non-adapted Phenylalanine Tyrosine cells cells adapted cells adapted cells a-Ketoglutarate + phenylalanine 0,302 0.000 0.040 0.008 a-Ketoglutarate + tyrosine 0,230 0.027 0.010 0.017 Pyruvate + phenylalanine 0.542 0.000 0.010 0,020 Pyruvate + tyrosine 0.538 0.026 0.040 0.041 Oxaloacetate + phenylalanine 0.176 0.000 0.010 0.030 Oxaloacetate + tyrosine ' 0,388 0.024 0,020 0.030 Control + phenylalanine^' 0.000 0.000 .0,000 ^ 0.010 2 Control + tyrosine^ ' 0,008 0.029 0.000 0P020 (1) optical density increase after 2 hours incubation at pH 7.6 (see text). (2) no a-keto acid. 3d Fissure 32. Autoradiographs of chromatograms prepared from the ether 14 14 extracts of C-phenylalanlne and C- tyrosine trans• aminase experiments with extracts of I. galbana and N. lncerta. Oxaloacetic acid was utilized as the a-keto acid ln each assay, u lOOb DL-fVe-l-'K Noh-ad»p. cells + 101. characteristic of the degradative routes for both amino acids were obtained (Figure 32). A correct interpretation of the endogenous a-keto acids used by these algae for transaminase activity (Table 22) is not possible because of the impure enzymes utilized ln these assays. Overall, a-ketoglutaric acid, oxaloacetic acid, and pyruvic acid Were utilized for the transamination shown by both species. Pyruvate resulted in the greatest activity for N. incerta. but no a-keto acid consistantly resulted in greater activity for I. galbana. The only other enzymatic factor studied was pH.' Only N. lncerta extracts were examined and the activity at pH 7.6 was greater than at either 7.1 or 8.0. The low values of doubtful enzyme activity in the .absence of an a-keto acid suggest that L-amino acid oxidase may not be present in either Xi galbana or N± incerta. The corresponding high values for galbana non-adapted cells with tyrosine appear to be due to products from other un• identified reactions. In all cases cinnamic or p-coumaric acids were never detected as products of these enzyme, tests, p-Hydroxybenzolc acid hydroxylase. This enzyme was only assayed for in N± lncerta. In two experiments, phenylalanine, tyrosine, and nitrate grown cells were examined, but no activity was detectable. Protein determinations. The Lowry method of protein estimation gave a value of 4 mg of protein per 20 mg of N.,lncerta freeze dried cells and 5 mg of protein per 20 mg of I. galbana freeze dried cells. The-cells examined were either cultured on nitrate or phenylalanine. 103. DISCUSSION The results Indicate that Isochrysls galbana and Navlcula lncerta were capable of assimilating L-phenylalanine and L-tyrosine against a concentration gradient?" The uptake for both amino acids was far superior for N. lncerta . when compared to I. galbana. Illumination generally stimulated the uptake rates of both amino acids by both spec- les. This effect of light (an energy source) and the assim• ilation against a concentration gradient1strongly suggested that active transport was involved. To date, only the up- 2 take of L-phenylalanlne in algae has been investigated. It was reported that Chlorella fusca. C. vulgaris, and Platymonas s u bcord'lf ormis a ss i ml la ted ' t h 1 s amino acid againstr a concen• tration gradient (Pedersen et a^., 1974} Van Sumere et al.. 1971i North et al.. 19^7) and light enhanced phenylalanine uptake in vulgaris "(Van Sumere et al., 1971). I. galbana and N. lncerta were able to •metabolize* L-phenylalanine and L-tyrosine when grown on those compounds, as the sole nitrogen source. Induction of one or more en• zymes was indicated for I. galbana by a four day extension of the lag period observed for cells previously cultured on nitrate as the nitrogen source. No increase in the lag period was ob• served for Navlcula which suggested that the initial enzyme or enzymes were constitutive. These amino acids were not the best source of nitrogen (1) see Addendum, p.159. (2) in terms of aromatic amino acids. 104. for growth of Navlcula as the growth constants were only one half the value obtained for nitrate (best N-source tested). Isochrysls utilized L-phenylalanine as effectively as nitrate, but severe growth inhibition resulted with L-tyroslne. A. similar growth inhibition by L-tyrosine was reported by Ingram and Jensen (1973) for Anacystls nidulans. but no attempt to ex• plain the cause of the Inhibition was undertaken. L-phenyl• alanlne was reported to inhibit the growth of Agmenellum quadrupllcatum (Ingram and Jensen, 1973)• but the addition of L-tyrosine to the medium completely reversed this inhi• bition. They found that L-phenylalanine inhibited 3-deoxy- D-arablno heptulosonic acid 7-phosphate synthetase (DAHP synthetase), the Initial enzyme in the shikimic acid pathway (Figure. 1). This prevented the biosynthesis of not only L- tyrosine but also L-tryptophan which inhibited normal growth. In an attempt to explain the effect of L-tyrosine on I. galbana. L-phenylalanlne was added to the medium containing L-tyrosine to determine if feedback inhibition was the cause of the reduced cell yield. No change in the cell yield was obtained. Normal growth of Isochrysls was also obtained on nitrate in the presence of L-tyrosine which confirmed that feedback inhibition was not involved as L-tyroslne was assimilated from the medium when nitrate was present. Growth inhibition should have resulted if L-tyrosine was Involved. These results suggested that one of the metabolic products from the degradation of L-tyrosine and not L-tyroslne itself caused the growth inhibition. No indication as to which product was reponsible was found, but both p-hydroxymandellc acid and p-hydroxybenzaldehyde resulted in reduced cell yields. The D-isomers of phenylalanine and tyrosine were also examined for their potential use as nitrogen sources. I.•galbana was unable to utilize either D-amino acid, but . N.•lncerta. after a lag period of about 11 days, utilized both D-amlno acids as a nitrogen source. The growth on D-phenylalanine was greater than on D-tyrosine. The ob• served low growth rates and pigmentation suggested that the D-amino acids were not as efficient a nitrogen source as the L-amino acids. Only two other algae have been re• ported to utilize D-phenylalanine, and one species to pos• sibly metabolize D-tyrosine. .. ' • Other reports indicated that D-phenylalanine was r not assimilated, if assimilated at all, until all the L-phenylalanine was removed from the medium (Pedersen et al., 1974; Van Sumere et al., 1971)*- It was also observed that D-phenylalanine at a ratio of 100il had no influence on the uptake of the L-isomer. This suggested that in the DL-mixtures, the L-isomer was the initial and possibly the only Isomer utilized. M: • The metabolic path of D-phenylalanlne and D-tyrosine has not been studied ln algae, but D-serine (4 species) and' D-methionine (24 species in 8 divisions) were studied by Ladeslc et al., (1971) and Pokokny et al., (1970) respectively Both were metabolized, but when D-methlonlne was administered. 