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The degradation of phenylalanine, tyrosine and related aromatic compounds by a marine diatom and a haptophycean… Landymore, Arthur Frederick 1976

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/ THE DEGRADATION OP PHENYLALANINE, TYROSINE, AND RELATED AROMATIC COMPOUNDS BY A MARINE DIATOM AND A HAPTOPHYCEAN ALGA by ARTHUR FREDERICK LANDYMORE B.Sc., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1968 M.Sc., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f Botany We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNTOHSITY OF BRITISH COLUMBIA March, 1976" In present ing th is t h e s i s in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L ibrary s h a l l make i t f r e e l y a v a i l a b l e for reference and Study. I fu r ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t i ves . It is understood that copying or p u b l i c a t i o n of th is thes is for f i n a n c i a l gain s h a l l not be al lowed without my wr i t ten permiss ion . ABSTRACT,, i i i . The d e g r a d a t i o n o f p h e n y l a l a n i n e and t y r o s i n e was ex-amined i n a x e n i c c u l t u r e s o f I s o c h r y s l s g a l b a n a Parke and N a v l c u l a l n c e r t a H u s t e d t . B o t h s p e c i e s were a b l e t o m e t a b o l i z e L - p h e n y l a l a n i n e and L - t y r o s i n e as t h e s o l e n i t r o g e n s o u r c e , but s e v e r e growth I n h i b i t i o n was o b s e r v e d f o r _ I . g a l b a n a . No growth o f I_. g a l b a n a was o b t a i n e d on the D-isomers of t h e s e two amino a c i d s , but N. l n c e r t a was a b l e t o u t i l i z e b o t h D-amino a c i d s a f t e r an extended l a g p e r i o d . A n a l y s i s o f t h e growth medium and t h e a l g a l c e l l s f r o m n o n - r a d i o a c t i v e and r a d i o a c t i v e e x p e r i m e n t s n e v e r r e v e a l e d c i n n a m i c o r p-coumaric a c i d s . T h i s s u g g e s t e d t h a t p h e n y l -a l a n i n e and t y r o s i n e ammonia-lyases (PAL and TAL) were not i n v o l v e d i n the i n i t i a l d e g r a d a t i v e s t e p o f e i t h e r t h e s e amino a c i d s . T h i s was c o n f i r m e d as no e n z y m a t i c a c t i v i t y f o r PAL was d e t e c t e d i n c r u d e enzyme p r e p a r a t i o n s . E n z y m a t i c a c t i v i t y f o r amino a c i d t r a n s a m i n a s e was ob-t a i n e d f o r b o t h a l g a l s p e c i e s . T h i s s u g g e s t e d t h a t p h e n y l p y r u v l c a c i d and p - h y d r o x y p h e n y l p y r u v i c a c i d were t h e I n i t i a l r e s p e c -t i v e p r o d u c t s from t h e m e t a b o l i s m o f p h e n y l a l a n i n e and t y r o s i n e . From t h e n o n - r a d i o a c t i v e and r a d i o a c t i v e e x p e r i m e n t s , a scheme f o r t h e d e g r a d a t i o n of L - p h e n y l a l a n i n e and L - t y r o s i n e was p r o p o s e d f o r b o t h a l g a l s p e c i e s . The compounds i n b r a c k e t s were not i d e n t i f i e d but were e x p e c t e d . The pathways were: L - p h e n y l a l a n i n e — p h e n y l p y r u v l c a c i d >-[phenylacetaldehyde] — y - p h e n y l a c e t l c a c i d — ^ - m a n d e l i c a c i d — ^ - b e n z o y l f o r m l c a c i d — ^ > [ b e n z a l d e h y d e ] b e n z o i c a c i d — * - p - h y d r o x y b e n z o i c a c i d ; and • ' _ • ( " L - t y r o s l n e — * - p - h y d r o x y p h e n y l p y r u v l c a c i d — ^ - J ^ p - h y d r o x y p h e n y l a c e t a l d e h y d e ] — ^ - p - h y d r o x y p h e n y l a c e t i c a c i d — * - p - h y d r o x y -m a n d e l i c a c i d — > - J j j - h y d r o x y b e n z o y l f o r m i c a c i d ] — p - h y d r o x y -b e n z a l d e h y d e —»-p-hydroxybenzoic a c i d and p - h y d r o x y b e n z y l -a l c o h o l . B e n z o i c a c i d was a l s o h y d r o x y l a t e d i n t h e o r t h o and meta p o s i t i o n s by b o t h a l g a l s p e c i e s . I n b o t h t h e s e schemes, t h e two C^-fragments removed from t h e s i d e c h a i n were i d e n -t i f i e d as CO^* A l s o , the carbon-3 o f t h e s i d e c h a i n o f b o t h p h e n y l a l a n i n e and t y r o s i n e was removed and t r a p p e d as COg. The r e l a t i o n s h i p o f t h e s e pathways t o o t h e r a l g a e i s a l s o d i s c u s s e d . E v i d e n c e s u g g e s t e d t h a t p - h y d r o x y b e n z o i c a c i d by b o t h I . g a l b a n a and N. l n c e r t a was (1) d e c a r b o x y l a t e d , p r o b a b l y r e s u l t i n g l n 1,4-dihydroxybenene, (2) b r o m l n a t e d t o 3-bromo-p - h y d r o x y b e n z o i c a c i d , and (3) e x c r e t e d i n t o the medium. I t was unknown i f t h e 1,^-dihydroxybenene and/or 3-bromo-p-' h y d r o x y b e n z o i c a c i d caused t h e browning o b s e r v e d m a i n l y i n c u l t u r e s o f b o t h s p e c i e s grown l n the p r e s e n c e o f t y r o s i n e . S a l t c y c l l c a c i d ( o r t h o - h y d r o x y b e n z o l c a c i d ) was a l s o de-c a r b o x y l a t e d , p r o b a b l y r e s u l t i n g i n c a t e c h o l (1,2-dihydroxy-b e n z e n e ) . R i n g c l e a v a g e o b s e r v e d f o r t y r o s i n e and f o r p h e n y l a l -a n i n e (Vose e t a l . . 1971) appeared t o i n v o l v e a C^-C^ com-pound, p r o b a b l y b e n z o i c a c i d o r one o f i t s h y d r o x y l a t e d p r o -d u c t s . R i n g c l e a v a g e does not appear t o be i m p o r t a n t i n t h e d e g r a d a t i o n o f e i t h e r amino a c i d . No d i h y d r o x y p h e n o l i c compounds were d e t e c t e d , b u t t h i s does n o t e l i m i n a t e t h e p o s s i b i l i t y o f t h e i r f o r m a t i o n . E v i -dence s u g g e s t e d t h a t b o t h s p e c i e s had problems h y d r o x y l a t i n g not o n l y t h e a r o m a t i c r i n g (eg. b e n z o i c a c i d ) but a l s o t h e s i d e c h a i n o f i n t e r m e d i a t e s i n t h e d e g r a d a t i v e pathway. B o t h a l g a l s p e c i e s degraded c i n n a m i c and p-coumaric a c i d s i n a s i m i l a r pathway t o t h a t r e p o r t e d i n h i g h e r p l a n t s The pathway i n v o l v e d 0 - o x i d a t i o n o f t h e s i d e c h a i n t o produc b e n z o i c and p - h y d r o x y b e n z o l c a c i d s from c i n n a m i c and p-c o u m a r i c a c i d s r e s p e c t i v e l y . The u p t a k e r a t e s o f b o t h p h e n y l a l a n i n e and t y r o s i n e and t h e e f f e c t o f o t h e r a r o m a t i c compounds on t h e growth c o n s t a n t s and l a g p e r i o d s o f b o t h a l g a l s p e c i e s a r e a l s o p r e s e n t e d . v i . TABLE OF CONTENTS. Page ABSTRACT i i i TABLE OF CONTENTS v i L I S T OF TABLES i x L I S T OF FIGURES x i i ACKNOWLEDGEMENTS x v i i l INTRODUCTION 1 LITERATURE REVIEW MATERIALS [ AND METHODS 16 • ' I . C u l t u r i n g . 16 A. The medium 16 B. A l g a l s p e c i e s u t i l i z e d 16 C. Maintenance o f s t o c k c u l t u r e s 18 De- C o n t a m i n a t i o n t e s t i n g 18 E. C e l l e n u m e r a t i o n 21 a,. Hemacytometer 21, b. O p t i c a l d e n s i t y 21 F. O p t i c a l d e n s i t y e x p e r i m e n t s and medium p r e p a r a t i o n 23 G. One l i t e r c u l t u r e e x p e r i m e n t s 2k H. Mass c u l t u r i n g 25 I.: C e l l c o l l e c t i o n and s t o r a g e 27 I I . C h e m i c a l s t u d i e s o f a r o m a t i c compounds 29 • A. Sou r c e s 29 B, Chromatography 30 c.. S p e c t r o s c o p y 31 v i i . Page D». M e l t i n g p o i n t s 31 E. C h e m i c a l p r e p a r a t i o n s o f n o n - r a d i o a c t i v e compounds « 32 F, C h e m i c a l p r e p a r a t i o n s o f r a d i o a c t i v e compounds 32 I I I o I s o l a t i o n o f p r o d u c t s 32 IV, R a d i o a c t i v e f e e d i n g e x p e r i m e n t s 33 A. S o u r c e of ^ C - i s o t o p e s 33 l 4 B. P r e p a r a t i o n and a d m i n i s t r a t i o n o f C-compounds 34 F i s s i o n o f the a r o m a t i c r i n g o f t y r o s i n e 3^ S i d e c h a i n d e g r a d a t i o n o f p h e n y l a l a n i n e and t y r o s i n e 35 14 D e g r a d a t i o n o f o t h e r C - l a b e l l . e d s u b s t r a t e s 37 Uptake e x p e r i m e n t s w i t h p h e n y l a l a n i n e and t y r o s i n e 38 14 C. D e t e c t i o n of C - p r o d u c t s 39 A u t o r a d i o g r a p h y 39 S c i n t i l l a t i o n c o u n t i n g 39 V, Enzyme a s s a y s 40 A» P h e n y l a l a n i n e ammonia-lyase 40 B. - Transaminase 4 l C. p-Hydroxybenzoate" h y d r o x y l a s e 4 l D» P r o t e i n d e t e r m i n a t i o n 42 RESULTS - ^3 I,. C u l t u r i n g ky A*. The e f f e c t o f p h e n y l a l a n i n e and t y r o s i n e 43 B, M e t a b o l i s m o f p h e n y l a l a n i n e and t y r o s i n e 59 C. . The e f f e c t and m e t a b o l i s m o f o t h e r a r o m a t i c compounds 62 - Page D e R e s u l t s o f r a d i o a c t i v e t r a c e r s 62 Uptake o f p h e n y l a l a n i n e and t y r o s i n e 62 The c a t a b o l i c f i s s i o n o f t h e a r o m a t i c r i n g o f t y r o s i n e 71 S i d e c h a i n d e g r a d a t i o n o f p h e n y l a l a n i n e and t y r o s i n e 7k D e g r a d a t i o n o f o t h e r C - l a b e l l e d s u b s t r a t e s 92 E.. R e s u l t s o f enzyme a s s a y s 96 P h e n y l a l a n i n e ammonia-lyase 96 Transaminase ^ 96 p - H y d r o x y b e n z o i c a c i d h y d r o x y l a s e 101 P r o t e i n d e t e r m i n a t i o n s ; 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 i x . L I ST OF TABLES> TABLE Page 1. The growth o f a l g a e on p h e n y l a l a n i n e when used as t h e s o l e N-source. 8 2. The growth o f a l g a e on t y r o s i n e when u s e d as t h e s o l e N-source. 9 3. T o t a l ^CC>2 measured as p r o d u c t o f c a t a b o l i s m from 2-weeks i n c u b a t i o n o f a l g a e w i t h r i n g l a b e l l e d ^ C - p h e n y l a l a n i n e . 10 A r o m a t i c compounds I d e n t i f i e d from a l g a e . 13 5* B r o m o p h e n o l i c compounds i d e n t i f i e d from a l g a e . 14 6. P h y t o p l a n k t o n c u l t u r e medium. 17 7. I n i t i a l s o u r c e o f a x e n i c a l g a l c u l t u r e s . 20 8. Growth c o n s t a n t s and c e l l y i e l d s f r om mass . c u l t u r e s . .44 9. The e f f e c t o f a r o m a t i c compounds on t h e growth c o n s t a n t s and c e l l y i e l d s from one l i t e r c u l t u r e s o f I s o c h r y s l s g a l b a n a . 45 10. The e f f e c t o f a r o m a t i c compounds on the growth c o n s t a n t s and c e l l y i e l d s from one l i t e r c u l t u r e s o f N a v l c u l a l n c e r t a . 45 1.1. A summary o f t h e e f f e c t and m e t a b o l i s m o f o t h e r a r o m a t i c compounds on I s o c h r y s l s  g a l b a n a and N a v l c u l a l n c e r t a . 6y 12. Uptake r a t e s o f p h e n y l a l a n i n e and t y r o s i n e by I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a a t "two s u b s t r a t e c o n c e n t r a t i o n s . 65 X . TABLE Page 13. T o t a l ^ ' C 0 2 measured as a p r o d u c t o f c a t a -b o l i s m from 2-weeks i n c u b a t i o n o f a l g a e w i t h u n i f o r m l y r i n g - l a b e l l e d ^ C - t y r o s i n e , 72 14. R a d i o a c t i v i t y i n p r o d u c t s I s o l a t e d f rom 14 u n i f o r m l y r i n g l a b e l l e d C - t y r o s i n e p r o d u c t s . 73 14 15* T o t a l COg measured as a p r o d u c t o f c a t a -b o l i s m f r o m i n c u b a t i o n o f p r e - a d a p t e d c e l l s o f I s o c h r y s l s g a l b a n a w i t h l a b e l l e d p h e n y l -a l a n i n e and t y r o s i n e . 