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The Degradation Op Phenylalanine, Tyrosine, And / 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.
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