106. only L-methionine was found Incorporated in the algal tis• sues (24 species in 8. divisions). This strongly Indicated that some of the D-isomer was racemlzed. They suggested this conversion proceeded via oxidative deaminatlon by D-amino acid oxidase, followed by an L-specific reamination or via enzymatic racemization of the D-isomer. Which route was in• volved was not determined. The extended lag period before either D-phenylalanine or D-tyrosine was utilized as a nitro• gen source by N_ lncerta can be explained by assuming that time is required for the induction of either of these en• zymatic systems. i The initial enzyme may be (A) non-oxidative-ammonia lyases, (B) oxidative-L-amino acid oxidase, or (C) transaminase- amino transferase in regard to the metabolic degradation of L-phenylalanine and L-tyroslne" by deaminatlon. In no 14 x C-experiment was cinnamic acid or p-coumaric acid detected for I_j_ galbana or N_j_ incerta which suggested that the am- 14 monia lyases were not present. In C-enzyme assays for phenylalanine ammonia lyase on eighteen other algal species, no activity was detected. Young et al. (1966), who examined four algal species for phenylalanine and tyrosine ammonia lyases also found no activity. When non-labelled L-tyroslne was fed to nineteen species of algae, p-coumaric acid was never detected. So far as is known, the ammonia lyases ap• pear to be solely restricted to vascular plants, certain fungi, and certain species of bacteria (see Towers and Subba Rao,, 1972) V 107. p In enzyme assays for the amino transferase where no a-keto acid was present, no activity was obtained for I» galbana and N.,lncerta. which suggested that the L-amino acid oxidase was also not active in either species. But when an a-keto acid was present activity was obtained for the amino transferase. Excellent activity was obtained for Navlcula. but pre-adapted cells were required before activity was ob• tained for Isochrysls. This suggested that the four day lag period observed before growth of Isochrysls on L-phenyl• alanlne or L-tyrosine as the sole nitrogen source was relat• ed to the induction of this enzyme. No activity was obtain• ed if the cells of Isochrysls were not pre-adapted to either of these amino acids. Amino transferase activity has been previously de• tected in algae for L-phenylalanine (Gassman et al., 1968. Jacobl, 1957 r Stenmark et al., 197*0 but no activity was reported for L-tyrosine. The amino transferase for phenyl• alanine and tyrosine has been demonstrated in plants, fungi, bacteria, and animals (see Towers and Subba Rao, 1972). The importance of this reaction in higher and lower plants has not been investigated thoroughly, possibly because of the interest in the ammonia lyases.. In Figure 33 a scheme elucidated by these studies for the degradation of L-phenylalanine for both I, galbana and N. lncerta Is presented. This scheme was based on the phenylalanine- C studies as well as the metabolism of non• radioactive aromatic compounds. The compounds in brackets were not identified in this study but were expected. 108. L-phenylalanine f\ CH2-CH-C00H RlH2 phenyllactic acid V (f\cH?-CH-C00H ;^NH3 ^ OH . phenylpyruvic acid <^>-CH2-^-C00H 0 >^co2 phenylacetaldehyde <£^-CH2-CHO phenylacetic acid mandelic acid CH-COOH OH benzoylformic acid (^-C-COOH [C?2] n catechol ,S-co2 benzaldehyde [0CH ]' CH Q J OH ;co2 o-hydroxy- benzoic acid benzoic acid COOH *- {^COOH I HO y m-hydroxy- 3-bromo- benzoic acid p-hydroxybenzoic COOH yc-Tv acid HO-O-COOH HO p-hydroxybenzoic HO-^! OHl acid B'I U-dihydroxybenzene P^^-0H] »- BROWN ? +COo semiquinone Figure 33. The degradative route of L-phenylalanlne in Isochrysls galbana and Navlcula lncerta. The compounds In brackets were not detected. 109. For both species, the transaminase reaction product phenyl- pyruvic acid and its reduction product, phenyllactic acid U \k 14 were detected when phenylalanlne-1- C, -2- Cf and -3- C Ik were fed. In the dark feedings with phenylalahine-1- Cf less, than 2,0% of the radioactivity for I_. galbana and 10% of the radioactivity for lncerta were located in these two aromatic acids. The remainder of the radioactivity from Ik Ik the metabolized phenylalanlne-1- C was in the trapped COg. The decarboxylation of phenylpyruvate would produce phenyl- acetaldehyde, but this aldehyde was not positively identi• fied. A radioactive spot was detected for both species where it should chromatograph (see Figures 28 and 29 and Appendix Ik B for location) when phenylalanine-2- C was fed. This alde• hyde ^as.s known to be oxidized to phenylacetic acid In air and it would be expected, in vivo, to be oxidized to phenylacetic acid. Phenylacetic acid was detected for both species when lk lk phenylalanine-2- C and -3- C were fed. This acid was found to be stimulatory to growth in concentrations at or below 0.5 mM for I± galbana and below 0.1 mM for Incerta. Similarly Algeus (19^6) observed concentrations below 1.0 mM stimulated the growth of Scenedesmus obliqus although he did not speculate on metabolism. If Inhibition above 1.0 mM occurred it was not stated. At the aforementioned concentrations for Isochrysls and Navlcula. growth inhibition was observed. With Isochrysls and Navlcula. remaining phenylacetic acid was 110 detected along with benzoic and p-hydroxybenzoic acids. In the same time period before extraction and chromatographic analysis, mandelic acid was totally metabolized to benzoic and p-hydroxybenzoic acids. This suggested that both species have difficulty ln hydroxylating the side chain of phenyl• acetic acid to produce mandelic acid. In I_. galbana the volatile product from phenylalanine detected In the KOH was Identified as a C^-Cg compound. From 14 the side chain cleavage studies of phenylacetic acid-1- C 14 and -2- C, very high backgrounds were obtained for the con• trol ln the KOH. This suggested that phenylacetic acid was the volatile product. Possibly some other Cg-C2 acid was in• volved, but generally their concentrations were very low. The experiments of Vose et al. (1971) indicated .that the .vol• atile compound was not carbon monoxide or phenolic or olefinic ln nature. They suggested that an aromatic compound was in• volved, which is supported by the above results. Mandelic and benzoylformic acids were detected in the 14 autoradiographs when phenylacetic acld-1- C was; fed to both 14 algal species. In the phenylalanine- C experiments, both these acids chromatographed with phenylpyruvic-phenylacetic acids; thus only one radioactive spot was obtained. Comparison of the percentage of radioactivity in phenylpyruvic-phenylacetic acid spots, when phenylalanine-l-^C was fed, with the same spot 14 when phenylalanine-2- C was fed showed that there was a de• C finite increase. Which suggested that these two £-C2 acids were present along with phenylpyruvic-phenylacetic acids. In the growth experiments, mandellc acid was found to be mildly inhibitory for both algal species at high con• centrations, but at low concentrations the growth of Isochrysls was stimulated. When the medium from these experiments was examined, mandelic acid was not detected but benzoic acid was present. This suggested that the observed effeot on growth may not be due to mandelic acid but to benzoic acid. The effect on the growth of many compounds may not in fact be due to the initial substance, but to one of its modified products. Benzoylformic acid would be expected to decarboxy- late to produce benzaldehyde and CO^. When phenylalanlne- ili 14 2- C and phenylacetic acid-1- C were fed to both species, 14 CO;, was trapped in the KOH which confirmed this decarboxy• lation. About 45/6 and J0% of the metabolized phenylalanine- 14 14 2- C in the dark was In the C02 for Isochrysls and Navlcula respectively. Benzaldehyde was not identified in these two species, but it should chromatograph in the same region as phenylacetaldehyde. No reports of benzaldehyde in phyto• plankton are known, but it has been identified in macroscopic marine algae (Katayama, 1962). It's bio-organic origin was unknown. Benzaldehyde was reported to be inhibitory to the growth of Chlorella vulgaris although it stimulated cellular respiration (Dedonder and Van Sumere, 1968, 1971). They also observed that it was rapidly oxidized by Chlorella to benzoic acid. This aldehyde undergoes fairly rapid 112. oxidation to benzoic acid in air. Benzoic acid was detected as a product from the met• abolism of DL-mandelic acid by both algal species. When - h 14 phenylalanlne-3- C was fed,benzoic acld-1- C was only de• tected for N. lncerta. This suggested that very little ben• zoic acid was produced in I. galbana or it was rapidly met• abolized. In the dark about yi% of the metabolized phenyl- 14 alanlne-3- C was trapped as COg as compared to 12$ for N. lncerta. This may have accounted for benzoic acid