76 14 16. T o t a l CO2 measured as a p r o d u c t o f c a t a -b o l l s m f rom i n c u b a t i o n o f p r e - a d a p t e d c e l l s o f N a v l c u l a l n c e r t a w i t h l a b e l l e d p h e n y l -a l a n i n e and t y r o s i n e . 77 1 4 17. R a d i o a c t i v i t y l n p r o d u c t s I s o l a t e d f r o m x C-. l a b e l l e d f e e d i n g s from i n c u b a t i o n o f p r e -adapt e d c e l l s o f I s o c h r y s l s g a l b a n a . 83 18. R a d i o a c t i v i t y I n p r o d u c t s I s o l a t e d f rom -Re-l a b e l l e d f e e d i n g s from i n c u b a t i o n o f p r e -adapt e d c e l l s o f N a v l c u l a l n c e r t a . 84 1 4 19« T o t a l C 0 2 measured as a p r o d u c t o f c a t a -b o l i s m l n t h e d a r k from i n c u b a t i o n o f non-a d a p t e d c e l l s w i t h l a b e l l e d a r o m a t i c compounds. 93 20. T o t a l ^ C 0 2 measured as a p r o d u c t o f c a t a -b o l l s m from a 12-hours d a r k i n c u b a t i o n o f x i . TABLE Page non-adapted c e l l s w i t h l a b e l l e d a r o m a t i c compounds. 9 5 2 1 . P h e n y l a l a n i n e ammonia-lyase a c t i v i t y l n marine a l g a e . 9 7 22.. Transaminase a c t i v i t y i n c e l l e x t r a c t s o f N a v l c u l a l n c e r t a and I s o c h r y s l s g a l b a n a . 9 9 2 3 . P h e n o l i c compounds d e t e c t e d from f e e d i n g non-r a d i o a c t i v e p h e n y l a l a n i n e and t y r o s i n e t o v a r i o u s a l g a e and t h e r e l a t i o n s h i p o f t h e p h e n y l a l a n i n e f e e d i n g s t o t h e m e t a b o l i s m o f p h e n y l a l a n i n e r i n g - l - ^ C . 121 x i i . . . L I S T OF FIGURES  FIGURE . . '._ Page 1. The s h l k i m l c a c i d pathway f o r t h e b i o -s y n t h e s i s o f a r o m a t i c amino a c i d s . 2 2. The p r e t y r o s i n e pathway i n b l u e - g r e e n a l g a e . 6 3« P h o t o m i c r o g r a p h s o f t h e a l g a l s p e c i e s u t i l i z e d i n t h i s i n v e s t i g a t i o n . 19 4. The l i n e a r range o f OD i n r e l a t i o n t o c e l l number f o r p l a n k t o n i c s p e c i e s . 22 5 . Diagram o f mass c u l t u r e a p p a r a t u s and t r a p f o r v o l a t i l e p r o d u c t ( s ) . 26 14 6. Diagram o f a p p a r a t u s f o r COg r e g e n e r a t i o n and r e t r a p p i n g , 36 7. Diagram o f t h e e f f e c t s o f L - p h e n y l a l a n i n e on t h e g r o w t h - c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . 46 8. Diagram o f t h e e f f e c t s o f L-ty.rosl.ne on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s  g a l b a n a and N a v l c u l a l n c e r t a . 46 9« Growth c u r v e s o f N a v l c u l a l n c e r t a on n i t r a t e and L - p h e n y l a l a n i n e w i t h and w i t h o u t n i t r a t e . 47 10. Growth c u r v e s o f N a v l c u l a l n c e r t a on n i t r a t e / and L - t y r o s i n e w i t h and w i t h o u t n i t r a t e . 48 11. Diagram o f t h e e f f e c t s o f L - p h e n y l a l a n i n e as t h e s o l e N-source on t h e growth c o n s t a n t and l a g p e r i o d of I s o c h r y s l s g a l b a n a and N a v l c u l a  l n c e r t a . 5 0 x i i i . FIGURE Page 12. Diagram o f t h e e f f e c t s o f L - t y r o s i n e as t h e s o l e N-source on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a  l n c e r t a . 50 13. Growth c u r v e s o f I s o c h r y s l s g a l b a n a on n i t r a t e and L - p h e n y l a l a n i n e w i t h and w i t h o u t n i t r a t e . 51 Ik,. Growth c u r v e s o f I s o c h r y s l s g a l b a n a on n i t r a t e and L - t y r o s l n e w i t h and w i t h o u t n i t r a t e . 52 15. The e f f e c t of L - t y r o s l n e w i t h and w i t h o u t L-p h e n y l a l a n i n e on t h e growth y i e l d s of I s o c h r y s l s g a l b a n a . 5k 16. Growth c u r v e s o f N a v l c u l a l n c e r t a on n i t r a t e and D - p h e n y l a l a n l n e . 56 17. Growth c u r v e s o f N a v l c u l a l n c e r t a on n i t r a t e and D - t y r o s l n e . , .57 18.. Diagram o f t h e e f f e c t s o f D - p h e n y l a l a n i n e and D - t y r o s i n e on t h e growth c o n s t a n t and l a g p e r i o d o f N a v l c u l a l n c e r t a . 58 19. A b s o r p t i o n spectrum i n e t h a n o l o f the p h e n o l i c a c i d t e n t a t i v e l y i d e n t i f i e d as 3-bromo-p-hydroxybenzolc a c i d I s o l a t e d from I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . 6l 20. - Time c o u r s e o f u p t a k e o f D L - p h e n y l a l a n i n e - 3 -C f o r I s o c h r y s l s g a l b a n a a t an I n i t i a l , p h e n y l a l a n i n e c o n c e n t r a t i o n o f 10 uM. 6k 21. Time c o u r s e of u p t a k e o f D L - p h e n y l a l a n i n e - 3 -. C f o r I s o c h r y s l s g a l b a n a a t an i n i t i a l x i v . FIGURE Page p h e n y l a l a n i n e c o n c e n t r a t i o n of 0.1 mM. 64 22. Time c o u r s e of u p t a k e o f D L - p h e n y l a l a n i n e - 3 -•^C f o r N a v l c u l a l n c e r t a a t an i n i t i a l p h e n y l -a l a n i n e c o n c e n t r a t i o n o f 10 uM. 66 23. Time c o u r s e o f u p t a k e of D L - p h e n y l a l a n i n e - 3 -14 C f o r N a v l c u l a l n c e r t a a t an i n i t i a l p h e n y l -a l a n i n e c o n c e n t r a t i o n o f 0.1 mM. 66 24. Time c o u r s e of u p t a k e o f D L - t y r o s i n e - 3 - 1 ^ C f o r I s o c h r y s l s g a l b a n a a t an i n i t i a l t y r o s i n e c o n c e n t r a t i o n of 10 uM. 68 25. Time c o u r s e o f u p t a k e o f D L - t y r o s i n e ^ - ^ C f o r I s o c h r y s l s g a l b a n a a t an i n i t i a l t y r o s i n e c o n c e n t r a t i o n o f 0.1 mM. 68 26. Time c o u r s e o f u p t a k e o f D L - t y r o s i n e - 3 - ^ C f o r N a v l c u l a l n c e r t a a t an I n i t i a l t y r o s i n e . c o n c e n t r a t i o n o f 10 uM. 69 14 27. Time c o u r s e o f u p t a k e o f D L - t y r o s l n e - 3 - C f o r N a v l c u l a l n c e r t a a t an i n i t i a l , t y r o s i n e c o n c e n t r a t i o n o f 0.1 mM. 69 28. A u t o r a d i o g r a p h y o f t h e e t h e r e x t r a c t s from 14 'C-slde c h a i n l a b e l l e d p h e n y l a l a n i n e and t y r o s i n e f e d t o I s o c h r y s l s galbana., 80 29.. A u t o r a d l o g r a p h s o f t h e e t h e r e x t r a c t s f rom 14 C - s l d e c h a i n l a b e l l e d p h e n y l a l a n i n e and t y r o s i n e f e d t o N a v l c u l a l n c e r t a . 81 A u t o r a d i o g r a p h s o f t h e e t h e r e x t r a c t s from C - s l d e c h a i n l a b e l l e d p h e n y l a l a n i n e and t y r o s i n e t o be f e d t o I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . A u t o r a d i o g r a p h s o f t h e e t h e r e x t r a c t s from l 4 1U c i n n a m i c a c i d - 2 - C and p-coumaric a c i d - 2 - C f e d t o I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . A u t o r a d i o g r a p h s o f t h e e t h e r e x t r a c t s f r o m 1U l 4 C - p h e n y l a l a n i n e and C - t y r o s i n e t r a n s -aminase e x p e r i m e n t s w i t h e x t r a c t s o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . The d e g r a d a t i v e r o u t e of L - p h e n y l a l a n i n e i n I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . The d e g r a d a t i v e r o u t e o f L - t y r o s i n e i n I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . The d e g r a d a t i v e r o u t e of c i n n a m i c and p-c o u m a r l c a c i d s i n I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . A b s o r p t i o n s pectrum o f J,5-dlbromo-p-h y d r o x y b e n z o l c a c i d and i t * s d e g r a d a t i o n p r o d u c t . A b s o r p t i o n s p e c t r u m o f J,5-dibromo-p-h y d r o x y b e n z a l d e h y d e . A b s o r p t i o n s pectrum o f p - h y d r o x y p h e n y l - . a c e t a l d e h y d e . A b s o r p t i o n spectrum o f p - h y d r o x y b e n z o y l f o r m i c a c i d and i t ' s spontaneous d e g r a d a t i o n p r o d u c t p - h y d r o x y b e n z a l d e h y d e . A b s o r p t i o n s p e c t r u m o f s y n t h e t i c p h e n y l h y d r a -c r y l i c a c i d . Diagram of the e f f e c t s o f p h e n y l a c e t l c a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s i s g a l b a n a and N a v l c u l a l n c e r t a . Diagram of the e f f e c t s o f p - h y d r o x y p h e n y l -a c e t l c a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a  l n c e r t a . Diagram of t h e e f f e c t s of DL-mandellc a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram o f t h e e f f e c t s o f DL-p-hydroxy-m a n d e l i c a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram o f t h e e f f e c t s o f b e n z o i c a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a Diagram o f the e f f e c t s o f p - h y d r o x y b e n z o i c a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram o f t h e e f f e c t s o f p - h y d r o x y b e n z a l d e -hyde on the growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram of the e f f e c t s o f 3 i 5 - d i b r o m o - p -h y d r o x y b e n z o l c a c i d on the growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram o f t h e e f f e c t s of 3.5-dibromo-p-h y d r o x y b e n z a l d e h y d e on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram o f the e f f e c t s of c i n n a m i c a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram of t h e e f f e c t s o f p-coumaric a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . A b s o r p t i o n spectrum i n e t h a n o l o f the p h e n o l i c t e n t a t i v e l y i d e n t i f i e d as p-. h y d r o x y p h e n y l h y d r a c r y l i c a c i d f r om I s o -c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram o f t h e e f f e c t s o f m-hydroxybenzoic a c i d on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . Diagram of t h e e f f e c t s o f o - h y d r o x y b e n z o l c a c i d on the growth c o n s t a n t and l a g p e r i o d °f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . x v i i i . ACKNOWLEDGEMENTS. I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o Dr. G. H. N. Towers and Dr. N. J . A n t i a under whose s u p e r v i s i o n t h i s s t u d y was c o n d u c t e d , and f o r t h e i r v a l u a b l e a d v i c e and c r i t i c i s m d u r i n g t h e r e s e a r c h and p r e p a r a t i o n o f t h i s m a n u s c r i p t . To Dr. C. K. Wat, Mr. J . Y. Cheng, and my c o l l e g u e s f o r t h e i r a s s i s t a n c e and h e l p f u l d i s c u s s i o n s d u r i n g t h i s r e s e a r c h . To Dr. J , S:. C r a i g i e r N. R. C. A t l a n t i c R e g i o n a l Lab., H a l i f a x , N.S, f o r h i s k i n d n e s s i n p r o v i d i n g samples o f s y n t h e t i c b r o m o p h e n o l i c compounds. To t h e members of my committee f o r t h e i r h e l p f u l comments on t h i s m a n u s c r i p t . To t h e F i s h e r i e s R e s e a r c h B o a r d of Canada f o r p r o v i d i n g f i n a n c i a l a s s i s t a n c e d u r i n g t h i s s t u d y . F i n a l l y , but f i r s t l y , t o my w i f e Marlene who t y p e d t h i s m a n u s c r i p t and w i t h s t o o d t h e many problems I had d u r i n g " t h e r e s e a r c h and p r e p a r a t i o n o f t h i s t h e s i s . INTRODUCTION : ' Both phenylalanine and tyrosine are important pre-cursors not only f o r protein "biosynthesis, but also a host of other compounds found i n 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) i s 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 eith e r t h e i r d i e t or the.-micro-, f l o r a within t h e i r alimentary canal. Axenlc cultures of algae do not require either of these two amino acids f o r autotrophic growth, suggesting that they possess the shikimic a c i d pathway* The metabolism of phenylalanine and tyrosine i s of s p e c i a l i n t e r e s t . For plants, fungi, bacteria, and animals the metabolic products and pathways are well described (Luckner, 1972; Towers and Subba Rao, 1972), but l i t t l e i s known f o r algae. Both of these amino acids can serve as the sole N-source f o r growth of c e r t a i n a l g a l species, but the metabolic path of t h e i r u t i l i z a t i o n has not been investigated. Vose et a l . (1971) reported that nine of twenty-two phyto-plankton species could metabolize the t o t a l carbon skeleton o f phenylalanine to C 0 2 y but they did not investigate the route of degradation. They also detected the presence of an u n i d e n t i f i e d v o l a t i l e product or products. To date, there COOH < j 3 0 0 H Phosphocnolpyruvatd —<~— HO— C—H + H—C—OH CHO I I H—,C—OH H—C—OH HQ. .COOH NADH,H+ NAD + Pi COOH CHjO—® D-Er>1hrose-4-phosphate H—C—OH CHjO® 3-Deoxy-D- arabinoheptulo sonic acid 7-phospbate OH 5- Dehydroquinic acid OH OH 5-Dehydroshilcimic acid NADP^,H4'^ADP f ^Ap'P - V ^ O H HOT OH Shikimic acid CH2CHNH2COOH L-Phenylalanine OH L-Tyrosine OH p-Hydroxyphenyl pyruvate HOOCV CH,CC00H o OH Prcphenic acid L-Tryptophaii —— — -©O T OH OH 5-Phospho shikimic acid PEP ceo CH, O-t OH COOH J-Enolpyruyyl shikimic acid 5-phosphate CH, O—C ZOOM Chorismic acid Figure 1. The shikimic acid pathway for the biosynthesis of aromatic amino aclds 0 a r e no f u r t h e r r e p o r t s on t h e m e t a b o l i s m i n a l g a e o f t h e s e two amino a c i d s . An e x a m i n a t i o n o f t h e i r m e t a b o l i s m i n a l g a e was t h e r e f o r e c o n s i d e r e d t o be worthy o f s t u d y . Two s p e c i e s , I s o c h r y s l s g a l b a n a Parke and N a v l c u l a l n c e r t a H u s t e d t were s e l e c t e d f o r t h i s i n v e s t i g a t i o n based on t h e r e s u l t s o f Vose e t a l . ( 1 9 7 1 ) . Upon f e e d i n g r i n g -I k l a b e l l e d C - p h e n y l a l a n i n e , t h e y r e p o r t e d I . g a l b a n a produced an u n i d e n t i f i e d v o l a t i l e compound(s) which c o n t a i n e d a l a r g e p e r c e n t a g e o f t h e r a d i o a c t i v i t y w h i l e t h e r a d i o a c t i v i t y o b t a -in l n e d i n C 0 2 was low. I n N. l n c e r t a , on t h e o t h e r hand, t h e r e v e r s e s i t u a t i o n was o b s e r v e d . Out o f twenty-two s p e c i e s t h e y examined, t h e g r e a t e s t r a d i o a c t i v e count c o l l e c t e d i n ^COg was from N. l n c e r t a w h i l e t h e g r e a t e s t r a d i o a c t i v e count c o l l e c t e d i n t h e u n i d e n t i f i e d v o l a t i l e C-compound was from I, g a l b a n a . B o t h t h e s e p l a n k t o n l c a l g a e were con-s i d e r e d t h e most promsing s p e c i e s t o u s e i n o r d e r t o i n v e s t -i g a t e t h e . d e g r a d a t i v e pathway o f n o t o n l y p h e n y l a l a n i n e but a l s o t y r o s i n e . T h i s d i s s e r t a t i o n w i l l p r e s e n t e v i d e n c e f o r t h e de-g r a d a t i v e r o u t e s o f p h e n y l a l a n i n e and t y r o s i n e i n b o t h a x e n i c p l a n k t o n l c a l g a l s p e c i e s . The m e t a b o l i c r o u t e s were examined w i t h s p e c i f i c a l l y l a b e l l e d r a d i o a c t i v e t r a c e r s . S e v e r a l enzymes, i n v o l v e d i n t h e d e g r a d a t i v e pathways, were i n v e s t i -g a t e d i n c e l l - f r e e e x t r a c t s f rom both, a l g a l s p e c i e s . The e f f e c t s on growth o f v a r i o u s a r o m a t i c compounds and t h e i r m e t a b o l i s m by b o t h a l g a l s p e c i e s were a l s o examined. LITERATURE REVIEW. 4. The b i o s y n t h e t i c pathway f o r a r o m a t i c amino a c i d s b e g i n s w i t h t h e c o n d e n s a t i o n o f p h o s p h o e n o l p y r u v i c a c i d and e r y t h r o s e - 4 - p h o s p h a t e ( F i g u r e l ) ' r t h e f i r s t o f seven enzym-a t i c r e a c t i o n s t h a t l e a d t o the f o r m a t i o n o f c h o r i s m i c a c i d . C h o r i s m a t e i s c o n v e r t e d t o p r e p h e n l c a c i d o r c a n be u s e d f o r t r y p t o p h a n s y n t h e s i s . So f a r as i s known, a l l organisms t h a t a r e a b l e t o s y n t h e s i z e L - p h e n y l a l a n i n e and L - t y r o s i n e u t i l i z e p r e p h e n a t e as t h e l a s t common i n t e r m e d i a t e o f a b r a n c h i n g pathway. T h i s pathway i s i n f e r r e d t o be t h e b i o -s y n t h e t i c r o u t e i n a l g a e . No i n v e s t i g a t i o n t o c o n f i r m t h i s has been c o n d u c t e d , a l t h o u g h ~ s e v e r a l key enzymes have been e x t e n s i v e l y s t u d i e d . The i n i t i a l enzyme, 3-deoxy-D-arablno h e p t u l o s o n i c a c i d 7-phosphate s y n t h e t a s e has been s t u d i e d in Agmenellum q u a d r u p l l c a t u m (Ingram .et a^,, 1973l J e n s e n e t a l . . 1974j Stenmark e t a l , , 197*0, A n a c y s t l s n i d u l a n s (Weber e t a l . , 1968j Stenmark e t a l . , 1974; Phares e t a l . , 1975)» Anabaena v a r i a b i l i s . O s c l l l a t o r l a t e n u i s . Nostoc s p . , Meso-taenlum s p . , A n k l s t r o d e s m u s s p . a n d Euglena. g r a c i l i s . (Weber et a l . , 1964). H i g h e n z y m a t i c a c t i v i t y was o b t a i n e d f o r t h i s enzyme and i t s c o n t r o l by p h e n y l a l a n i n e and t y r o s i n e was examined. The bran c h p o i n t enzyme, c h o r i s m i c a c i d mutase has a l s o been I n v e s t i g a t e d i n A n a c y s t l s n i d u l a n s , Zygnema sp,, Chlamydomonas r e l n h a r d t l and A n k l s t r o d e s m u s s p , , (Weber e t a l . , 1969). E u g l e n a g r a c i l i s (Weber e t a l , . . 1964, 1970), A c ^ e n e l l u r a q u a d r u p l l c a t u m and A n a c y s t l s n i d u l a n s (Stenmark e t a l , , 1974). A g a i n , c o n t r o l o f t h i s enzyme by p h e n y l a l -a n i n e and t y r o s i n e was s t u d i e d . ; '"•y.;;£:'":jv.Htgh s p e c i f i c a c t i v i t y f o r . p r e p h e n a t e d e h y d r a t a s e and a n a r o m a t i c transaminase'was d e t e c t e d f o r Agmenellum  q u a d r u p l l c a t u m and A n a c y s t l s n i d u l a n s , however no a c t i v i t y / f o r p r e p h e n a t e dehydrogenase was o b t a i n e d (Stenmark e t a l , . 1974). I n b l u e - g r e e n a l g a e ( o r b a c t e r i a ) a new pathway f r o m p r e p h e n i c a c i d t o t y r o s i n e was r e p o r t e d (Stenmark e t a l , . 1974| J e n s e n and P l e r s o n , 1975? J e n s e n and Stenmark, 1975). T h i s pathway has been shown by enzym a t i c s t u d i e s t o be p r e s e n t I n Agmenellum q u a d r u p l l c a t u m , A n a c y s t l s n i d u l a n s , C o c c o c h l o r i s e l a b e n s , Nostoc muscorum, Lyngbya l a g e r h e l m l l , and O s c l l i l a t o r l a s p , and i n v o l v e s a t r a n s a m i n a t i o n t o g i v e p r e t y r o s l n e f o l l o w e d by d e h y d r o g e n a t i o n - d e c a r b o x y l a t i o r t t o g i v e L - t y r o s l n e ( F i g u r e 2), I n t h e a n a l y s i s o f v a r i o u s a l g a e , b o t h t h e s e amino a c i d s were found n o t o n l y i n p r o t e i n s (Fowden, 1962j Schmld, I969J Kempner e t a l . . 1974) but a l s o i n t h e f r e e s t a t e (Fowden, 1951» Watanabe, 1951J Schmld, 1969? Kempner e t a l . , 1974» I m p e l l l z z e r l , 1975). I n one c a s e , i t . i s r e p o r t e d , t h a t p h e n y l -a l a n i n e i s e x c r e t e d by C a l o t h r l x s c u p u l o r u m I n t o t h e medium ( S t e w a r t , 1963K The pr e s e n c e o f p h e n y l a l a n i n e and t y r o s i n e i n a l g a e and o f enzymes i n v o l v e d i n t h e i r s y n t h e s i s as w e l l as t h e .lack o f e i t h e r amino a c i d f o r g r o w t h , l e a d s , one t o c o n c l u d e t h a t a l g a e as a . r u l e , can s y n t h e s i z e b o t h amino a c i d s , p r o b -a b l y by. t h e s h i k i m i c a c i d pathway. : A l t h o u g h t h e m e t a b o l i s m o f p h e n y l a l a n i n e and t y r o s i n e -has been p o o r l y i n v e s t i g a t e d , t h e i r u t i l i z a t i o n as t h e s o l e s o u r c e o f n i t r o g e n has been e x t e n s i v e l y s t u d i e d . I n H O O D 0 CH2-C-COOH HOOC prephenate transaminase NH2 CH2-CH-COOH CO-^ H 2 CH2-CH-COOH OH OH pretyrosine, dehydrogenase NAD+ OH Prephenic Acid Pretyrosine L-tyrosine Figure 2 . The pretyrosine pathway In blue-green algae. Table 1 a l i s t i s compiled of a l g a l species examined f o r t h e i r a b i l i t y to u t i l i z e the nitrogen of phenylalanine and a corresponding l i s t f o r tyrosine i s shown i n Table 2. Approxim-at e l y one-third of the species were able to metabolize phenyl-alanine and tyrosine^ The growth i n most cases was never comp-arable to cultures grown on n i t r a t e . Several species could me-tabo l i z e D-phenylalanine, although D-tyrosine never served as a nitrogen source. No a l g a l species capable of heterotrophic growth on e i t h e r phenylalanine or tyrosine are known. In a l l the nitrogen u t i l i z a t i o n studies, the f a t e of the carbon skeleton of the amino ac i d was disregarded. The only i n v e s t i g a t i o n of t h i s problem was that by Vose et a l . l i t 14 (1971) who Incubated DL-phenylalanine- C (ring-1- C l a b e l -led) with 22 axenic phytoplankton species. In the l i g h t , nine species metabolized a f r a c t i o n of the t o t a l carbon skeleton to COg whereas i n the dark only four species retained t h i s ca-pacity (Table 3). It must be noted that t h i s study only indicated the species that have the c a p a b i l i t y to degrade the aromatic r i n g . No Indications as to which species can p a r t i a l l y metabolize the carbon skeleton or which species are able to u t i l i z e phenylalanine as a N-source were obtained. The rate of uptake of phenylalanine by various a l g a l species has been investigated. In Meloslra nummuloldes (Mel-3), 0.1 mM phenylalanine was taken up at 2,7 x 10 umoles/ mln/cell f o r the f i r s t ten minutes; when averaged over s i x -9 hours however, the rate decreased to 0.14 i 10 jimoles/min/ c e l l (Hellebust et a l . . 1967). Platymonas subcordlformis (Wille TABLE 1 The Growth of Algae on Phenylalanine When Used as the Sole N-source. Alga Concentration Isomer fed (mM) Growth Reference Chlorophyta Anltlstrodesmus amalloides » : a " angustus " falcatus var stlnltatus • » i» * if " nanoselene » « Brachlinonas subaiarlna Chlorella sp_. (F. B. Wann's No. 11) pyrenoldosa vulgaris (strain 11) " "Delft" « It • . Strain 260 m m « " Strain Z6\ m a n Strain 262 • n n Strain 263 « it n Chlorogonlun elona;atum " euchlorum  Eaematoooccus P l u y l a l l s " " (2 strains) Kannochlorls oculata Strain No.66 Prototheca elferrl 60-49 1. n n " morIformis 60-50 Stephanosphaera pluvlalls  S'jglenophyta Saglena anabaer.a var minor * deses " gracilis " fclebsli " plsclformis " 9 t e l l a t a L D L D L 0 L D ? ? DL DL L DL L D L D L D L D :L D DL DL 1 1 ? L D L D ? 21.1 21.1 21.1 21.1 21.1 21.1 21.1 21.1 0.24 12.5 ii.5 6.1 ^.o 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 9.1 9.1 ? 0.24 ? 