not being detected. In the metabolism of benzoic acid, the para- and meta-hydroxylated derivatives were detected for both species, but salicylic acid was only detected from Nj, lncerta. This may possibly be due to a very active decarboxylase in I. galbana for salicylic acid (see Table 20). It was un• known if the decarboxylation of benzoic acid or it's hydroxy- 14 lation oroducts or both accounted for the CO trapped from 14 d phenylalanine-3- C. The presence of benzoic acid in the medium was in• hibitory on growth of both species at high concentrations. Similar results were observed for Chlorella vulgaris (Dedonder and Van Sumere, 1968, 1971). They reported no growth stim• ulation at lower concentrations but the growth of both I. galbana and N. Incerta was stimulated with low concentrations of benzoic acid. Dedonder and Van Sumere (1968, 1971) also observed that cell respiration was increased by benzoic acid but they did not examine the medium for any metabolic pro• ducts. Benzoic acid was observed to give a high radioactive count in the control when it was C- labelled. This sug• gested that, for similar reasons to phenylacetic acid., ben• zoic acid was the volatile product produced by N^ lncerta. The presence of o-hydroxybenzoic acid (salicylic acid) had very little effect on growth of either galbana or N. •:' lncerta. except at high concentrations. Similar results were observed for Chlorella vulgaris (Dedonder and Van Sumere, 1971) and Skeletonema costatum (McLachlan and Craigle, 1966) while no effect on Crypotomonad 3-C. Monochrysls lutherl. and Dunallella tertlolecta was observed (McLachlan and Craigle, 1966). This acid also Increased the respiration in C. vulgaris (Dedonder and Van Sumere, 1971) and increased the ATP level and 0^ output in Scenedesmus obtuslusculus (Tillberg/ 1970). No' reports as to the metabolism of o-hydroxybenzoic appear ln the literature but both I. galbana and N. lncerta decarboxylated this acid. In a species of Chlorella. this acid was decarboxylated to catechol which underwent ring cleavage to produce CO (B. Ellis, personal communication). Whether ring cleavage was extra- or intra-diol was not known. This decarboxylation was also reported to occur In higher plants (see Ellis, 1974), Very little effect of m-hydroxybenzoic acid was observed on the growth of either algal species. Similar- results were reported for other planktonlc algal species (Dedonder and Van Sumere, 1971 r McLachlan and Craigle, 1966). p-Hydroxybenzoic acid was not inhibitory on the growth of N. lncerta or four other algal species (McLachlan and Cralgle, 1966), but high concentrations Inhibited the growth not only of I_. galbana but also of Chlorella vulgaris (Dedonder and Van Sumere, 1971 )• Evidence suggested that both I. galbana and lncerta (1) decarboxylated this acid probably resulting ln 1,4-dlhydroxybenzene, (2) bromlnated this acid to 3-bromo-p-hydroxybenzoic acid, and (3) excreted this acid Into the medium. When both species were mass cul• tured on either phenylalanine or tyrosine, p-hydroxybenzoic acid was observed in the medium after all cells were removed. It was not detected ln the medium of the control cultures. It must be kept in mind that p-hydroxybenzolc acid, a pre• cursor of ubiquinone biosynthesis, can be formed directly from chorismic acid (see Figure 1) (Luckner, 1972) which may explain Its common, appearence in all cell extracts. Decarboxylation^of p-hydroxybenzolc acid also occur• red in both Ij galbana and N. lncerta. The product, 1,4- dlhydroxybenzene, was reported to be inhibitory on the growth of Chlorella vulgaris (Dedonder and Van Sumere, 1971)) possib• ly due to the formation of p-benzoquinone which was reported to be extremely toxic for eight algal species (Dedonder and Van Sumere, 1971.; McLachlan and Craigie, 1966). It was not known if the algal culture medium promoted this reaction. The oxidation of a hydroquinone to a quinone, and the re• verse reaction, proceeds through a semi quinone (Mlchaelis,, I96l). These semlquinones are known to be coloured com• pounds and it was observed that ln the growth experiments using C^ vulgaris which contained p-benzoquinone, the medium (1) see Addendum. 115. showed a pronounced reddish colour formatlon(Dedonder and Van Sumere, 1971). . Whether this together with the photo- lytic degradation of 3-bromo-p-hydroxybenzoic acid caused the browning ln the cultures of I_, galbana and N± lncerta was not established. The CO^ trapped from tyrosine-3- C was greater 14 than for phenylalanlne-3- C for both algal species (Tables 14 l4 17 and 18). The CO^ from tyrosine-3- C was probably de• rived from the decarboxylation of p-hydroxybenzoic acid while 14 for phenylalanine-3— C from benzoic acid or the three mono- hydroxybenzoic acids. Thus from tyrosine, a large amount of 1,4-dihydroxybenzene would be formed which probably resulted in browning of the medium. The contribution of 3-bromo-p- hydroxybenzoic acid towards the browning was not determined, but the amount of this acid was always less in the light than in the dark for the tyrosine feedings. This suggested that it contributed to the browning. The content of bromine (65 mg/L,, Home, 1969) was sufficient to have allowed enough 3-bromo-p-hydroxybenzolc acid to be formed to account for the browningIn Figur. e a scheme elucidated by these studies for the degradation of L-tyrosine by both I. galbana and N. lncerta is presented. This scheme was based on the tyrosine-1*^ studies as well as the metabolism of non-radioactive aromatic compounds. The compounds in brackets were not identified in this study but were expected. p-Hydroxyphenylpyruvic acid was only detected for Isochrysls galbana. but the side chain reduction product, p-hydroxyphenyllactic acid was detected for both 116. L-tyrosine HO-c^-CHo-CH-COOH IvJH 2 HO-^CH2-CH-co0h JSNH3 - sjf OH p-hydroxy- /f- p-hydroxy- phenylpyruvic acid HO'-^^-CH^fr-COOH phenyllactic acid 0 >co2 p-hydroxy phenylacetaldehyde H0-^Jj)-CH2-ch0 p-hydroxy- ^ phenylacetic acid HO-^J^CH^COOH p-hydroxy- mandelic acid HO-^2VCH-COOH OH p-hydroxy yuiuxy- p benzoytformic acid H0-v_v-C-c00h L | 6 J p-hydroxy- y-L ^2 benzaldehyde HO-^_^CHO 'HO-^-CHJDH p-hydroxy• p-hydroxy benzyl- alcohol benzoic acid -HO-^^-COOH and 1^-dihydroxybenzene [HO^OH]+CO2 3-bromo-p- hydroxy- i benzoic ac id HC-^)-COOH 0 Br ^ polymerization ^^[^O "] semiqumone BROWN ? Figure Jk. The degradatlve route of L-tyrosine in Isochrysls galbana and Navlcula lncerta. The compounds In brackets were not detected. 117 e I. galbana and.N. lncerta. The presence of this acid con• firmed that p-hydroxyphenylpyruvic acid was produced by both species. The effect of p-hydroxyphenylpyruvate was examined on the growth of Gonlotrlchum alslall (Fries, 197*0 and it was found to stimulate growth at low concentrations. Ex• periments with this acid and with phenylpyruvate must be carefully Interpreted because they are unstable at alkaline ,pH values producing the respective C -C aldehyde and a C0 ol fragment. This was not observed to have occurred iri vivo * lk as less than J% of the metabolized tyrosine-1- C was in the ether, phase while for I± galbana 75$ of tyrosine-2-1^C ^ lk and for N. Incerta 31$ of tyroslne-2- C was in the ether phase. If a C -fragment was lost, these percentages for the ether phases would all be expected to be around 3$» The C^- fragment also should be rapidly metabolized to produce COg, 14 lk therefore the values for C02 trapped from tyroslne-1- C and lk -2- C should be the same If a Cg-fragment was removed. This lk was not observed as 97$ of the metabolized tyrosine-1- C was lk trapped in CO^ for both species while for I_. galbana 25$ and for lncerta 69$ of the metabolized tyrosine^-^C lk was in the . CO,,. These findings confirmed that a Cg- fragment was not produced. Decarboxylation of p-hydroxyphenylpyruvic acid would result in p-hydroxyphenylacetaldehyde. This aldehyde was not lk lk detected in the tyrosine-2- C and -3- C experiments because it probably chromatographed with p-hydroxyphenyllactic and p-hydroxymandelic acids. A very faint spot ln the correct 118 o positions was observed after a 2k hour feeding with tyro- sine-U-^C. Whether p-hydroxyphenylacetaldehyde spontaneously oxidized to p-hydroxyphenylacetic acid, was not established,, p-Hydroxyphenylacetlc acid was detected in the tyro- ik lk sine-2- C and -3- C'feeding experiments. In the growth experiments, high concentrations were inhibitory on growth,, Chromatographic analysis from the growth experiments, re• vealed remaining p-hydroxyphenylacetic acid, p-hydroxyben• zoic acid, and p-hydroxybenzylalcohol. In the same time period, p-hydroxymandelic acid was totally metabolized to p-hydroxybenzoic acid and p-hydroxybenzylalcohol. This sug• gested that both species had difficulty in hydroxylating the side chain of p-hydroxyphenylacetic acid to produce p-hydroxy- mandellc acid. p-Hydroxymandelic acid was detected in the p-hydroxy- lk lk phenylacetic acld-1- C and -2- C feedings. In the growth experiments at high concentrations I. galbana was almost totally inhibited while only mild inhibition was observed for N. lncerta. The other cg~C2 phenolic compound, p-hydroxy- benzoylformic acid was not detected. The unknown compound lk #2 in Tables 17 and 18 for the tyrosine-2- C feeding may be this compound. The chromatographic location was approximately where it theoretically should have chromatographed. This acid when synthesized, was found to be unstable and spontaneously produced p-hydroxybenzaldehyde and probably CO^o In the lk tyrosine-2- C feedings, 59% of the tyrosine metabolized by lb M lk N. incerta was in the C02, while only 2% was in the CO2 119 c for I. galbana. p-Hydroxybenzaldehyde was detected when tyrosine-2- \h 14 C and p-hydroxyphenylacetic acid-2- C were fed to both __ galbana and N. lncerta. This aldehyde was very inhibi• tory on the growth of five other phytoplankton (Dedonder and Van Sumere, 1971; McLachlan and Craigie, 1966) but at low concentrations one species, similar to N^, lncerta. was found to be stimulatory to growth (Fries, 1974). The metabolism of p-hydroxybenzaldehyde produced mainly p-hydroxybenzoic acid and traces of 3-"bromo-p-hydroxybenzoic acid and p-hydroxy• benzylalcohol for both Isochrysls and Navlcula. At no time for either Isochrysls galbana or Navlcula lncerta were any dlhydroxyphenolic compounds detected. This does not mean they were not produced, but only that they were not observed. This suggested that any hydroxylation to pro• duce a dlhydroxyphenolic compound proceeded at a rate slower than the rate for the ring cleavage of such a compound. When a hydroxylation of the side chain of phenylacetic and p- hydroxyphenylacetic acids or the ring of benzoic acid was required, these reactions were very slow and were possibly rate limiting. Thus addition of a second hydroxyl group into the aromatic ring should also be rate limiting, even though both Isochrysls and Navlcula possess the potential for hydroxy• lation of the ortho-, meta-, and para- positions of benzoic acid. With the production of "^CC^ from benzoic acid-(U)- 14 C-ring labelled, it was suggested that the route 120, for ring cleavage was through a Cg-C-^ compound. Unlike the 14 situation in higher plants, benzoic acid-(U)- C-rlng labelled produced no ^CO (Berlin et al., 1971J Ellis et al., 1970), It was established that horoogentisic acid (2,5-dihydroxy- phenylacetic acid) was an intermediate in higher plants under• going ring cleavage in the degradation of tyrosine to C02 (Ellis, 1973). This was the same pathway originally thought to be only restricted to microbial and animal metabolism (Meister, 1965). In the degradation of the aromatic ring of phenylalanine in higher plants, 2,3-dihydroxyphenylacetic acid was believed to undergo ring fission (see Towers and Subba Rao, 1972). In a survey of the degradation of unlabelled tyro- sine by 19 planktonlc species (Table 23), both p-hydroxy• phenylacetic and p-hydroxybenzolc acids were detected. When 14 C-tyrosine-(U)-rlng labelled was fed to seven species of phytoplankton, both p-hydroxyphenylacetic acid and p-hydroxy- " benzoic acid were also radioactive (Table 14). This suggested that the pathway of tyrosine degradation in Figure 34 was present in most if not all algae. Phenylalanine, however, may not be degraded by all species. No phenolic compounds, other than p-hydroxybenzolc acid were detected. When this 14 acid was detected, phenylalanine-rlng-1- C was also degraded 14 to C02< This suggested that the pathway in Figure 33 was the degradative route for phenylalanine. The great number of species that did not degrade phenylalanine to p-hydroxyben• zolc acid or C02 may be the result of poor or no hydroxylase (1) undertaken for part of this dissertation. 121. TABLE 23 Phenolic Compounds Detected from Feeding Kon-radloactlve Phenylalanine and Tyrosine to Various Algae and the Relationship of the Phenylalanine Feedings to the Ketabollsm of Phenylalanine- (ring-l-l'+C). Algal species Control Tyrosine Phenylalanine Phenylalanine rlng-l-1^1* Chlorophyta (2) Brachlmonas submarlna 3(+) K+).2( + ).3( + ) unknown Dur.allella tertlolecta 1(?) K + ),2( + ) Nannochlorls oculata K + ) Haotophyta Coccollthus huxleyl l(+),2(+) Isochrysls galbana 1(+) K++).2( + ),3( + ) Monochrysls lutherl 1(?) K?) Baclllarophyta Amphlprora paludosa 3(+) K+) K?) 1(+) Navlcula lncerta K3+).2(3+),3( + ) K++) Skeletonema costatum 3(?) K + ),2(?) K?) Thalassiosira fluvlatllls 1(+) l(+),2(+) i(?) Cryptonhyta Chroompnas sallna 1(+).3(+) K+).2( + y,3( + > K + ) Cryptomonas str. WHI 1(+)J3(+) K + ).2( + ).3( + ) unknown K + ),2( + ),3( + ) PrrrroPhyta ^VOdlnlum carterl 1(?) l(4+),2(3+),3(+) l(+),2(+) +(light) 3(++) (+).2(?) ghodii5I^Hla_L l 6 3(?) K++),2(+),3(?) unknown fgigg guadrupllcatum 1(?) l(+),2(?) +(light) 1(+) l(+).2(+) 0 v^thnphvta S Hetej^thrl* P> M ).2{ + ) HonaVlaritus sallna K + unknown 1(+) unknown (1) from Table 3. mP S (2) n^i^? r^ ^tected In e*her extract!. 1 = p-hydroxybenzoic acid, 2 = p-hydroxy phenylacetic acid, 3 = P-hydroxybenzylalcohol. Intensity ln brackets. ? =* doubtfult + - trace, ++ = low concentration, 3+ = medium concentration, and k+ = hl?h concentra• tion as determined from colour Intensity on thin layer chromatograms. ooncentra- 122. activity. p-Hydroxybenzoic acid appeared to be a common intermediate in a pathway leading to ring fission in algae. Ring fission in algae does not appear to be important 14 as very, low radioactive counts in the COg were always ob• tained. The main degradative product, p-hydroxybenzoic acid, was always detected. The excretion of this compound into the medium was observed in mass culturing and. when open ocean seawater was analyzed, this phenolic acid was detected (Degens et al., 1964). When the sediments (dead algal cells) were analyzed, o-hydroxybenzoic, m-hydroxybenzoic and p-hydroxy• phenylacetlc acids were also detected. A pathway for the degradation of phenylalanine to benzoic acid appears to be present in higher plants but no clear evidence has been obtained. . In higher plants, fungi, and bacteria o-hydroxyphenylacetic acid was formed from phenyl- pyruvic acid in a similar reaction to the formation of homo- gentislc a.cld from p-hydroxyphenylpyruvlc acid. The aromatic ring was further hydroxylated. then ring fission occurred (see Towers and Subba Rao, 1972). Generally, if phenylacetic acid was fed, it was hydroxylated in the ortho- or para- positions. These products were either (A) further hydroxylated followed by ring cleavage, (B) further hydroxylated followed by the reduction of the side chain to one carbon before ring cleav• age, or (C) the side chain was reduced to one carbon followed by further hydroxylation then ring cleavage (see Towers and Subba Rao, 1972). In animals, phenylalanine was hydroxylated to form tyrosine which was degraded via p-hydroxyphenylpyruvic 123. cinnamic acid <^CH=CH-COOH phenylhydracrylic benzoylacetic acid ^C-CH2C00H Y benzoic acid ^>-COOH p-hydroxy- ,—v benzoic acid HO-<^)-COOH CH=CH_C00H p-coumaric H0^3" acid N—' p-hydroxy- . phenylhydracrylic HOvJ^H-CH2~COOH acid w OH °r HO-^>-CHO + [ch^COOH p-hydroxy- r " -S* p-hydroxy- benzoylacelic H0-f7-C-CH^C00H benzaldehyde acid L >=/ 5 2 J Figure 35. The degradative route of cinnamic and p-coumaric acids in Isochrysls galbana and Navlcula lncerta. The compounds In brackets were not detected. 