3.0 3.0 3.0 3.0 0.24 NG NG NG NG NG ? NG NG 55* <2 NG ? ) (2)' +++ ++++ KG NG ++ ? NG ++ NG. ? + + NG NG NG +++ +++ +++ +++ NG Den Dooren (I969) Droop (1961) Ludwlg (1938) (hosh et al. (1950] Arnow"Tl953) Ballard (1966) Den Dooren (I967) (1969) DL 12.1 ++++ Dus DL 12.1 NG n DL 12.1 ++++ DL 12.1 + DL 12.1 NG DL 12.1 NG DL 12.1 ++ Loefer (1932) n n • Droop (1955) (1961) (1955) La Hue et al. (1966) Droop (1-961) l (1931)(1933b) (1933a) (1933b) (1931)(1933b) Chrysophyta  Kor.ochr,y3l3 lutherl Prysr.eslnm paryum Syracosphaera elongata Ba^mgrloohyta Msloslra nujimjTold33 Clone Mel-3 Cryotpphyta P.'ndoohyta  rihoden.a roaculata Xanthonhyta £ i a o . ' 3 l i 2 aubterraneus Trlbonema aequale T L DL DL DL 0.1 0.5 0.42 0.31* 0.25 NG +++ ? Droop (1955) KG Hellebust et al. (1967) NG Antla et al. (1968) NG Turner (1970) NG ,,, Killer et a l . (1958) 8iy2' Belcher ej al. (1958) Cyanophyta Amtnellum quail ru pi lea turn ' 3traln P!l-6 0.9 50* (2) Kapp s i al. (1975) (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. Alga Concentration Isomer fed (mM) Growth Reference Chlororhyta Anklatro'jesmus amallolde3 • ~ •* " angU3tu3 * m " falcatus var -s'tlpltatus m- m m m " nano3elene • n Brachlesnaa submarlna  Chlorella sp. (F. B. Wann's Ko. 11). " vulgaris • " "Delft" . . . ' • • » » • " Strain 260 m m ' m m " " Strain 261 • • " Strain 262 m it m n • " Strain 263 ' m m m m Chlamydomona3 pulvlr.ata " euchlorua  Haeniato^ occus o l u v l a l l 3 ( 2 strains) 'Lobonor^ s sp. PolvtoTa uvella  Prototheca zopfIt  Stephar.olpfaaera pluvlalls Euglenophyta Euglena aniiaana var minor * ~~ deses " alaollls • '• fclebsll " plsclformis " atellata Chrysonhyta Hymenoronas sp. 156 Baclllarlophvta Melo3lra nummuloldes Clone Mel-3 Crypto^hyta  Bemlselals vlre3cen3 Rhodor>h7ta  Rhodella maculata Xantho^hvta Monodu3 subterraneus Trlbongia aequale Cyanop'-.yta Agmenellum quadrupllcatum Strain PR-6 L D L D L D L D ? ? L L 0 L D L D L D L D ? L L ? ? 1 ? ? L L L L L L L L L L L 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 0 . 2 1 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 1 0 . 8 2 7 . 0 ' < 9 . 1 < 9 . 1 0 . 2 1 27.0 2 7 . 0 2 7 . 0 0 . 2 1 1 1 . 0 U . O 1 1 . 0 1 1 . 0 1 1 . 0 1 1 . 0 1 1 . 0 0 . 5 ^ . ^ 0 . 1 0 . 5 0 . 3 8 0.17 0 . 2 5 0 . 1 - » - 2 . 1 NG NG NG NG NG NG NG NG-( 1 ) Den Dooren ( I 9 6 9 O 6k% 1 ? NG NG NG NG NG NG NG NG ++ ++++ '++ NG ++ NG ++++ NG NG ++ NG NG NG NG ( 2 ) Droop ( 1 9 6 1 ) Ludwig ( 1 9 3 8 ) Arnow ( 1 9 5 3 ) Den Dooren ( 1 9 6 7 ) ( 1 9 6 9 ) ( 1 9 6 9 ) Bond (1933) Loefer ( 1 9 3 2 ) M I. Droop (1961) . Bond (1933) Bond (1933) N II-Droop (1961) Dual (1931) (1933b) II N H (1931a)' (1931b) (193D(1933b) ++ Printer et a l . (1963) NG Hellebust et al. (1967) NG Antla et al. (1968) NG Turner (1970) + (2) Miller et al. ( 1 9 5 8 ) 8 5 # Beloher et al. ( 1 9 5 8 ) NG Kapp et a l . (1975) ( 1 ) NG-no growth: 7-doubtfuli +-poori ( 2 ) of control. ++-faln +++-goodi ++++-excellent. , TABLE 3 10. T o t a l l*COz m e a s u r e d a s p r o d u c t o f c a t a b o l i s m f r o m 2 - w e e k s ' i n c u b a t i o n o f a l g a e w i t h ring-labeled i 4 C - p h e n y l a l a n i n e • -. L i g h t D a r k A l g a " C O : , d p r a X 1 0 - 3 C h l o r o p h y t a Dunaliella tertiolecta Nannochloris oculata 0 0 0 : - . C h r y s o p h y t a .'• Isochrysis galbana ' Prymnesium parvum Monochrysis lutheri Coccolithus huxleyi 1 . 0 8 ( 0 . 0 5 % ) * 2 . 9 4 ( 0 . 1 3 % ) 0 o • 0 2 . 6 1 ( 0 . 1 2 % ) •o -0 B a c Q I a r i o p h y t a P e n n a t e d i a t o m s Amphiprora paludosa Cylindrotheca fusiformis Phaeodactylum tricornutum Navicula incerta Nitzschia angularis C e n t r i c d i a t o m s Cyclotella nana Chaeloceros gracilis Skeletonema costatum Thalassiosira fluvial His 0 8 . 2 2 ( 0 . 3 7 % ) 2 . 5 6 ( 0 . 1 2 % ) 3 2 . 9 4 ( 1 . 5 0 % ) 1 3 . 2 5 ( 0 . 6 0 % ) 0 0 0 o 0 0 2 . 6 2 ( 0 . 1 2 % ) 1 0 . 0 7 ( 0 . 4 6 % ) 0 • 0 0 o . 0 : . C r y p t o p h y t a Chroomonas salina Rhodomonas lens Hemiselmis virescens 0 1 8 . 9 2 ( 0 . 8 6 % ) 0 0 7 . 6 6 ( 0 . 3 5 % ) .'• o V y r r o p h y t a Amphidinium carteri 1 . 6 6 ( 0 . 0 7 % ) •' 0 R h o d o p h y t a Porphyridium omentum 0 0 C y a n o p h y t a Anacystis marina Agmenellum quadruplicatum 0 1 . 0 5 ( 0 . 0 5 % ) •: o 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 f i s s i o n of the aromatic ring of phenyl-alanine by marine planktonlc algae. Can. J. Bot., ^9:259-261, • • ' • • ; \-- • • i i . ; . Hazen a s s i m i l a t e d 50% o f t h e s u p p l i e d p h e n y l a l a n i n e o v e r a c o n c e n t r a t i o n range o f 0.2-1.0 uM /1 ( N o r t h e t a l . , 1967). and a t a c o n c e n t r a t i o n o f 0.7 ;u;M/l, a n a c c u m u l a t i o n r a t i o o f 670:1 was o b t a i n e d ( N o r t h e t a i . , 1969). I n more d e t a i l e d s t u d i e s , C h l o r e l l a f u s c a showed an a p p a r e n t K M o f 5 MM, a V ^ y o f 0.1 nmoles/min / 1 0 ' c e l l s and a t a c o n c e n t r a t i o n o f 2 juM. an a c c u m u l a t i o n r a t i o o f 1000:1 ( P e d e r s e n e t a l . , 197*01 whereas C h l o r e l l a v u l g a r i s had a Kffl o f 7.2 uM o r 15.0 uM f o r L - p h e n y l a l a n l n e - l - ^ C o r 10.0 uM f o r L - p h e n y l a l a n l n e - 4 - T (Dedonder e t a l . , 1968; Van Sumere e t a l . , 1971)• I t was a l s o o b s e r v e d t h a t C_. f u s c a has t h e a b i l i t y t o a s s i m i l a t e . D - p h e n y l a l a n i n e ( P e d e r s e n e t a l . , 1974). The m e t a b o l i c r o u t e s f o r t h e d e g r a d a t i o n o f b o t h p h e n y l a l a n i n e and t y r o s i n e a r e unknown. P h e n y l a l a n i n e and t y r o s i n e ammonla-lyases c o u l d n o t be d e t e c t e d i n A l a r l a  e s c u l e n t a (L.) G r e v l l l e , U l v a l a c t u c a . Rhodymenla p a l - . mata ( L . ) G r e v l l l e , and P o l y s l p h o n l a l a n o s a (L*) Tandy, (Young e t a l . , 1966). I n s e v e r a l a l g a e an a r o m a t i c amino, a c i d t r a n s a m i n a s e has been d e t e c t e d . I n C h l o r e l l a vulgaris»» t h e amino group was t r a n s f e r r e d f rom L - p h e n y l a l a h i n e t o d l o x o v a l e r l c a c i d (an a - k e t o a c i d ) (Gassman e t a l . , 1968) w h i l e l>n U l v a l a c t u c a p y r u v a t e and a - k e t o g l u t a r a . t e were u t i -l i z e d as t h e a - k e t o a c i d ( J a c o b i , 1957). No t r a n s a m i n a s e a c t i v i t y .was d e t e c t e d l n U, l a c t u c a f o r L - t y r o s i n e w i t h e i * t h e r o f t h e s e a - k e t o a c i d s . I n Agmenellum q u a d r u p l l c a t u m and A n a c y s t l s n i d u l a n s , a t r a n s a m i n a s e was r e p o r t e d i n v o l v i n g L -p h e n y l a l a n i n e and a c t i v i t y w i t h s e v e r a l a - k e t o a c i d s (Stenmark e t a l , , 197*0* A t p r e s e n t no o t h e r enzymes have been examined w h i c h c o u l d be I n v o l v e d l n t h e d e g r a d a t i o n o f p h e n y l a l a n i n e a nd/or t y r o s i n e . The f i s s i o n o f t h e a r o m a t i c r i n g by a l g a e has a l s o •.- ----- ik . ' .. been s t u d i e d by C r a i g l e e t a l . , 1965 • C - p h l o r o g l u c l n o I •(•'1»3!i 5 - t r l hydroxybenzene ) was added t o n i n e a x e n l c s p e c i e s o f a l g a e . S i x were a b l e t o m e t a b o l i z e t h e r i n g c a r b o n s t o C02« F i s s i o n o f t h e a r o m a t i c r i n g was enhanced s e v e r a l f o l d by p r e c o n d i t i o n i n g each s p e c i e s t o p h l o r o g l u c i n o l . No I n -d i c a t i o n as t o t h e pathway o f r i n g f i s s i o n was g i v e n * I n t h e c h e m i c a l a n a l y s i s o f a l g a e , many a r o m a t i c •'. compounds have been i d e n t i f i e d . They r a n g e . f r o m s i m p l e t o l a r g e m u l t i - r i n g e d s t r u c t u r e s . I n T a b l e k % l i s t i s p r e s e -n t e d o f i d e n t i f i e d and n o n - I d e n t i f i e d compounds a l o n g , w i t h t h e a l g a l s p e c i e s from which t h e y were i s o l a t e d . Only b e n z a l d e h y d e can be h y p o t h e t i c a l l y i m p l i c a t e d I n t h e d e g r a d a t i o n o f a r o m a t i c amino a c i d s , whereas t h e o t h e r s based on p h l o r o g l u c i n o l , a r e a l m o s t c e r t a i n l y d e r i v e d f rom a n o t h e r 1 p a t h w a y • .An i n t e r e s t i n g group o f p h e n o l i c s c o n t a i n ^ bonded bromine have, been i d e n t i f i e d * They a r e f o u n d i n t h e Hhodophyta, m a i n l y i n t h e Rhodomelaceae, a l t h o u g h s e v e r a l have been r e p o r t e d i n two o t h e r a l g a l d i v i s i o n s (see T a b l e 5 )•• I n T a b l e 5 a r e l i s t e d t h e a l g a and t h e b r o m o p h e n o l l c compounds c o n t a i n e d - i n each a l o n g w i t h t h e r e f e r e n c e s and c h e m i c a l .. , s t r u c t u r e s . The e t h y l and m e t h y l e t h e r s a r e now c o n s i d e r e d , t o be a r t i f a c t s o f i s o l a t i o n and o n l y t h e c o r r e s p o n d i n g a l d e h y d e was p r e s e n t ( P e d e r s e n e t a l . , 197^1 G l o m b i t z a e t a l . , 197^1 13 : • TABLE 4 ';. Aromatic Compounds Identified Prom Algae. Arotnatlo compound Algal species Reference Benzaldehyde 0 " C H ° Phlorogluclnol H 0 < H HO-<^0-£V)H ^ O H W 0 H . Ulva pertusa. Enteromorpha sp., Codlum fragile. Sargassum sp., Laminaria sp., P_arp.hy.ra temerar Dlgenea simplex Cladostephus sponglosus. C. vertl-cllUtus. DletTQta dlchototya, .Fucus  veslculosus. F. serratus, F, spir- alis . Hlmanthalla elongata. Cysto-selra tamarlsclfolla. C_. baecata. C. granulata, C. dlscors. C_. myrloohyl- loldes. Elfurearla blfuroata. Hall-dry_s slllguosa. Chorda fHum. Lamin-aria oehruleuea. Saccorhlza.polysch-ldes Fucus veslculosus  Blfurcarla blfurcata HalIdrys slllguosa Fucus veslculosus Bl-and polyphenols (2-lf phlorogluclnol units) Bl-and polyphenylethers (2 or 3 phlorogluclnol units)' Mixed polyphenyl-polyphenol ethers (Z-U- phlorogluclnol units Polyphenol ether (2-7 units) (non galloyl hydroxylatlon pattern) Sargalin C20H2«v08Nl»K Phenolic (unknown) • Fucus veslculosus Fucus veslculosus Cystoselra tamarlsclfolla C_. baecata Fucus veslculosus Halldrvs slllguosa. Blfurcarla  rotunda Fucus veslculosus Sargassum confusum Fucus veslculosus Alarla esculenta. Aacophyllum nodosum. Chorda f 1'1'lum. Chord-aria flagelllformls. Ectocarpus sp. Laminaria agardhll. dlgltata  Sargassum natans Katayama (1962) Glombltza et al. U973b7 Bagan et al. (1975) Glombltza et al. (19?^r Glombltza et al. (I973al Glombltza £_£ al. (1975) Bagan ejt ;iai^ V<1975) Glombltza e£ al. (1975) Glombltza e£ al. (197H Glombltza et al. (1975) Glombltza al. (1975) Glombltza e£ al. (1974*) Bagan et al. (1975) Glombltza fi£ al. (197*) Bagan et al. (1975) Salto et.al. (1951) HcLachlan ej ,al..,(196'0 Cralgle et al. (1964) Sleburth et al. (1965) TABLE 5 Bromophenollc Compounds Isolated Prom Algae. Algal species Bromophenollc oompound ln table (Reference 1 ) Cytnophy.ta  Calothrlr brevlsslma lEhweoohyta Fucus veslculosus Rhodoohyta Antlthitsnlon piuaula CeraTtum .rubrum Coralllna officinalis Cystoslonlua purpureum Halopltys lncurvus ~ Odonthalla dentata ." corymblfcra PhycoArys rubens Polyslphonla brodlacl " elongata * frutlculosa " . lanosa " morrowl'l " nigra " nlgrescens " thuyolfles .. " urceolata " violacea Bhodonela confervoldes larlx " subfusca  Vldalla volubllls ?(A) , 14(A)2. 15(A)2, 1 6 ( A ) 7(B), 13(B), 16(B) 11(c), 13(c) 11(c). 13(C) 11(C) 10(C.J) 1(D), 7(D). 16(K.P) 2(C). 3(C.G) 4(C). 5(C). 6(C), 7(C). 10(C). ll(C.G) 12(C). 13(C) 11CQ). 12(S.Q). 9?(D. 16(Q.) 13(C) 1(D), 3(C.D). 6(C), 7(C.D). 10(C)., ll(C.D), 12(C), 16(D , 0 ) 6(D)..10(D), 11(D). l6(D.O) 1(D), 10(D), 11(D), 16(D ,0 ) 3(D), 6(D,K), 7(D,M), 10(D,H). ll(D.L) 15(D), 16(D,H.H,0). 17(D) 6(F) 1(D), 10(D), 11(D).. 16(D , 0 ) 3(C,D), 6(C), 7(D), 10(D). U(C,D), 12(C), 16(D ,0) 10(D), 11(D), 16(D) 1(D), 6(C,D), 7(C,D), 10(C)., 11(C),. 12(C) 10(D). 11(D), 16(D) 3(C.G), 10(C). lKC.P.G), 12(C) 10(E). 12(E) 10(D,B), 11(D), 12(H), 15(D), 1 6(D , 0 . 