124. acid to homogentislc acid. No evidence for the hydroxylatlon of phenylalanine to form tyrosine was found for I± galbana or N. Incerta. The other possible degradative pathways are through phenylalanine and tyrosine ammonia lyases. These enzymes have not been found ln algae, but their products are degraded ln a similar pathway to that reported ln higher plants (see Ellis, 1974). In Figure 35 the proposed scheme for their degradation is presented and involves a 0-oxidatlon of the side chain. In the degradation of cinnamic acid, phenyl• hydracrylic acid and benzoic acids were detected and in the degradation of p-coumaric acid, p-hydroxyphenylhydracrylic acid, p-hydroxybenzaldehyde, and p-hydroxybenzolc acid were detected. Whether acetate was removed from the hydracrylic acid or it's oxidized product Is unknown. Acetate was re• ported to be lost directly from p-hydroxy-3-methoxyphenyl- hydracrylic acid (Toms et al., 1970). The degradative routes for phenylalanine and tyro• sine in algae do not result in any energy yielding reactions. Therefore, heterotropic growth on either of these amino acids appears to be Impossible. This explains why heterotropic growth has never been observed. The metabolism of phenyl• alanine and tyrosine appear to be for obtaining nitrogen, the carbon skeleton being eliminated in a non-toxic form. 125 LITERATURE CITED. Algeus, S. 1946. 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ROUTINE STERILITY CHECKS : The technique of enrichment of possible microbial contami• nants (bacteria and molds) by heterotrophic growth In a seawater medium enriched with organic material was used for checking all algal cultures for such contamination. The STP and ST^ media of Tatewakl and Provasoli (1964) were used for such contamination checks. One to two drops of the algal culture growth medium were added to a tube of each medium and Incubated about three o weeks In the dark at room temperature (22-25 C) for a contami• nation check. The appearance in either medium of cloudiness and turbidity typical of bacterial growth or of discolouration and tuft-formation typical of mold growth was taken as evidence of possible contamination. From a positive tube, 1-2 drops were subcultured in the same medium, for confirmation, and 1-2 drops were examined microscopically. In the absence of any visible changes in the sterility check tube, the algal culture tested was considered to be free from contamination. 135. APPENDIX B. SPRAY REAGENTS The spray reagents employed in the detection of various aromatic compounds separated by paper and thin layer chroma• tography are« (A) Phenollcs. Diazotized p-nitroaniline reagent (Ibrahim and Towers, I960): 5 ml of 0,J%p-nitroaniline, 1 ml of 5% sodium nitrite and 15 ml of 20% sodium acetate were com• bined, in that order, just prior to use. After spray• ing with this solution the chromatogram was allowed to dry briefly and then oversprayed with a 5% aqueous NaOH solution. Phenolic compounds appear as various• ly coloured spots. Ferric chloride reagentJ 2% ferric chloride in 95$ ethanoli detects or- thodihydroxy phenolic compounds as brownish or reddish spots, (B) Aldehydes. 2,4-dlnitrophenylhydrazine reagent (Dawson et al.« 1969. p. 517)* 0ek% 2,4-dinitrophenylhydrazihe in 2N HC1; de• tects aldehydes and ketones as yellow spots which turn red-brown when oversprayed with 10$ aqueous NaOH solution. Appendix B (cont. ) 136. (C) Acids. Methyl red-bromothymol blue reagent (Handbook of Chromatography. Vol. 2, 197^, p. 132): 0.2 gm methyl red and 0.2 gm bromothymol blue ln a mixture of 100 ml formaldehyde and kOO ml of 96% _ ethanol and adjust pH to 5.2 with 0.1 N NaOH; detects organic acids as yellow spots on a pink background. Upon exposure to NH^ vapour, spots become red-orange on a green background. Chromatographic positions of various 0^-0^ aromatic compounds in solvent system C s isopropanol:conc. NH^OH: water (8il»l, V/V/V). solvent front p-hydroxybenzylalcohol O 3,5-dibrorao-p-hydroxybenzylalcohol benzoic acid O 2,4-dihydroxybenzolc acid O m-hydroxybenzoic acid 0~ p-hydroxybenzoio acid 3.4- dihydroxybenzolc acid O O 3.5- dibromo-p-hydroxybenzolc acid C •3,5-dibromo-p-hydroxybenzoic acid mono- potassium sulfate + + origin ester Appendix B (contd.) 137. Chromatographic positions of various aromatic compounds. Ai 2% aqueous formic acid; B: Benzene:HOAci(10i7:3, v/v/v) 1, Benzaldehyde and phenylacetaldehyde; 2. trans-clnnamlc acid; 3. cls-clnnamlc acid; h, benzoic acid; 5. phenylacetic acid; 6. ? benzoylacetlc acid ?; 7. hydracryllc acid; 8. benzoyl- formlc acid; 9. mandelic acid; 10. phenylpyruvlc acid; 11. phenyllactlc acid; 12, p-hydroxyphenylacetlc acid; 13. p- hydroxybenzaldehyde; lk. cls-p-coumarlc acid; 15. p-hydroxy• benzolc acid; 16. 3-bromo-p-hydroxybenzolc acid; 1?, trans-p- coumarlc acid; 18. protochatechulc acid; 19. p-hydroxyphenyl- pyruvlc acid; 20. p-hydroxyphenylhydracryllc acid; 21. p- hydroxyphenylacetaldehyde; 22. p-hydroxyphenyllactlc acid; 23. p-hydroxymandellc acid; Zh. p-hydroxybenzylalcohol; 25. unknown #2. 138. . APPENDIX C. CHEMICAL- PREPARATIONS OF NON-RADIOACTIVE COMPOUNDS Preparation of 3.5-dlbromo-p-bydroxybenzolo acid. 1.10 gm p-hydroxybenzoic acid was dissolved In. 2.5 .ml glacial acetic acid. To this, 1 ml Br2 in 1 ml glacial acetic acid was carefully added. The reaction mixture was heated for one hour, then the excess Br^. HBr, and acetic acid were .driven off under a stream of nitrogen. The white product was recrystalized from dilute acetic acid. The yield was 2.24 gm, (94$ of the theoretical yield) and the melting point was 266-268°C (Lit. 268°C, Leulier et al. 1927). NMR spectrum in dg acetone was 5.50 (S, -OH) and 7.10 (S, 2H), and the MS was m/e = 298 (45), 296'(100), 294 (48), 281 (27), 279 (56), 277 (32),, 253 (4.9), 251 (7.9), and 249 (4.9). The UV-spectrum is presented in Figure 6. It was found that this bromophenolic compound was unstable over a long period of time and degraded to give the spectrum shown ln Figure 6. Preparation of ?.5-dlbromo-p-hydroxybenzaldehyde. 1.00 gm of p-hydroxybenzaldehyde was dissolved in 1.5 ml glacial acetic acid and to this 1 ml Br2 in 1 ml glacial acetic acid was carefully added. This reaction mixture was heated for one hour, then under a stream of nitrogen, the ex• cess Br2, HBr, and acetic acid were removed. The white pro- duct was recrystalized from an acetic acid water mixture and the final yield was 2.21 gm, 96$ of the theoretical yield and the melting point was 181-183° C. The NMR ;spectrum in dg 139. Appendix C (oont.) 140. Appendix C (cont.) acetone was 3.55 (S, -OH), 8.06 (S, 2H) and 9.83 (S, -CHO) and the MS was m/e = 282 (38), 281 (54), 280 (77), 279 (100),. 278 (42), 277 (52), 253 (9.5), 251 (19.0), and 249 (105). In Figure 7 the UV spectrum Is presented. Preparation of 3.5-dlbromo-p-hydroxybenzylalcohol. Two methods of synthesis were used, (1) from •p-hydroxy• benzylalcohol and (2) from 3»5-dibromo-p-hydroxybenzaldehyde. 