3 ) 16(N) ( 1 ) letter refers to reference, while number refers to chemical structure. ( 2 ) .Isolated from medium. A. B C D E P G H I J K L R' K 0 P Q Pedersen, Pedersen, Pedersen, Glombltza, Katsul, K. Salto, T., Cralgle, J Hodgkln, J Katsumoto, Pedersen, Augler, J Kastagll, Stoffelen, Augler, J. Glombltza, Peguy, M. Kurata, K. Kurata, K, !K. arid E. J. Da Silva (1973), K. and L. Fries (1975). K., P. Saenger, and L. Fries (197*0. K. H. Stoffelen, U. Murawskl, J. Blelaczek, and H. Egge (1974) , Y. Suzuki, S. Kltamura, and T. Irle (1967) and Y. Ando (1955) S.. and D. E. Gruenlg (1967) H., J. S. Cralgle, and A. G. Kclnnes (1966) T., and S. Kagawa (1964) K. (1974) , and P. Kastagll ( 1 9 5 6 ) P., and J. Augler ( 1 9 4 9 ) E., K. -W. Glombltza, U. Murawskl. J. Blelaezek, and H. Egge ( 1 9 7 2 ) , and K. H. Henry ( 1 9 5 0 ) K. -W., and H. Stoffelen ( 1 9 7 2 ) • ( 1 9 6 4 ) , T. Anlya, and-K. Yabe ( 1 9 7 3 ) , and T. Araiya ( 1 9 7 5 ) H20H M i Bt^s^-OH OH © CH 2OCH 3 OH © CH2OCH3 OH © CH0 B,6Br BrS^-OH OH © C^CX^Hg OH CH20H B r _ \ ^ 0 H OH CH2OCH3 © © ^ 2 O C 2 H S C H 0 B r - \ ^ - B r B OH Br. B CH0 OH @LAN0S0L @ CH20H ^OCHj rS^OH B OH OH © -or-CH20H B r , ^ Br-© © CH20S0^K+ ChL^ OCjHy Br '* ; ^0S0 3 -K + Br^s^-OH . Br\^OH OSOj-K* OSO^K* REF.-H REF.-O OH 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 i n 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 i t 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 w i l l be introduced. ; * MATERIALS AND METHODS 16. I . C u l t u r i n g . A l l methods were based on s t a n d a r d p r i n c i p l e s o f a s e p t i c t e c h n i q u e s used i n c u l t u r i n g p h y t o p l a n k t o n . A. The medium The c o m p o s i t i o n of t h e p h y t o p l a n k t o n c u l t u r e medium used I n t h i s i n v e s t i g a t i o n o f a r o m a t i c m e t a b o l i s m i s p r e s e n t e d I n T a b l e 6. The medium i s based on t h a t u s e d by Dr. N. J.. A n t i a f o r mass c u l t u r i n g p l a n k t o n l c a l g a l s p e c i e s ( A n t i a and K a l m a k o f f , 1965). Open ocean seawater (see M c A l l i s t e r et a l . i960) was alw a y s u s e d . A l l t h e i n o r g a n i c c h e m i c a l s u s e d f o r the c u l t u r e medium were 'Baker A n a l y z e d ' Reagents o b t a i n e d f r o m J . T. Bak e r C h e m i c a l Co., P h l l l i p s b u r g , N.J. The pH was 7.9-8.1 a f t e r a u t o c l a v i n g o r 7.7-7.9 a f t e r f i l t e r s t e r i l i z a -t i o n . The s a l i n i t y was c a l c u l a t e d t o be 27$°. B. A l g a l s p e c i e s u t i l i z e d . A x e n i c c u l t u r e s o f N a v l c u l a l n c e r t a Hustedt and I s o c h r -y s l s g a l b a n a Parke were o b t a i n e d from Dr. N.J. A n t i a , Vancouver L a b o r a t o r y o f F i s h e r i e s and Marine S e r v i c e o f Environment Canada, N a v l c u l a l n c e r t a H u s t e d t was I n i t i a l l y o b t a i n e d as s t r a i n Lewin #66-M from Dr. J o y c e L e w l n . Dr. R a l p h L e w i n i s o l a t e d t h i s s t r a i n (73-M) from a s a l i n e p o o l i n San F r a n c i s c o , C a l i f o r n i a . I s o c h r y s l s g a l b a n a Parke was o b t a i n e d f r o m Woods Hole Oceanography I n s t i t u t e as c l o n e " I s o " by Dr. J . D. H. S t r i c k l a n d . Dr. Mary Parke i s o l a t e d t h i s s t r a i n ( F l a g e l l a t e I ) from a f i s h pond a t t h e Marine B i o l o g i c a l S t a t i o n , P o r t E r i n , I s l e o f Man, U n i t e d Klngdon.- L i g h t 17. TABLE 6 P h y t o p l a n k t o n C u l t u r e Medium K N 0 5 126.0 mg ( 1.25 mmoles) NaH 2PO^'H 20 40.2 mg ( 0.40 mmoles) Na 2S10 3'9H 20- 1- 268.8 mg ( 0.96 mmoles) V l t a m i n s : Thiamine*HC1 0.96 mg ( 2.84 umoles) B i o t l n 1.92 wg ( 7.8 nmoles) B 1 2 (Cyanocobalamin) 3.84 ug ( 2.57 nmoles) T r a c e M e t a l s ( c h e l a t e d ) : Na 2*EDTA*2H 20 8.1 mg (21.8 umoles) P e C l 3 ' 6 H 2 0 2.7 mg (10.0 umoles) MhS0^»4H 20 1.125 mg ( 5.0 ;umoles) ZnSO^.'7H20 0.575 mg ( 2.0 umoles) Na 2MoO^*2H 20 0.243 mg ( 1.0 umole ) CuSO^'5H 20 0.025 mg ( 0.1 umole ) CoSO^*7H 20 0, 014 mg (50.0 nmoles) B u f f e r : T r i s ' H C l (pH 7.5-7.6)2 1.0 gm ( 8.3 mmoles) Sea Water: Open ocean, s a l i n i t y 33&> -767.0 ml D i s t i l l e d Water t o 1 l i t e r (1) one h a l f t h i s c o n c e n t r a t i o n was u t i l i z e d l n mass c u l t u r e s o f I s o c h r y s l s g a l b a n a . (2) t h i s g i v e s a pH o f 7.9-8.1 I n t h e medium a f t e r a u t o c l a v i n g (15 minutes a t 120°C). 18. m i c r o s c o p e photographs o f b o t h s p e c i e s a r e p r e s e n t e d i n F i g u r e 3. Other p h y t o p l a n k t o n s p e c i e s u t i l i z e d i n v a r i o u s ex-p e r i m e n t s were a l s o o b t a i n e d f rom Dr. A n t i a . T h e i r i n i t i a l s o u r c e i s l i s t e d i n T a b l e 7. N o n - p l a n k t o n i c a l g a l s p e c i e s were c o l l e c t e d f r o m S t a n l e y Park, Vancouver, B.C. ne a r Lumber-m a n ^ A r c h . C, Maintenance o f s t o c k c u l t u r e s . N. l n c e r t a and I± g a l b a n a were m a i n t a i n e d i n 125 ml sc r e w cap E r l e n e m y e r f l a s k s c o n t a i n i n g kO ml o f t h e c u l t u r e medium i n T a b l e 69 The s i l i c a t e c o n c e n t r a t i o n used was one h a l f t h e v a l u e s t a t e d i n t h e t a b l e f o r o n l y s t o c k c u l t u r e s . These two s p e c i e s were s u b c u l t u r e d e v e r y two months o r when e x p o n e n t i a l l y g r o w i n g c e l l s were r e q u i r e d f o r an e x p e r i m e n t . These c u l t u r e s were m a i n t a i n e d a t 19-21°C and a t a p p r o x i m a t e l y 100 f o o t - c a n d l e s (Weston F o o t c a n d l e L i g h t Meter) under c o n -t i n u o u s i l l u m i n a t i o n from c o o l - w h i t e f l u o r e s c e n t l i g h t s (No, F1^T12/CW., S y l v a n i a E l e c t r i c Canada, LTD.). The o t h e r p h y t o p l a n k t o n s p e c i e s were o b t a i n e d i n t h e e x p o n e n t i a l growth phase from Dr. A n t i a ' s c o l l e c t i o n . D. C o n t a m i n a t i o n t e s t i n g . A c o n t a m i n a t i o n t e s t was performed whenever a s t o c k c u l t u r e was u t i l i z e d t o I n i t i a t e an experiment and a t t h e end o f t h e same e x p e r i m e n t . One o r two drops o f t h e a l g a l c u l t u r e growth medium were added t o a screw capped c u l t u r e t u b e c o n t a i n i n g t h e s t e r i l i t y t e s t medium (Appendix A ) . The A B F i g u r e 3. P h o t o m i c r o g r a p h s by i n t e r f e r e n c e c o n t r a s t o f t h e a l g a l s p e c i e s u t i l i z e d i n t h i s i n v e s t i g a t i o n , (A) I s o c h r y s l s g a l b a n a Parke and (B) N a v l c u l a l n c e r t a H u s t e d t . TABLE 7 Initial Source of Axenlc Algal Cultures Algal species Initial sourc C h l o r o p h y t a B r a c h l m o n a s a u b m a r l n a ( B o h l l n ) D r o o p v a r p u i s I f e r a 7 / 2 a D r o o p D u n a l l e l l a t e r t l o l e c t a B u t c h e r N a n n o c h l o r l s o c u l a t a D r o o p H a p t o p h y t a E m l l l a n l a h u x l e y l ( L o h m . ) H a y & M o h l e r P a v l o v a l u t h e r l ( D r o o p ) G r e e n ( 3 ) ' B a c l l l a r i o p h y t a A a p h l ' p r o r a p a l u d o s a W. S m i t h v a r d u p l e x D o n k , T h a l a s s i o s i r a p s e u d o n a n a ( H u s t e d t ) H a s l e & H e l m d a l S k e l e t o n e a a c o s t a t u m ( G r e v . ) C l e v e T h a l a s s i o s i r a f l u v l a t l l l s H u s t e d t C r y p t o o h y t a C h r o o i c o n a s s a l l n a ( W i s l o u c h ) B u t c h e r C r y p t o m o n a d s t r . : / f f l l R h o d o m o n a s l e n s P a s o h e r a n d R u t t n e r M i l l p o r t s t r a i n D r . M . R . D r o o p W o o d s H o l e C l o n e " D u n " , D r . J . D . H . S t r i c k l a n d M i l l p o r t s t r a i n # 6 6 , D r . M . R . D r o o p ( 2 ) W o o d s H o l e C l o n e " B T - 6 " , D r . T . R . P a r s o n s M i l l p o r t s t r a i n # 6 0 , D r . J . D . H . S t r i c k l a n d W o o d s H o l e " L e w i n # 7 3 - K " , D r . J . L e w l n W o o d s H o l e C l o n e " 3 H " , D r . R . R . L . G u l l l a r d W o o d s H o l e C l o n e " S k e l " , D r . R . R . L . G u l l l a r d W o o d s H o l e C l o n e " A c t i n " , D r . J . L e w l n W o o d s H o l e C l o n e " 3 C " , D r . F . T . H a x o H a s k l n s W H l , D r . L . P r o v a s o l i H a s k l n s L a b o r a t o r i e s , D r . L . P r o v a s o l i P y r r o p h y t a A m p h l d l n i u m c a r t e r l H u l b u r t R h o d o o h y t a P o r p h y r l d l u c i c r u e n t u m N a g e l l R h o d e l l a m a c u l a t a E v a n s C y a n o p h y t a A g t n e n e l l u m q u a d r u p l l c a t u m ( K e n e g . ) B r e b i s s o n A n a c y s t l s m a r i n a ( H a n s g . j D r o u e t & D a l l y X a n t h o p h y t a H e t e r o t h r l T s p . M o n a l l a n t u s s a l l n a B o u r r e l l y W o o d s H o l e C l o n e " A m p h l 1 " , D r . L P r o v a s o l i V l s c h e r ' s s t r a i n # 1 0 7 , D r . F . T . H a x o M i l l p o r t s t r a i n # 2 0 7 , D r . M . F . T u r n e r V a n B a a l e n ' s s t r a i n " P R - 6 " , D r . C . V a n B a a l e n V a n B a a l e n ' s s t r a i n " 6 " , D r . C . V a n B a a l e n . S . M . E . M a r s e i l l e s t r a i n . D r . S . Y . M a e s t r l n l S . M. E . M a r s e i l l e s t r a i n . D r . S . X. M a e s t r l n l ( 1 ) o r i g i n a l I s o l a t e a n d n a m e o f s p e c i m e n - c u l t u r e d o n o r . ( 2 ) t a x o n o m l c a l l y r e v i s e d f r o m C o c s o l l t h u s h u x l e y l ( L o h m . ) K a m p t n e r , ( s e e H a y e t a l . , 1 9 6 7 ) . ( 3 ) t a - x o n o m l c a l l y r e v i s e d f r o m K o n o c h r y s l s l u t h e r l D r o o p , ( s e e G r e e n , 1 9 7 5 ) . (6) t a x o n o m l c a l l y r e v i s e d f r o m C y c l o t e l l a n a n a H u s t e d t , ( s e e H a s l e e t a l . , 1 9 7 0 ) . If t u b e was i n c u b a t e d about t h r e e weeks i n t h e d a r k a t room t e m p e r a t u r e . C o n t a m i n a t i o n was e x t r e m e l y r a r e and when d i s -c o v e r e d t h e d a t a t h e r e f r o m was d i s c a r d e d . I n Appendix A, t h e p r o c e d u r e u n d e r t a k e n when p o s s i b l e c o n t a m i n a t i o n was s u s p e c t e d i s o u t l i n e d , ? E, C e l l e n u m e r a t i o n , . C e l l numbers were d e t e r m i n e d d i r e c t l y by c o u n t i n g t h e c e l l s w i t h a hemacytometer o r i n d i r e c t l y by m e a s u r i n g t h e o p t i c a l d e n s i t y o f a c e l l s u s p e n s i o n , a. Hemacytometer. A c o u n t i n g chamber (AO S p e n c e r b r i g h t - l i n e ) was em-p l o y e d when an e s t i m a t e o f c e l l number was r e q u i r e d . Samples °f N, l n c e r t a o r I . g a l b a n a were n o t r e q u i r e d t o be p r e s e r v e d f o r c o u n t i n g ( t h e m o t i l e s t a g e o f I . g a l b a n a was n o t e n c o u n t -e r e d i n e x p o n e n t i a l l y g r o w i n g c u l t u r e s ) . However, when m o t i l e p l a n k t o n l c s p e c i e s were t o be c o u n t e d , samples were t r e a t e d w i t h one d r o p o f 5% f o r m a l i n p e r ml f o r c a , 5 n i n . b e f o r e f i l l i n g t h e hemacytometer chambers. F o r each sample, a t l e a s t two r e p l i c a t e f i e l d s were c o u n t e d t o o b t a i n an e s t i m a t e o f t h e c e l l number. F o r v e r y s m a l l p l a n k t o n l c s p e c i e s a P e t r o f f - H a u s s e r b a c t e r i a c o u n t e r c o u n t i n g chamber (C, A. H a u s s e r and Son) was u s e d , b. O p t i c a l d e n s i t y . O p t i c a l d e n s i t y (O.D.) was u s e d as an e s t i m a t e o f c e l l number when growth e x p e r i m e n t s were c o n d u c t e d l n 8 ml c a p a c i t y . o p t i c a l l y ^ c l e a r , , s c r e w capped ....culture tubes,. The c o n t e n t s o f 4.0x107-h 1.0x# 4.0x1cPf CELL NUMBER PER ml 1.0x1064r 40x105f i i i i-111 1—I—( i t l i l j 1 1—1 1 M i l • — • I s o c h r y s i s galbana Navicula incerta loxicr 4.0x10n-b—H-i Q004 0.010 0.040 0.100 0.400 OPTICAL DENSITY at 600 nm. 1.000 F i g u r e k> , The l i n e a r range o f OD i n r e l a t i o n t o c e l l number f o r p l a n k t o n l c s p e c i e s . 23. e a c h c u l t u r e t u b e was f i r s t t h o r o u g h l y mixed w i t h a V o r t e x J r 0 m i x e r . The tube.was t h e n I n s e r t e d i n t o a S p e c t r o n l c 20 C o l o r i m e t e r ( B a u s c h and Lamb, I n c . , R o c h e s t e r , N.Y.) and t h e OD r e a d a t 600 nm. . The r a n g e w i t h i n w h i c h OD was l i n e a r l y r e l a t e d t o c e l l number was d e t e r m i n e d by u s i n g c o n c e n t r a t e d c u l t u r e s o f N. l n c e r t a and Xi g a l b a n a o b t a i n e d by c e n t r i f u g a t i o n . A d i l u -t i o n s e r i e s was p r e p a r e d and t h e OD o f each d i l u t i o n was measured. I n F i g u r e 4, t h e r e l a t i o n s h i p o f OD t o c e l l number i s p r e s e n t e d . A d d i t i o n a l e x p e r i m e n t s I n d i c a t e d t h a t t h e r e -l a t i o n s h i p between c e l l number and OD was a l m o s t c o n s t a n t t h r o u g h o u t t h e e x p o n e n t i a l phase. F . O p t i c a l d e n s i t y e x p e r i m e n t s and medium p r e p a r a t i o n . When t h e s i m u l t a n e o u s u s e o f many compounds o v e r a l a r g e c o n c e n t r a t i o n range was r e q u i r e d f o r an e x p e r i m e n t , t h e u s e o f l a r g e c u l t u r e volumes was i m p r a c t i c a l and s m a l l Kimax s c r e w capped t e s t t u b e s ( K i m b l e No 45042, 15x125 mm) were u s e d i n s t e a d . These t u b e s were s e l e c t e d f o r ease o f measure-ment w i t h o u t s a m p l i n g e v e r y t u b e because t h e y f i t d i r e c t l y i n t o t h e S p e c t r o n l c 20. T h e r e f o r e t h e r e was l i t t l e chance o f c o n t -a m i n a t i o n r u i n i n g t h e e x p e r i m e n t . By u s i n g t h e a l g a l c u l t u r e medium, 2.1 mM s t o c k s o l u -t i o n s o f each a r o m a t i c compound were p r e p a r e d ^ t h e n f i l t e r s t e r i l i z e d (0.2-um p o r e - s i z e Gelman M e t r i c e l f i l t e r i n • s t e r i l e d i s p o s a b l e p l a s t i c u n i t s f r o m N a l g e Co., R o c h e s t e r N.Y.). A d i l u t i o n s e r i e s (2.1, 1.05, 0.525, 0.262, 0.105, 0.0525, and 0.0262 mM) was prepared by ase p t i c a l l y pipetting . Into p r e - s t e r i l l z e d tubes the required volume of stock solu-t i o n and s t e r i l e culture medium to give a f i n a l volume of 4 m l 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 d i l u t i o n in each tube, caused by the Inoculum, decreased the i n i t i a l 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 l i g h t placed h o r i -.zoritaily above them. ' The temperature was maintained^ between 19 -21° C and the l i g h t intensity was estimated • at ca. 100-120 foot-candles. Growth measurements were made at 1-2 d a y . 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 i n OD (expon-ential growth phase) was also calculated from the same plot. At the termination of the experiment, each tube was ac i d i f i e d to pH~2 then extracted with peroxide free diethylether for analysis of metabolic products. G. One l i t e r culture experiments. In 2,8 l i t e r capacity Erlenmeyer flasks, 1 l i t e r growth experiments were conducted at 19-21°C under continuous [ ' 25. i l l u m i n a t i o n from cool-white fluorescent lamps. The i n t e n -s i t y of i l l u m i n a t i o n was estimated at ca. 280-310 foot-candles. Each culture was incubated stationary and every day was thoroughly mixed by vigorous sw i r l i n g of the f l a s k . The growth of each culture was followed by taking c e l l counts, then p l o t t i n g 1 ° S 1 0 E^/EQ against growth period (t hr), where N Q and Nj. denote c e l l concentrations at time of inoculation and t hr_ respectively. N Q was calculated d i r e c t l y from the c e l l 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 l o g 1 0 N t/N 0 = K ( 1 Q ) • t and log 1 0.2 = K ( l 0 ) • tg The slope of the s t r a i g h t - l i n e portion of the growth curve was used to derive K(io)» H. Mass c u l t u r i n g . In 6 l i t e r capacity Erlenmeyer f l a s k s , modified f o r mass cu l t u r i n g phytoplankton, 5 l i t e r growth experiments were conducted. In Figure 5# the design of the mass culture v e s s e l which i s s i m i l a r to that used by Antia and Kalmakoff (I965) i s outlined. The temperature was maintained at 19-o 20 C by means of an incubator. Cool-white fluorescent lamps provided continuous i l l u m i n a t i o n at an i n t e n s i t y estimated at ca. 1200-1500 foot-candles.. Each culture was kept i n constant motion by means of a magnetic s t i r r i n g bar and was aerated d a i l y f o r 3-6 hours with a source of compressed a i r (95$ a i r and 5% C0 2) through the f r i t t e d - g l a s s o u t l e t . Growth parameters f o r each culture were determined as Diagram of mass c u l t u r e a p p a r a t u s and t r a p f o r v o l a t i l e p r o d u c t ( s ) . A, s t r e a m o f gas c o n t a i n i n g ' 95% a i r and 5% C O 2» S l a s s h o l d e r packed w i t h c o t t o n w o o l . C, a u t o c l a v a b l e r u b b e r t u b i n g . D, ground g l a s s f i t t i n g f o r a e r a t i o n t u b e . E, 6 - l i t e r E r l e n m e y e r c u l t u r e v e s s e l . F, f r i t t e d - g l a s s gas o u t l e t . G, magn e t i c s t i r r i n g b a r . H, magnetic s t i r r i n g b l o c k . I, c o t t o n wool ( n o n - a b s o r b e n t ) p l u g wrapped i n gauze*. J , c o t t o n wool p l u g wrapped i n gau z e , r K, a u t o c l a v e d r u b b e r s t o p p e r f i t t e d w i t h g l a s s 27. F i g u r e 5 ( c o n t . ) . t u b i n g ( t o r e p l a c e t h e c o t t o n wool p l u g ( J ) i n o r d e r t o c o n n e c t t h e gas b u b b l e r t o t h e c u l t u r e v e s s e l ) . L, gas b u b b l e r c o n t a i n i n g r e a c t a n t f o r v o l a t i l e p r o d u c t ( s ) . * r e p l a c e d by an a u t o c l a v e d r u b b e r s t o p p e r when gas b u b b l e r was c o n n e c t e d . d e s c r i b e d i n P a r t G f o r one l i t e r c u l t u r e e x p e r i m e n t s . A f t e r f i l l i n g w i t h 5 l i t e r s o f t h e a l g a l c u l t u r e medium th e c u l t u r e v e s s e l was f i t t e d w i t h t h e a e r a t i o n a p p a r a t u s t h e n a u t o c l a v e d as a u n i t . A f t e r c o o l i n g , t h e g a s - i n l e t l i n e was con n e c t e d t o t h e s o u r c e o f COg-enriched a i r and t h e sys t e m was a l l o w e d t o s t a b i l i z e 1-2 days b e f o r e i n o c u l a t i n g w i t h g e n t l e a e r a t i o n and m i x i n g a t 19-21° C. J u s t p r i o r t o i n o c u l a t i o n , s t e r i l e v i t a m i n s and n i t r a t e - p h o s p h a t e s o l u t i o n s were added a s e p t i c a l l y t o t h e c u l t u r e v e s s e l . To t r a p t h e v o l a t i l e m a t e r i a l r e p o r t e d by Vose e_t a l . (1971) from I . g a l b a n a , when grown on L - p h e n y l a l a n i n e , t h e gas b u b b l e r was c o n n e c t e d t o t h e c u l t u r e v e s s e l and t h e c o t t o n wool p l u g was r e p l a c e d by an a u t o c l a v e d r u b b e r s t o p p e r . The r u b b e r s t o p -p e r w i t h t h e g l a s s t u b e and t h e c o n n e c t i n g r u b b e r t u b i n g were a u t o c l a v e d b e f o r e c o n n e c t i n g t h e c u l t u r e v e s s e l t o t h e gas b u b b l e r . Two t r a p p i n g s o l u t i o n s were u s e d : 2,k-dinitrophenyl-h y d r a z i n e (100 mg/100 ml i n 2N HC1, see Dawson e t a l . 1969) and 5N KOH. I . C e l l c o l l e c t i o n and s t o r a g e . When r e q u i r e d , c u l t u r e s were h a r v e s t e d towards t h e end o f t h e e x p o n e n t i a l phase o f growth. A l l o n e . l i t e r C u l t u r e s ^ were haves t e d a s e p t i c a l l y by c e n t r i f u g a t i o n ( S e r v a l l RC-2 model c e n t r i f u g e ) i n a u t o c l a v e d 250 ml c e n t r i f u g e b o t t l e s a t ca.. 6,000 rpm f o r 20 min. Then t h e c e l l s were washed w i t h t h e s t e r i l e a l g a l c u l t u r e medium t o remove any r e s i d u a l a r o -m a t i c compound i n i t i a l l y added t o the medium. The medium was a n a l y z e d f o r a r o m a t i c p r o d u c t s , and t h e p r e - a d a p t e d c e l l s were used f o r t r a c e r e x p e r i m e n t s . The mass c u l t u r e s were h a r v e s t e d by u s i n g t h e c o n t i n u o u s f l o w system ( S z e n t - G y o r g l and Blum) f o r t h e S e r v a l l RC-2 c e n t r i f u g e . A speed o f 10,000 rpm was m a i n t a i n e d t h r o u g h o u t t h e c o l l e c t i o n . The c o n t i n u o u s f l o w system was r i n s e d a t t h e end o f t h e h a r v e s t w i t h 3.0$ NaCl t o remove exc e s s s e a w a t e r , t h e n t h e c e l l s were packed f o r a n o t h e r 20 min. To p r e v e n t c e l l l y s i s , t h e i n -t e r n a l t e m p e r a t u r e o f the c e n t r i f u g e was h e l d between 17-21 C f o r h a r v e s t i n g a l l c u l t u r e s . The c o l l e c t e d c e l l s f r o m t h e mass c u l t u r e s o r fr o m t h e C - t r a c e r e x p e r i m e n t s were d r i e d w i t h P 2 O 5 a t room temp-e r a t u r e i n a d e s i c c a t o r which was c o n t i n u o u s l y evacuated; by a h i g h vacuum pump. A f t e r t w e l v e h o u r s , t h e P 2 O 5 was changed and the d e s i c c a t o r was ev a c u a t e d t o 100-150 t o r r t h e n s t o r e d a t 0-Vc f o r s e v e r a l days d u r i n g which t i m e t h e P 2 O 5 was changed u n t i l t h e c e l l s were t o t a l l y d r y . The d r i e d c e l l p e l l e t s were ground t o a powder i n a mortar and p e s t l e , 0 weighed, and s t o r e d a t -30 C i n v i a l s w i t h i n a d e s i c c a t o r e v a c u a t e d t o 100-150 t o r r . These c e l l powders can be s t o r e d i n d e f i n i t e l y u nder t h e s e c o n d i t i o n s and were u s e d f o r a l l 2 9 . enzyme a s s a y s . The f o u r mass c u l t u r e s o f . 