0.05 gm of p-hydroxybenzyalcohol was dissolved in 1 ml glacial acetic acid. 0.5 ml Br2 in 0.5 ml glacial acetic acid was added, and the reaction mixture heated for one hour. Under a stream of nitrogen, the excess Br2, HBr, and acetic acid were removed, leaving a white crystaline product which was a tribromo substituted compound (see Auwers et al., 1899). This was dissolved Inacetohe arid water was added uht 11 a .faint turbidity appeared which was redlssolved by adding more ace• tone. After 5-6 hours at room temperature, water was added and the precipitate filtered off. Following recrystalllzation from dilute acetic acid-, 0.023 gm of product (20$ yield) was obtained which had a melting point of 113-114° C (Lit. ll6-H7°C). Method ( 2 ) Involved reduction of 3»5-dibromo-p- hydroxybenzaldehyde with lithium aluminum hydride. Some pro• duct was obtained but in very poor yield. Preparation of 3-bromo-p-hydroxybenzolc acid (Leuller et al., 1972). m 3.5 S p-hydroxybenzolc acid was added to 80 ml H202 solution (ll.3-.ml-of 30$ H202 diluted to 100 ml - 3.4 gm H202/ 100 ml). To this 4.15 ml HBr (48$ HBr in HOAc) was added and Appendix C fcont.) l4l. left 24 hours. The product was collected by filtration, and recrystallized from boiling water. A yield of 4..91 gm of a mixture was obtained. Approximately 60% was the monobromo,, 35% the 3,5-dlbromo, and 5% a trlbromo derivative (probably 3,5-dlbromo-p-hydroxybenzylbromlde). The major MS peaks for the monobromo were m/e = 218 (83), 216 (89). 201 (95), and 199 (100). Preparation of p-hydroxyphenylacetaldehyde (Greensteln et al., I96D. To 100 mg tyrosine dissolved in water, 200 mg ninhydrin was added. This was boiled for 10 minutes, cooled, and ex• tracted with dlethylether. The ether extract was evaporated to dryness and p-hydroxyphenylacetaldehyde was purified by sublimation at 150* C In vacuo; followed by chromatography in solvent system A. The Rf value in solvent system A and B aret Solvent system A Solvent system B p-hydroxyphenylacetaldehyde 0.74 0.15 p-hydroxyphenylacetic acid 0.67 0.25 p-hydroxybenzaldehyde 0.64 0.39 p-Hydroxyphenylacetaldehyde when sprayed with diazotized p- nltroaniline produces a yellow-red colour and when sprayed with 2,4-dinitrophenylhydrazine a yellow colour. In Figure 8 the UV-spectrum is presented. Very little product was ob• tained, thus further characterization was impossible. - The method of Langheld (1907) using sodium hypochlorite to synthesize p-hydroxyphenylacetaldehyde was tried but again very little product was obtained. Preparation of p-hydroxybenzoylformlc acid. The method described for the oxidation of mandelic 143. Appendix C (cont.) acid to benzoylformic acid was tried (Organic Synthesis, Vol. I., p,24l) but the only product recovered was p-hydroxyben- aldehyde. In place of KMnO^ as the oxidizing reagent, al• uminium isopropoxlde was prepared (Vogel, 19&7) and used. 50 mg DL-p-Hydroxymandelic acid was dissolved in anhydrous acetone and 20 ml aluminium isopropoxlde was added. This was allowed to react for one hour, then the reaction was fil• tered and evaporated to dryness. The residue was dissolved in dilute HC1 and extracted into ether. A very poor yield was obtained. In Figure 9 the UV-spectrum of the product is presented. It was found that this product rapidly decarboxy- lates to give p-hydroxybenzaldehyde, (see Figure 9). This was also confirmed by chromatography. No other data pertain• ing to this compound was obtained, but 'because of its in• stability it is not likely to be present in extracts from any metabolic studies. Preparation of phenylhydracrylic acid. - This was prepared using the method of Wright et al., 1958. 1.65 gm cinnamic acid was dissolved ln 3 ml of glacial acetic acid containing 30$ HBr. After reacting for three days the acetic acid and HBr was removed under a stream of nitrogen. The residue was dissolved and recrystalized from benzene. The crude solid was boiled in 8 ml water for 15 min, cooled, then extracted into ether. The ether was evaporated and the re• sidue was recrystalized twice from chloroform to give a yield of 0.29 gm or 40$ of the theorectlcal yield. The melting point was 92-94°C (Lit. 92-93°C) and the UV-spectrum Is presented in Figure 10. The MS was found to be m/e 166 (37), 148 (10.8), 147 (121), 107 (100), 106 (325), 105 (295), 79 (61..5), 77 (50), 144. Appendix C (cont.) Figure 39. Absorption spectrum of p-hydroxybenzoylformic acid and Its spontaneous degradation product - p-hydroxybenzaldehyde. Both spectra are ln ethanol. p-hydroxybenzoylformic acid — p-hydroxybenzaldehyde OPTICAL DENSITY 250 275 300 WAVELENGTH (nm) Figure 4,0. Absorption spectrum of synthetic phenylhydracryllc acid ln ethanol. 16 1.2 I OPTICAL DENSITY Ci8| 0.0 r 200 225 250 275 300 325 350 WAVELENGTH (nm) Appendix C (cont.) . — Is-5 • and 71 (23.5), while the NMR ln dg acetone was 2.68 (d, 2H -CH2~, J = 7Hz), 5.11 (t, 1H, -CH-, J = 7Hz), 6.25 (m, IH, H of -OH and -COOH), and 7.30 (m, 5H, aromatic). Preparation of p-hydroxyphenylhydracryllc acid. The method used to synthesize phenylhydracrylic acid was tried and found to be unsuccessful. The. same method was used with p-acetoxycoumarlc acid replacing p-coumaric acid. Some product was obtained but hydrolysis of the ester to the free acid was unsuccessful because the phenolic hydrogen ln the presence of base forms a quinone which causes dehydration back to p-coumaric acid. A similar problem was encountered ln the attempted synthesis of 4-hydroxy-3-methoxyphenylhydra- crylic acid (Toms e_t al., 1970). They believe that enzymatic synthesis is probably the only way to make their product and the same is likely true of p-hydroxyphenylhydracrylic acid. Preparation of sulfate esters of phenolic compounds. The method outlined by Hodgkin et al., (1966) was used to prepare the sulfate esters of p-hydroxybenzoic acid, 3i5- dibromo-p-hydroxybenzoic acid, 315-dibromo-p-hydroxybenzal- dehyde and p-hydroxybenzaldehyde. 1.2 umoles of the phenol was dissolved ln 10 ml water containing 0.5 gm of potassium car• bonate, 0.24 gm sulfur trioxide-pyridine complex (Aldrich Chemical Co., St. Louis, Missouri) was slowly added with stlr- ring. The solutions were cooled to 0 Cfor 24 hours and the resulting crystals which were collected were recrystalized from water.. The yields were approximately 60% for both acids but for the aldehydes the yields were reduced to 40$. This was due to decomposition or side reactions which resulted in an Initial brown to black coloured product for both aldehydes. 146. APPENDIX D. CHEMICAL PREPARATIONS OF RADIOACTIVE COMPOUNDS Preparation of L-tyroslne - uniformly ring-^C. The procedure used was based on that of Ellis et al. (1973). One liter of medium (0.2$ L-tyrosine, 0,2$ KH2P0^, 0.1$ MgS02^7H20, 0.0001$ FeSO^«7H20, 0.01$ pyridoxine, 0.6$ glycerol, 0.5$ succinic acid, 0.1$ DL-methlonlne, 0.2$ DL- alanine, 0.05$ glycine, 0.1$ L-phenylalanine and 0.5$ hydro- . lyzed soybean protein in distilled water with pH adjusted to 7.3) in a 2,8 liter Erlenmyer flask was cooled after auto- claving and inoculated from a fresh slant culture of Erwlnla herblcola (ATCC 21434). The flask was then placed on a re- o clprocatlng shaker at 30C for 14-16 hours. The cells were harvested, washed, and then resuspended ln 5 ml buffer 0.1 M KH2P0ij, pH 6.0 (with 5 n>M mercapteothanol) per gram of cells. The cells were disrupted by sonification for 5 minutes, cen• trifuged and the supernatant was fractionated with ammonium sulfate. The protein precipitating between 30$ and 70$ saturation was dissolved ln the above buffer, then dialyzed overnight against the same buffer. 