1* g a l b a n a grown on L - t y r o s i n e were c o l l e c t e d w i t h t h e c o n t i n u o u s f l o w system, but t h e s t e e l t u b e s c o n t a i n i n g t h e c e l l p e l l e t s were s t o r e d f r o z e n a t - 3 0 ° C , These c e l l s were m a i n t a i n e d a t t h i s t e m p e r a t u r e u n t i l e x t r a c t -ed f o r t h e b r o m o p h e n o l l c compound. I I . . C h e m i c a l s t u d i e s o f a r o m a t i c compounds. A, S o u r c e s . The a r o m a t i c compounds used i n t h i s s t u d y were r e -ag e n t grade and were used w i t h o u t f u r t h e r p u r i f i c a t i o n . L-p h e n y l a l a n l n e and L - t y r o s l n e were " g o l d l a b e l * p r o d u c t s from C a l b i o c h e m , Los A n g e l e s , C a l i f o r n i a . p - H y d r o x y p h e n y l a c e t i c a c i d was a l s o o b t a i n e d from C a l b i o c h e m . D - P h e n y l a l a n i n e and D - t y r o s l n e were o b t a i n e d from A l d r i c h C h e m i c a l Co., S t . L o u i s , M i s s o u r i . . P h e n y l a c e t i c a c i d was s u p p l i e d by N u t r i t i o n a l B i o -c h e m i c a l Corp., C l e v e l a n d , Ohio, whereas BDH L a b o r a t o r i e s , P l a l n v i e w , N.Y. s u p p l i e d DL-p-hydroxymandelic a c i d and F i s h e r -c h e m i c a l Co., F a i r l a w n , N.J., s u p p l i e d o - h y d r o x y b e n z o i c a c i d ( s a l i c y l i c a c i d ) , Coumarin, DL-mandelic a c i d and p-hydroxy-b e n z o i c a c i d were o b t a i n f rom J . T. B a k e r C h e m i c a l Co., P h l l l l p s b u r g , N. J . The s o u r c e o f m-hydroxybenzolc a c i d was unknown, but I t was a c h r o m a t o g r a p h i c a l l y homogeneous compound as were a l l t h e a r o m a t i c s f o r m e t a b o l i c s t u d i e s . P h e n o l , o b t a i n e d from F i s h e r C h e m i c a l Co., was r e -d i s t i l l e d b e f o r e use and p-h y d r o x y b e n z a l d e h y d e , o b t a i n e d from J,- T. B a k e r C h e m i c a l Co., was r e c r y s t a l i z e d . B o t h t h e s e 30. p u r i f i e d compounds were c h r o m a t o g r a p h i c a l l y homogeneous. A l l o t h e r a r o m a t i c compounds, used f o r as chroma-t o g r a p h i c s t a n d a r d s , were o b t a i n e d f r o m v a r i o u s c h e m i c a l com-p a n i e s o r were s y n t h e s i z e d whenever p o s s i b l e . The f o l l o w i n g bromophenol s t a n d a r d s were s u p p l i e d by Dr. J . S. C r a l g l e , N . R» C. A t l a n t i c R e g i o n a l Lab., H a l i f a x , N.S, : 3 , 5-dibromo-^-h y d r o x y b e n z o i c a c i d , 5 - b r o m o v a n i l l i n , 6 - b r o m o v a n i l T i n , 3,5-d i b r o m o - ^ - h y d r o x y b e n z y l m e t h y l e t h e r , 3-bromo - ^ , 5-dihydroxy-b e n z a l d e h y d e , 3 . 5 - d i b r o m o - ^ - h y d r o x y b e n z y l a l c o h b l , and" 2 , 3 -d i b r o m o - ^ , 5 - d l h y d r o x y b e n z y l a l c o h o l . . B. Chromatography.. Paper chromatography e m p l o y i n g Whatman No. 3MM p a p e r , was used f o r two d i r e c t i o n a l s e p a r a t i o n o f p h e n o l i c a c i d s I n Ik t h e C - r i n g l a b e l l e d L - t y r o s i n e e x p e r i m e n t s . These chromato-grams were d e v e l o p e d by d e s c e n d i n g s o l v e n t f l o w a t room temp-e r a t u r e . The . f i r s t d i r e c t i o n ( w i t h t h e paper m a c h i n i n g ) was d e v e l o p e d by u s i n g s o l v e n t system AiZ% aqueous f o r m i c a c i d (V/V) and t h e second d i r e c t i o n by u s i n g s o l v e n t system B i t h e o r g a n i c upper l a y e r o f benzene: a c e t i c a c i d : w a t e r (10:7*3r V/V/V). T h i n l a y e r chromatography (TLC), e m p l o y i n g "0.5 mm A v i c e l TG-101 c e l l u l o s e l a y e r s and s o l v e n t systems A and B, was us e d f o r most s e p a r a t i o n s . P r e p a r e d c h r o m a t o g r a p h i c s h e e t s (#1325^ c e l l u l o s e w i t h f l u o r e s c e n t i n d i c a t o r #6065) f r o m Eastman Kodak Co., R o c h e s t e r , N.Y. were used f o r t h e s e p a r a t i o n o f C r-C, a r o m a t i c compounds i n s o l v e n t system C: l s o p r o p a n o l t 6 1 Cone. NR4OH1 water (8:1:1, V/V/V). _ ' _ : P h e n o l i c compounds were d e t e c t e d by s p r a y i n g w i t h d i a z o t i z e d p - n i t r o a n i l i n e r e a g e n t . o r f e r r i c c h l o r i d e r e a g e n t • . . '.. '31. '• ( s e e Appendix B ) , Aldehydes were d e t e c t e d w i t h 2 , 4 - d i n l t r o -p h e n y l h y d r a z i n e r e a g e n t (Appendix B) a n d a c i d s by m e t h y l r e d -bromothymol b l u e r e a g e n t (Appendix B ) . To a i d l n i d e n t i f i c a t i o n o f t h e v a r i o u s d e g r a d a t i v e p r o d u c t s , s p o t s were e x c i s e d from t h e chromatograms, a UV-spectrum was o b t a i n e d , and t h e compound was co-chromatographed w i t h a u t h -e n t i c s t a n d a r d s . T h i s was u n d e r t a k e n f o r b o t h r a d i o a c t i v e and n o n - r a d l o a c t l v e e x p e r i m e n t s . >. Gas l i q u i d chromatography o f a r o m a t i c a c i d s was a c -c o m p l i s h e d by a T r a c o r 550 Gas Chromatograph w i t h a column c o n t a i n i n g ]$% OV-1 on 80/100 mesh Chromosorb W-HP. T r i m e t h y l -s i l y l d e r i v a t i v e s o f t h e a c i d s were p r e p a r e d w i t h T r i - s i l from P i e r c e C h e m i c a l Co., R o c k f o r d , 111. Temperature programming, i n c r e a s i n g a t 2°/minute from 90°C t o 200°C, was us e d t o s e p a r a t e the- : S i i y l a t e d - p r o d u c t s ' w h i c h • were d e t e c t e d by f l a m e 1 o h i z a t i o n . C. S p e c t r o s c o p y . U l t r a v i o l e t s p e c t r a were o b t a i n e d u s i n g a Unlearn model SP .800 r e c o r d i n g s p e c t r o p h o t o m e t e r . N u c l e a r magnetic r e s o n a n c e (NMR) s p e c t r a were o b t a i n e d on a V a r l a n HA 100-100 •MHz s p e c t r o p h o t o m e t e r . Mass s p e c t r a (MS) were .obtained on a 1290G N u c l e l d e 90° S e c t o r I n s t r u m e n t . Only t h e major c h a r a c t e r -i s t i c MS peaks a r e p r e s e n t e d w i t h t h e r e l a t i v e peak I n t e n s i t y l n b r a c k e t s 1.. e. ( ) g i v e n as a p e r c e n t a g e o f t h e g r e a t e s t peak. D. M e l t i n g p o i n t s . M e l t i n g p o i n t s were t a k e n l n a Thomas Hoover U n i -m e l t C a p i l l a r y m e l t i n g p o i n t a p p a r a t u s . The v a l u e s o b t a i n e d a r e u n c o r r e c t e d . 32. E. C h e m i c a l p r e p a r a t i o n o f n o n - r a d l o a c t l v e compounds. I n Appendix C, t h e methods f o r the s y n t h e s i s o f v a r i o u s a r o m a t i c compounds used i n t h i s i n v e s t i g a t i o n a r e p r e s e n t e d . The methods used were based on p r e v i o u s l y r e p o r t e d s y n t h e t i c r o u t e s . The a r o m a t i c compounds s y n t h e s i s e d were,* 3• '5-dTbromo-p-hydroxybenzoic a c i d 3»5-dibromo-p-hydroxybenzaldehyde 3 t 5 - d i b r o m o - p - h y d r o x y b e n z y l a l c o h o l 3-bromo-p-hydroxybenzoic a c i d . p - h y d r o x y p h e n y l a c e t a l d e h y d e p - h y d r o x y b e n z o y l f o r m i c a c i d p h e n y l h y d r a c r y 1 1 c a c i d p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d ( u n s u c c e s s f u l ) s u l f a t e e s t e r s o f p h e n o l i c compounds F-, C h e m i c a l p r e p a r a t i o n s o f r a d i o a c t i v e compounds. I n Appendix D, t h e methods f o r t h e s y n t h e s i s o f 14 C - l a b e l l e d compounds, not c o m m e r c i a l l y a v a i l a b l e , a r e p r e s e n t e d . The compounds s y n t h e s i s e d w e r e i L-.t yr.os 1 n e - ( U ) - r i ng- l l fC 14 p - h y d r o x y p h e n y l a c e t i c a c l d - 1 - C 14 p- . h y d r o x y p h e n y l a c e t i c a c i d - 2 - C 14 p-coumaric a c l d - 2 - C I I I . I s o l a t i o n o f p r o d u c t s . I n a l l c a s e s t h e medium, w i t h o r w i t h o u t c e l l s , was a c i d -i f i e d t o pH 2 w i t h cone. H C l f t h e n e x t r a c t e d w i t h r e d i s t i l l e d p e r o x i d e f r e e d l e t h y l e t h e r . The e x t r a c t s were washed w i t h 0/5 N HC1 t h e n a l l o w e d t o e v a p o r a t e t o d r y n e s s a t room temp-e r a t u r e i n a fume hood. The r e s i d u e was r e d i s s o l v e d i n e t h -.anol o r e t h e r f o r c h r o m a t o g r a p h i c s p o t t i n g . I n t h e e x t r a c t i o n o f I_. g a l b a n a c e l l s f o r t h e bromo-p h e n o l i c compound B.bl gmwet c e l l s were suspended i n 100 ml 1.5 N HC1 t h e n d i s r u p t e d i n a F r e n c h p r e s s . T h i s s u s p e n s i o n -was e x t r a c t e d 'With e t h e r and t h e r e s u l t i n g e m u l s i o n .was . c e n t r i -f u g e d t o s e p a r a t e t h e p h a s e s . The p h e n o l i c a c i d s ,ln t h e e t h e r were e x t r a c t e d i n t o 5% NaHCOy whi c h was a c i d i f i e d and r e -e x t r a c t e d w i t h e t h e r . T h i s e x t r a c t was e v a p o r a t e d t o a s m a l l volume, banded on Eastman p r e p a r e d c e l l u l o s e p l a t e s w i t h f l u -o r e s c e n t I n d i c a t o r , and chromatographed i n s o l v e n t s y s t e m C. The band was s c r a p e d o f f and e l u t e d w i t h e t h a n o l f o r UV and MS a n a l y s i s . I V . R a d i o a c t i v e f e e d i n g e x p e r i m e n t s . Ik ' A. Source o f C - l s o t o p e s . From New E n g l a n d N u c l e a r , B o s t o n , Mass. t h e - f o l l o w i n g lk lk C-compounds were purchased* L - t y r o s i n e - 1 - C ( s p e c i f i c a c -lk t i v l t y ( S A ) , 58.2 uCi/mmole), D L - t y r o s i n e - 3 - C (SA, 15.5 p C l / lk , mmole), L - p h e n y l a l a n l n e - 1 - C (SA, 52.6 uCi/mmole)., D L - p h e n y l -\k lb a l a n i n e - 3 - C (SA, 1^.5 uCi/mmole), p h e n y l a c e t i c a c l d - 2 - <C (SA, 2.8^ uCi/mmole), b e n z o i c a d d - u n i f o r m l y r i n g - ^ C (k$ u C l / Ik 1 mmole), and s a l i c y l i c a c i d - 7 - C (SA, 2.3 uCi/mmole). The R a d i o c h e m i c a l C e n t e r , Amersham, En g l a n d s u p p l i e d t h e f o l l o w i n g DL-tyrosine-2- l 2 |'C (SA, 50.0 uCi/mmole), L - t y r o s i n e - U - ^ C (SA, (1) s i d e c h a i n . 10 juCi/mmole), D L - p h e n y l a l a n i n e - 2 - i q C (SA, 32 juCl/mmole), L-p h e n y l a l a n l n e - U - ^ C (SA, 9^2 juCi/mmole), and p h e n y l a c e t i c a c i d -l - ^ C (SA, 59 yuCi/mmole). p - H y d r o x y b e n z o i c acid-7- l i*'C 1(SA, 55#000 uCi/mmole) was o b t a i n e d f rom Schwarz/Mann and c i n n a m i e acld-2- 1^C (SA, 1.4 juCi/mmole) from ICN C h e m i c a l and R a d i o -i s o t o p e D i v i s i o n , • lb ••: B. P r e p a r a t i o n and a d m i n i s t r a t i o n o f C-compounds. lb A l l t h e C-compounds used f o r m e t a b o l i c s t u d i e s were d i s s o l v e d I n d i s t i l l e d w a t e r , f i l t e r s t e r i l i z e d , and s t o r e d o f r o z e n a t -20 C u n t i l r e q u i r e d . F i s s i o n o f t h e a r o m a t i c r i n g o f t y r o s i n e . To a s t e r i l e E r l e n m e y e r f l a s k f i t t e d w i t h a, c e n t e r w e l l , 1 u C l (20 pg) o f L - t y r o s i n e - u n i f o r m l y r i n g - ^ C (SA, 9.3 u C i / mmole) was-added a s e p t i c a l l y o u t s i d e t h e c e n t e r w e l l and 0.5 ml 10 N KOH w i t h a f i l t e r p a p e r w i c k (Whatman #1) f o r t r a p p i n g C 0 2 I n s i d e t h e w e l l . A 15 ml a l i q u o t o f a 2 week o l d c u l t u r e was t r a n s f e r r e d a s e p t i c a l l y o u t s i d e t h e c e n t e r w e l l . The 0 f l a s k was i n c u b a t e d s t a t i o n a r y f o r 2 weeks a t 19-21 C, u n d e r c o n s t a n t i l l u m i n a t i o n (200-250 f o o t - c a n d l e s ) . A second f l a s k w i t h t h e same c o n t e n t s was I n c u b a t e d f o r t h e same p e r i o d i n c o n t i n u o u s d a r k n e s s . The c o n t e n t s o f t h e c e n t e r w e l l were t r a n s f e r r e d t o 15 ml s c i n t i l l a t i o n f l u i d , and a f r e s h paper w i c k Impregnated w i t h two d r o p s o f 10 N KOH was p l a c e d I n t h e c e n t e r w e l l . The medium was a c i d i f i e d w i t h two d r o p s o f $ N lb HC1 t o r e l e a s e any r e m a i n i n g COg. A f t e r 2 hours o f s h a k i n g , t h e paper w i c k was removed and p l a c e d i n s c i n t i l l a t i o n f l u i d a l o n g w i t h a l l washings o f t h e c e n t e r w e l l . The medium was (1) s i d e c h a i n . e x t r a c t ed;.an^ f o r autoradiographic a n a l y s i s . ' 14 . A f t e r the i n i t i a l counting, the CO2 was regenerated and retrapped i n cc-phenylethylamlne (PEA) using the apparatus i n Figure 6. The s c i n t i l l a t i o n f l u i d and paper wicks were placed l n the regeneration chamber (B) and nitrogen was pass-ed through the system such that the PEAtB^O ('3il, V/V) (G) r i s e s Into the exchange column (H), and the bubbling e f f e c t was moderate but constant. Then 20 ml of 4 N R^SO^ was slovr-T4 l y i n j e c t e d Into the s c i n t i l l a t i o n f l u i d releasing the CO2 which was c a r r i e d across and into the PEA. Nitrogen was • passed through the system f o r 15 minutes, a f t e r which the PEA was drained from the column into the 20 ml t e s t tube. The column was washed with 4 ml ethanol which was combined with the former PEA, mixed, and a 1 ml a l i q u o t was removed f o r s c i n t i l l a t i o n counting. Side chain degradation of phenylalanine and tyrosine. To s t e r i l e Erlenmeyer f l a s k s with center wells, 2-uCi of the ^ C r l a b e l l e d precursor (L-Phe-l-^C, DL-Phe-2- l I fC, DL-Phe-3-ll,'C, L - T y r - l - ^ C , D L - T y r ^ - ^ C , or D L - T y r ^ - ^ C ) was added a s e p t l c a l l y along with enough s t e r i l e c o l d E-lsomer to give a f i n a l concentration of 0,05 mM. Each substrate was i n -cubated i n duplicate, under continuous i l l u m i n a t i o n (280-210 foot-candles) or i n continuous darkness f o r 6 and 12 hour periods at 19-21 C. The pre-adapted c e l l s from one l i t e r c ultures (preadapted: on 0.1 mM of eit h e r L-phenylalanine o r L-tyroslne - c o l l e c t i o n and washing of the c e l l s was described under ."