2 ml portions of this o enzyme preparation were frozen and stored at -20 C until re• quired. The activity of the enzyme was measured by incub• ation of 0.1 ml enzyme for 30 minutes at 30°C with 5 umoles S-methyl-L-cystelne (SMC), 0.5 umoles pyridoxal phosphate and 200 umoles of pH 7.8 KH2P0ij. buffer in a total volume of 1 ml. Appendix D (cont.) LZ*7* After measuring the formation of pyruvate (Friedemann, 196?), one unit (U) of activity corresponded to the formation of 1 umole of pyruvate/minute. For the synthesis of L-tyros ine-ring-^C, phenol-U-^C (New England Nuclear) with a specific activity of 9.3 mCi/mM was used. 26 uCi were placed in a tube and the ether was re• moved under a stream of nitrogen. To this tube 400 ;umoles SMC, 1,5 jumoles pyridoxal phosphate and 800 mmoles of pH 7.8 KR^POi), buffer was added. After addition of 160 roU of the en• zyme preparation, the reaction (total volume 4.0 ml) was in• cubated at 30°C for three hours. The reaction mixture was spotted onto several sheets of Whatman 3MM chromatography paper and developed in solvent system C. The tyrosine-^C was located by autoradiography, and eluted with ethanol. The eluate was spotted on Avicel plates and chromatographed further in pyridine: isoamylalcohols glacial acetic acid: water (8:4:1:1, V/V/V/V). The yield of L-tyrosine -uniformly ring-^C was 20 uCi or 76% of theoretical value. Preparation of p-hydroxyphenylacetlc acld-l-^C and -2-^C. The method is based on that of Kristensen (1973). 6 ml dlalyzed enzyme (3 ml L-amino acid oxidase #12993 from Cal• biochem containing 2 mg/ml protein and 3 ml D-amlno acid oxi• dase #129852 from Calbiochem containing 0.5 mg/ml protein) in Tris-HCl buffer (0.4 M, pH 7.8) with 12 ;uCl DL-tyrosine^-^C and 0.1 mg of cold DL-tyrosine in 1 ml buffer were incubated Appendix D (cont.) at 30° C for 24 hours. In a similar reaction 12 uCi of DL- 14 tyrosine-3— C was used to prepare p-hydroxyphenylacetic 14 acid-2- C. Both reactions were stopped by adding cone. HC1, cen- trifuging, and extracting three times with ether.. The ex• tracts were washed with IN HC1, evaporated to dryness, redls- solved in ether, and chromatographed on Avlcel plates ln sol• vent system A. Each product was located by autoradiography and eluted with ethanol. The yield of p-hydroxyphenylacetic acld-l-^C (specific activity, 16.9 uCi/mmole) was 1.46 uCi and for p-hydroxyphenylacetlc acld-1- C (specific activity, 15.5 uCi/mmole) it was 0.33 uCi.. Unlike the results in 14 Kristensen's paper, other C-products were observed in the autoradiographs. 14 Preparation of p-coumarlc acid-2- C„ This preparation is based on that of Austin and Meyers (1965). Into 3 ml pyridine containing 3 drops piper- dine, 110 mg p-hydroxybenzaldehyde, 100 mg malonic acid, and 14 , 100 uCi malonic acid-2- C (ICN Chemical and Radioisotope division, 15 uCi/mmole) were dissolved. The reaction was heated 2 hours on a steam bath, cooled, acidified with cone. HC1, made up to 50 ml with water and then extracted with ether. The ether was extracted with 5% NaHCO^, which was acidified and extracted with ether. This extract was purified by chroma• tography ln solvent system B yielding 70 uCi p-coumaric acid- . 14_ APPENDIX E. THE EFFECT AND METABOLISM OF OTHER AROMATIC COMPOUNDS In Figures 4l to 54 are presented the effect of various aromatic compounds on the growth constant and lag period of Isochrysls galbana and Navlcula lncerta. The aromatic com• pounds studied were: phenylacetic acid Figure 4l Page 150 p-hydroxyphenylacetic acid 42 150 mandelic acid 43 151 p-hydroxymandelic acid 44 151 benzoic acid ^5 152 p-hydroxybenzolc acid 46 152 p-hydroxybenzaldehyde 47 153 3,5-dibromo-p-hydroxybenzoic acid 48 153 3,5-dibromo-p-hydroxybenzaldehyde 49 154 cinnamic acid 50 ' 154 p-coumaric acid (brief note on p.:155 ) 51 156 m-hydroxybenzoic acid 53 158 o-hydroxybenzoic acid • 5^ 158 % INHIBITON ol GROWTH CONSTANT LAG PERIOD growth constant (AOD/day) (days) a Navicula incerta (x—x) 9 a o o OOO P 1 o o o C3L o g O OOO a IVJ —• —. LP Ln en s jq o ro a O Ul O cn o cn o cn p o » •s3- < a o O o o c Isochrysis galbana (• f a3 B rt rt ro —• P 3" H" 3 CB o cn 3 cr oCC P ' t» rt 03, a B o • M ct P (K o •CoD •a - H1* a o p. 3 O B >-» O a M rf 01 0 O o 3" 0 Ho> ca P. ca o 3 » ct a1 a3- » P3 •i) t* -p- ro • Navicula incerta (x—x) p> o a o o 0 3 r» o o o o >-> 0 o o o o o o rf . ct cn cn cn cn o p 0 S i O O LP 3 CO 0 —I I 1— 0 s in 0 i o 3 o -l> Isochrysis galbana (• ) P. ct rt O p O O O p O S3 3" O cn o cn p cn o o p o o o P, ° 0 CO —* —* —. _i ro ro ro <) O cn oo co o * NJ o p i-» CO 0 -I 1 1 1— O 3 "> c ca "1 t-J -t CO o 0 30 ct h* rt ca 3 O 0 o (0 3 "> •1 p. ct •o VT 0 1 0 3 w CN VS O p. d •1 ^ S. CD oM o £- v; m o •o p. CO 3 —I un to* vs 0 ca o o CO •—I c. o ct 3 h~ o >_ •1 o < ca 0 t* o ca t-* P- X, w V.. INHIBITON of e GROWTH CONSTANT LAG PERIOD a growth constant • • (AOD/day) (days) Navicula incerta (x—x) V xt o~> (9 :o1 t- O i 01 p. o o 31 ! "1 p. a (H H CO •* Q o O 3" 0 •1 o •< H- CO i—i cn (J- to o 3 2 np ct CC o, CO 3 P CD 9i !•» in V. INHIBITON of GROWTH CONSTANT LAG PERIOD c 1 growth constant • (AOD/day) CO (days) •e- *• • Navicula incerta (x—x) B 0 P 3 o o o o H io o b b b ct IH CO CO ro ro _. » J T cn o cn a cn cn CD P cn o 3 —I— —l— B g SO >~i o 3 (rOt Isochrysis galbana ( — --•) p. s. ct ct OONIOILI^WM-. o o o p o p a p •3 3" OOOOOOOOO b b b b b b b so CD o <» o o o o QD br o o CD ai oo o ro o 3 ~> 01 <-> -i—i 1—i 1 r- ct SO SO O 3 cCDt en ct 01 3 o SO o CO 3 •1 p. ct SO H a O o. • P I O ^ TO •a Z o, rr •o O J> (0 *<: 1 a m •* o o•i p. X N << 5 o B "> P 3o£ 3 M P. 01 CD 5^ o H o t* i—c cn •3 O C! o •1 | § P 2 01 o s, —' OJ if 3 01. P. ro fc> 2 V. INHIBITON of GROWTH CONSTANT LAG PERIOD growth constant (AOD/day) (days) Navicula incerta (x—x) o P o p O O o P . p o o o CD o o r cn + + • + + CD O IS) cn CD rsj • i CJ o °P . p o o o tn r1 s o a -r -r- IP *-»• o 3 P Isochrysis galbana (• ) o '3 CO CK f V-1 «* to to n rx) (0 •l - t* o p. . o - P : 3 -P. Vo INHIBITON of cn GROWTH CONSTANT LAG PERIOD . c growth constant t» (AOD/day) (days) CO - -p- ON - • Navicula incerta (x—x) p (R .a -1* pppp p p pp p3 . - o1 P E M cn p in o o 55' ct >-> o o P P —I 1 1— —r— -r— T T- -4 a t* O o o o '-•) Isochrysis galbana (•— - cH * -. C3Q P - ct ct - p 3 CO 3 ct o • CO CO • P ••"> c•1t r- 3p . CD . p • O -fJ ct • P - 01 cn O •a o p. o o p t-1 4.0 rX X— o o »—* 3.0 CC ~ 3.5 / UJ 100 - ' 10.0 2.5x10s 5x105 10"4 2.5x1Cr" 5xl0/) 10~3 2Dx1(? s U 3 CONCENTRATION (M) 2.5x10 5x105 \6 2.5x:6Z' 5x10'' 1fJ 2J0X1O3 Figure l*7. Diagram of the effects of p-hydroxybenzaldehyde on the CONCENTRATION (M) Figure k8. Diagram of the effects of 3,5-dlbromo-p-hydroxybenzolc growth constant and Jag period of Isochrysls galbana acid on the growth constant and lag period of Isochrysls and Kavlcula lncerta. galbana and Kavlcula lncerta. • S*' *0 V. INHIBITON of GROWTH CONSTANT LAG PERIOD •a growth constant (AOD/day) (days) Navicula incerta (x—x) X 3- a o o o » V4 h* o o P o o g CD b b b § r-l a *~ OJ on oo rt a CD co on ro _> o o b So •1 CD O o o o o P —r- B 3 a 03 ct o 3 3" -» Isochrysis galbana ( ) a a ct cn-F~oJrvj_. o co o ° P p p p •z oooooo b o b b b o o g P wi CO <4 O CD -tV—i 1 1 1 r- o t "1 c •» H CD ro to •> O "on 3 ct x, •» 3 CO 3 CO o rt O a SO 3 ct cf P • O P 3 1 o P- a zz M o* o P m m o 5 01 rj o zz\ CD 1 •j TJ h- 1 O • •3i— i on P. 'A O & o a•j !-•> o •A H «S CO cf • o, o CD ' oo o 3 3" N ro •J P 'a •< M x, CO H- CaD 9o CO 1 t* oq V. INHIBITON of c GROWTH CONSTANT LAG PERIOD •» CD growth constant (AOD/day) (days) On o • Navicula incerta (x—x) S3 CJ a P 0 y p o O o o 3 P CO 0) s b b b b —• -» ro 071 O ct 1 on cn p P o o o o on O cn o on 6 H 3 B i I r P ct O p Isochrysis galbana ( ) 3 3 + + + O a ct o o o o p o p o o CD 3 —* —• ro oo 1 H CD b b b b b b o b b o o o o o OJ ct P ro ro ro ro ro CO P W CD cn cn ^o oo CO o —' ro cn o XI on —r- CD CD 1—r -J— •1 O ro I—" ct on 1 o 01 a o i Y o "1 I » o i i H l-» CO 3 on i / 3 o p i p- B o, r» '< CO o H- p ro 0) o t* )<1 P a H o 3 i—i cn if ct O £- 3 h e P Z a n -—\ m 2 3, a — oo to 3T b So i ' i 155. p-Coumarlc acid Chromatography of the ether extracts of the medium from the OD tubes and the one liter cultures revealed the cis- and trans-p-coumaric acids, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and an unidentified spot which re• acted with PNA to give a purple colour. This compound was tentatively identified as p-hydroxyphenylhydracrylic acid and chromatographed directly below the cis-p-coumaric acid (see Appendix <©). When this compound was isolated and again chromatographed, it was observed that a portion chromatographed as before, but a portion remained at the origin (purple colour with PNA), In Figure 52 the absorp• tion spectrum of p-hydroxyphenylhydracrylic acid from both chromatographic locations is presented. Both spectra were identical, but why a portion remained at the origin was unknown. When p-hydroxyphenyllactic acid and p-hydroxy• phenylhydracrylic acid were chromatographed in solvent system A two distinct spots (both purple with PNA) were obtained. The 'lactic acid' chromatographed in front of the 'hydracrylic acid' (see Appendix B), In Figure 52 the spectrum of p-hydroxyphenyllactic acid is presented for comparative purposes. No comparison of the p-hydroxy• phenylhydracrylic acid to a synthetic sample was possible because all.attempts at synthesis were unsuccessful (see Appendix B). 3.8 Q 3.6 O i—• ^-control / 2.0 3A -*—-—# K / LU <{> 1.5 3.2 < control 0.028 0.027 0.067 _ 0.026 T 0.066 j 0.025 LO ~ y >- rt0. 0 0.065 c 2U rt 8^ _Q Oj Q (_) 0.064 0.023 X O C rt oil 0.063 0.022 _rt i/) cr >~ co o 0.062 x: 0.021 *> u ^* o rt 0.061 0.020 0.019 0.018 ° c +10 +.10 2 rt Q + 5 0 i— c 0 10 20 -o O 30 IS ° CT) 2.5X105 5x105 104 2.5x10^ 5x10^ 103 2.0x103 - CONCENTRATION (M) Figure 51. Diagram of the effects of p-counarlc acid on the growth constant and lag.period of Isochrysls galbana and Navlcula lncerta. Figure 52. Absorption spectrum in ethanol of the phenolic acid tentatively identified as p-hydroxyphenylhydracrylic acid from Isochrysls galbana and Navlcula lncerta. p-hydroxyphenylhydracrylic acid (Rf=0.6) p-hydroxyphenylhydracrylic acid (from origin),! p-hydroxyphenyllactic acid OPTICAL DENSITY 250 275 WAVELENGTH (nm) 5.0 4.0 o o U in 4.5 3.0 02 4.0 2.0 control 0.072 0.032 0.070 „ 0.030 0.068 i 0028 LO 0.065 ro0.02 6 roc JQ • 0.06/, 0.024 : o a, 0.062 ai 0.022 or o 0060 0.020 O058 0X118 + 5 ° c 0 0 2 ro O ^ 5 10 S S 10 20 15 30 ~... $O 20 40 s 5 3 3 2.5x16 5x10 10* 2.5x10'' 5x10^ 10~ 2.0X10 2.5x10s 5x105 IO4 2.5x10* 5x10* 10~3 2J0X1CTB CONCENTRATION (M) CONCENTRATION (M) Figure 53. Diagram of the effects of m-hydroxybenzoic acid on the Figure $k. Diagram of the effects of o-hydroxybenzoic acid on the growth constant and lag period of Isochrysls galbana growth constant and lag period of Isochrysls galbana' and Kavlcula lncerta. H and Navlcula lncerta. v_n CO • 159. ; ADDENDUM Page 8. Table 1. Bac1liar1ophyta Phaeodactylum trlcornutum D-phenylalanine 2 mM Growth 88$ of control 4 mM Growth 85$ of control Hayward (1965) Page 9. Table 2. Chlorophyta Chara zeylanlca Tyrosine (isomer ?) 0.07 and 0.7 mM negative effect on growth Forsberg (1965) Bacillariophyta Phaeodactylum trlcornutum L-tyrosine 2 mM Growth 30$ of control 4 mM Growth 19$ of control Hayward (1965) Page 11. Line 14. L-Phenylalanine ammonia lyase has been reported in chloroplast preparations from Dunallella marina (Lbffel- hart et al.,1973). They reported an activity of 0.003 nMol/hr/mg protein which was associated mainly with the thylalcoid membrane. Page 12, and Table 4 (Page 13). Both phenylacetic acid and p- hydroxyphenylacetlc acid have been isolated from Undarla plnnatlfIda (Abe et al., 1974). Both these acids can be hypo- thetlcally implicated ln the degradation of Phe or Tyr. Page 13, Table 4. Iodlnated amino acids (3-iodotyrosine, 2,5- dlodotyroslne, and 3',3»5-triiodothyronlne) have been isolated from Rhodymenla palmata (Scott,1954). Also, ln 10 species of algae over 6 divisions, lunularic acid has been detected (Pryce, 1972). Page l4„ Table 5. From Halopltys lncurvus. 3,5-dlbromo-4-hydroxy- benzolc acid (Aug!er et al., 1956f Chantraine et al.. 1973) and 2-hydroxy-3(3',5'-dibromo-4*-hydroxyphenyl)-acrylic acid l6o. (Chantraine et ai., 1973) have also been isolated. Also in Table 5» under Rhodophyta Brongnlartella byssoides #7,l6 (Fries, 1973) Rhodomela larlx #16 (Weinstein et al.. 1976) Page 103. Internal and external amino acid concentrations for the uptake of L-phenylalanlne and L-tyroslne. Amino acid External Internal pool cone. (mM)^ - and cells pool cone. I. galbana N. incerta utilized (mM) 2 Phe Lt 0.1 0.7 59. non-adap Dk 0.1 0.5 49. cells Lt 0.01 0.2 18. Dk 0.01 0.1 14. Phe Lt 0.1 1.1 54. pre-adap Dk 0.1 0.5 48. cells Lt 0.01 0.03 8. Dk 0.01 0.02 3. Tyr Lt 0.1 1.0 183. non-adap Dk 0.1 0.6 161. cells Lt . 0.01 0.1 38. Dk 0.01 . 0.04 36. Tyr Lt 0.1 1.2 73. pre-adap Dk 0.1 0.3 43. cells Lt 0.01 0.05 3. Dk 0.01 0.04 3. (1) after one hour's incubation. (2) Lt = lightt Dk = darkj and adapt - adapted. The above internal concentrations comprise the soluble and insoluble pools. In each case the internal cellular conce• ntrations of amino acid was greater than the external conc• entration. This suggested both species can assimilate these amino acids against a concentration gradient. The degradat• ive rates for both L-phenylalanine and L-tyrosine for both species was less.than their uptake rates. 161. Page 114. Decarboxylation of p-hydroxybenzolc acid may be accom• plished by an oxidative or a non-oxidative mechanism. In the schemes (Figures 33, and 34), the production of 1,4- dlhydroxybenzene would be obtained as a result of oxidative decarboxylation while phenol would be produced by non-oxida• tive decarboxylation. No evidence was obtained as to which route was used by both species. Page H5t Line 5« "Cells of both species produce a brown ether- insoluble compound". This may also be a result of peroxida• ses acting on simple phenols to give polymeric compounds. Page 124, Line 5« The presence of phenylalanine ammonia lyase was detected in Dunallella marina (Loffelhart et al.. 1973) and the presence of lunularic acid in algae, including a Navlcula sp. (Pryce, 1972) suggests this enzyme may be widely distributed in algae. The biosynthesis of lunularic acid is thought to be through a Cv-C_acid (cinnamic or p-coumaric 6 3 acid) with 3 malonic acid units. Unlike in higher plants, where the ammonia lyases are the major enzymes and the transaminases are minor, the reverse is probably true for algae. In time, from cells cultured under the correct physiological conditions, the ammonia lyases will be det• ected in algae. Page 131. Ragan and Craigle, 1975 see Ragan and Cragle, 1976. ADDENDUM LITERATURE CITED Abe, HY, M. Uchlyama, and R. Sato. 1974. Isolation of phenyl• acetic acid and its p-hydroxy derivative as auxin-like substances from Undarla pinnatlfIda. Agr. Biol. Chem., \ 381 897-898. 16.2. Augler, J., and P. Mastagli. 1956. Sur un compose phenol!que brome extrait de I'algue rouge Halopltys incurvus. Comptes Rendus., Acad. Sci, Paris, ser. D, 242:190-192. Chantraine, J., G. Combaut, and J. Teste. 1973. Phenols bromes d'une rouge, Halopytls incurvus: acides carboxyliques. Phytochemlstry, 12s 1793-1795. Forsberg, C. 1965. Nutritional studies of Chara in axenic cultures. Physiologia Plantarum, 18J 275-290. Fries, L. 1973. Growth stimulating effects of the bromophenol, -lanosol, on red algae in axenic culture. Experientia, 29« 1436-1437. Hayward, H. 1965. Studies on the growth of Phaeodactylum trlcornutum (Bohlin) I. The effect of certain organic nitrogenous substances on growth. Physiologia Plantarum, 18« 201-207. Loffelhardt, W., B. Ludwlg, and H. Klndl. 1973. Thylakoid-gebund- ene L-Phenylalanin-Ammoniak-Lyase. Hoppe-Seyler's Z. Physiol. Chem., 354: 1006-1012. Pryce, R. J. 1972. The occurrence of lunularic and abscsic acids in plants. Phytochemlstry, 11: 1759-1761. Ragan, M. A., and J. S. Craigie. 1976. Physodes and the phenolic compounds of brown algae. Isolation and characterization of phloroglucinol polymers from Fucus veslculosus (L.).-. Can. J. Biochem.,54: 66-73. Scott, R. 1954. Observations on the iodoamino acids of marine algae using lodine-131. Nature, 173: 1098-1099. Weinstein, B., T. L. Rold, C. E. Harrell, Jr., M. W. Burns III, and J. R. Waaland. 1975. Reexamination of the bromophenols in the red alga Rhodomella larlx. Phytochemlstry, 14: 266?- 2670.