•Cell c o l l e c t i o n and storage*?. 27) were suspended i n enou s t e r i l e a l g a l culture medium to add 10 ml a l i q u o t s , of known 36. F i g u r e 6. Diagram o f a p p a r a t u s f o r ^00% r e g e n e r a t i o n and r e t r a p p i n g . A, s t r e a m o f n i t r o g e n . B, r e g e n e r a t i o n 14 " chamber. Ct. C 0 2 - K 0 H - s c i n t i l l a t i o n f l u i d . D, 30 ml s y r i n g e w i t h n e e d l e . E, 4N H2S02j.. F, r u b b e r s t o p p e r s . G, a - p h e n y l e t h y l a m i n e and w a t e r (3*1) i n a 20 ml t e s t t u b e . H, 25 ml v o l u m e t r i c p l p e t c o n t a i n i n g g l a s s beads. c e i l number, a s e p t i c a l l y t o each f l a s k . A t t h e end o f t h e i n c u b a t i o n p e r i o d , 2 d r o p s o f 5 N 14 H C 1 were added t o t h e medium t o r e l e a s e any r e m a i n i n g CO^. A f t e r 6 hours o f I n t e r m i t t e n t s h a k i n g , t h e paper w i c k was r e -moved and, a l o n g w i t h a l l washings o f t h e c e n t e r w e l l , was p l a c e d i n s c i n t i l l a t i o n f l u i d . A f t e r t h e i n i t i a l c o u n t i n g , l4 •'' t h e C O 2 was r e g e n e r a t e d and r e t r a p p e d f o r r e - c o u n t i n g (method d e s c r i b e d a b o v e ) . The medium was e x t r a c t e d and chroma-t o g r a p h e d f o r a u t o r a d i o g r a p h i c a n a l y s i s , 14 D e g r a d a t i o n o f o t h e r C - l a b e l l e d s u b s t r a t e s . The E r l e n m e y e r f l a s k s f i t t e d w i t h t h e c e n t e r w e l l were u s e d f o r e x p e r i m e n t s w i t h t h e f o l l o w i n g r a d i o a c t i v e compounds} p h e n y l a c e t i c a c i d - l - ^ C , p h e n y l a c e t i c a c l d ^ - ^ C , p-14 h y d r o x y p h e n y l a c e t l c a c l d - 1 - C , p - h y d r o x y p h e n y l a c e t l c a c i d -14 14 14 2 - C, b e n z o i c a c i d - u n i f o r m l y r i n g - C, s a l i c y l i c a c l d - 1 - C, Tit 14 and p - h y d r o x y b e n z o l c a c i d - 1 - C. A l l t h e s e C-compounds, a l o n g w i t h enough s t e r i l e c o l d compound t o g i v e i n each c a s e a f i n a l c o n c e n t r a t i o n o f 0 . 0 5 mM, were added a s e p t i c a l -l y t o t h e p r e - s t e r i l i z e d f l a s k s . 1 0 ml a l i q u o t s c o n t a i n i n g a known number o f non-adapted c e l l s , c o n c e n t r a t e d f r o m one l i t e r c u l t u r e s (see s i d e c h a i n d e g r a d a t i o n o f Phe and T y r ) 14 were added a s e p t i c a l l y t o each f l a s k . The C- p h e n y l a c e t i c . a c i d s and ^ C - p - h y d r o x y a c e t l c a c i d s were I n c u b a t e d f o r 6 and 1 2 hour p e r i o d s i n c o n t i n u o u s d a r k n e s s a t 1 9 - 2 1 ° C w h i l e t h e . o t h e r a c i d s were i n c u b a t e d f o r o n l y a 1 2 hour p e r i o d . The 14 14 t e r m i n a t i o n , c o l l e c t i o n o f C 0 2 , r e g e n e r a t i o n o f t h e C 0 2 . ( l ) s i d e c h a i n l a b e l l e d and e x t r a c t i o n o f t h e medium f o r chromatography was the same as d e s c r i b e d above l n t h e s i d e c h a i n d e g r a d a t i o n o f p h e n y l -a l a n i n e and t y r o s i n e . i h The d e g r a d a t i o n o f c i n n a m i c a c i d - 2 - • C and p-c o u m a r i c a c i d - 2 - ^ C was s t u d i e d w i t h p r e - a d a p t e d c e l l s . The c e l l s were c o l l e c t e d from a l i t e r c u l t u r e , washed (as d e s c r i b e d under ' c e l l c o l l e c t i o n and s t o r a g e ' p. 27), and r e s u s p e n d e d i n 5 ml a l g a l c u l t u r e medium. These c e l l s were i n c u b a t e d f o r Zk h o u r s under c o n t i n u o u s i l l u m i n a t i o n (280-310 f o o t - c a n d l e s ) o lh a t 19-21 C w i t h 2 u C i o f e i t h e r C - s u b s t r a t e l n a 50 ml E r l e n m e y e r screw capped f l a s k . The i n c u b a t i o n was t e r m i n a t e d by c e n t r i f u g i n g down th e c e l l s and d e c a n t i n g t h e s u p e r n a t a n t . The c e l l s were washed w i t h f r e s h a l g a l c u l t u r e medium, c e n -t r i f u g e d down, and t h e s u p e r n a t e n t d e c a n t e d . The c e l l s were t r e a t e d as d e s c r i b e d under ' c e l l c o l l e c t i o n and s t o r a g e ' p. 27 and t h e medium w i t h t h e r e s p e c t i v e washing was a c i d i f i e d t o pH 2 and e x t r a c t e d f o r c h r o m a t o g r a p h i c a n a l y s i s . Uptake e x p e r i m e n t s w i t h p h e n y l a l a n i n e and t y r o s i n e . I n t o p r e - s t e r l l i z e d 50 ml E r l e n m e y e r f l a s k s 2 ;uCi o f Ik lh D L - p h e n y l a l a n i n e - 3 - C or D L - t y r o s i n e - 3 - C was added a s e p -t i c a l l y a l o n g w i t h enough L-lsomer o f t h e r e s p e c t i v e amino a c i d t o g i v e a f i n a l c o n c e n t r a t i o n o f 0.1 o r 0.01 mM. With each c o n c e n t r a t i o n , 10 ml a l i q u o t s c o n t a i n i n g a known number o f non-adapted c e l l s were p l a c e d i n two f l a s k s , one t o be i n -c u b a t e d under c o n t i n u o u s i l l u m i n a t i o n (280-310 f o o t - c a n d l e s ) and t h e o t h e r i n c o n t i n u o u s d a r k n e s s . L i k e w i s e , w i t h a n o t h e r two f l a s k s a t t h e same c o n c e n t r a t i o n , 10 ml a l i q u o t s o f p r e -a d a p t e d c e l l s were added a s e p t i c a l l y . The p r e - a d a p t e d c e l l s 39. were c o l l e c t e d f r o m a-known volume o f media c o n t a i n i n g 0,1 mM o f - e i t h e r L - p h e n y l a l a n i n e " o r L - t y r o s i n e , w a s h e d , and r e -suspended i n t h e same volume of f r e s h a l g a l c u l t u r e medium f o r use i n t h e s e u p t a k e s t u d i e s . From each f l a s k , 1 ml samples were removed a t 0, 10, 30, 60, 120, 240, 360, 5^0, and 720 minute i n t e r v a l s . Each sample was f i l t e r e d u s i n g a p r e - m o i s t e n e d ( w i t h a l g a l c u l t u r e medium) R M i l l i p o r e HA (0.45-micron p o r e s i z e ) f i l t e r , g e n t l y s u c k e d • d r y , t h e n washed w i t h 5 nil a l g a l c u l t u r e medium, and a g a i n s u c k e d d r y . The f i l t e r was t h e n p l a c e d i n 15 ml s c i n t i l l a t i o n f l u i d a l o n g w i t h 1 drop 0.1 N HC1 t o d e s t r o y t h e p i g m e n t s . The samples were l e f t 24 hours b e f o r e b e i n g counted,, C. D e t e c t i o n o f -^'C-products. A u t o r a d i o g r a p h y . The d e v e l o p e d chromatograms, d r i e d o v e r n i g h t , were p l a c e d i n l i g h t t i g h t x - r a y f i l m e x p o s u r e h o l d e r s w i t h a s h e e t o f Kodak B l u e Brand M e d i c a l X - r a y f i l m ( E s t a r Base) f o r two t o t h r e e weeks d e p e n d i n g on t h e number o f dpm(s) a p p l i e d . A t t h e end o f t h e e x p o s u r e p e r i o d , t h e x - r a y f i l m was removed and de-v e l o p e d under a r e d - y e l l o w s a f e l i g h t . The ^ C - s p o t s l o c a t e d by t h e a u t o r a d i o g r a p h were s c r a p e d from t h e TLC p l a t e s and p l a c e d d i r e c t l y i n t o 15 ml s c i n t i l l a t i o n f l u i d . S c i n t i l l a t i o n c o u n t i n g . The s c i n t i l l a t i o n f l u i d u t i l i z e d i n a l l t h e s e s t u d i e s c o n s i s t e d o f 6 gm o f PPO, 0,4 gm o f P0P0P d i s s o l v e d i n 412 ml o f t o l u e n e and 688 ml o f e t h a n o l . The samples were c o u n t e d i n a N u c l e a r - C h i c a g o 720 s e r i e s o r U n l l u x I I l i q u i d • 4 0 . s c i n t i l l a t i o n s p e c t r o m e t e r . D u a l c h a n n e l c o u n t i n g p e r -m i t t e d c a l c u l a t i o n o f e f f i c i e n c y from a quench c u r v e p r e p a r e d f o r each I n s t r u m e n t e m p l o y i n g a s e r i e s o f v a r i a b l y quenched samp l e s . The dpm v a l u e s o b t a i n e d f o r samples c o n t a i n i n g a g a known number of c e l l s were e x p r e s s e d as dpm p e r 10 c e l l s . V. Enzyme a s s a y s . A. P h e n y l a l a n i n e ammonla-lyase. (Young e t a l . , 1 9 6 6 ) . To each t u b e , 10 mg c e l l powder (see ' c e l l c o l l e c t i o n and s t o r a g e ' f o r p l a n k t o n l c s p e c i e s p. 27), 0.5 ml o f K - T r i c i n e b u f f e r (0.2 M), and 0.4 ml d i s t i l l e d w ater were added. Each o t u b e was c o v e r e d w i t h P a r a f i l m , t h e n s o n i c a t e d a t 0-4 C f o r 5 minutes i n a Raytheon 1 0 - k c y c l e m a g n e t o s t r i c t i v e o s c i l l a t o r a t a maximum c u r r e n t o u t p u t o f 1.1 A. These s o n i c a t e s were us e d d i r e c t l y f o r enzyme a s s a y s , w i t h o u t f u r t h e r t r e a t m e n t . Each r e a c t i o n was i n i t i a t e d by a d d i n g 10 umoles of L - p h e n y l a l a n i n e 14 c o n t a i n i n g 1 >uCi o f L - p h e n y l a l a n i n e - U - C (The R a d i o c h e m i c a l C e n t e r , Amersham). A t t h e end o f t h e i n c u b a t i o n p e r i o d (2 h o u r s a t 3 0°C), t h e r e a c t i o n was s t o p p e d w i t h 1 ml 0.5 N HC1 t h e n c e n t r i f u g e d a t 5.000 rpm f o r 30 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d and t h e p e l l e t was washed w i t h 0.5 N HC1. The s u p e r n a t a n t s were combined and e x t r a c t e d t w i c e w i t h 4 ml e t h e r , t h e n t h e aqueous phase was d i s c a r d e d . The e t h e r was washed w i t h 10 ml o f 0.5 N HC1 and t h e e t h e r was d e c a n t e d I n t o a s c i n t i l l a t i o n v i a l and e v a p o r a t e d t o d r y n e s s a t room t e m p e r a t u r e under a j e t o f n i t r o g e n . The c o n t e n t s o f t h e v i a l , a f t e r a d d i n g 15 ml s c i n t i l l a t i o n f l u i d , was t h e n counted,, 41,, B. T r a n s a m i n a s e . A m o d i f i c a t i o n o f t h e a s s a y d e s c r i b e d by Dlamondstone (1966) was used. T h i s was a f i x e d - t i m e a s s a y based on t h e a l k a l i - c a t a l y z e d o x i d a t i o n o f p - h y d r o x y p h e n y l p y r u v l c a c i d o r p h e n y l p y r u v i c a c i d t o r e s p e c t i v e l y p - h y d r o x y b e n z a l d e h y d e o r be n z a l d e h y d e . The r e a c t i o n m i x t u r e c o n t a i n e d 5 umole o f t y r o s i n e o r p h e n y l a l a n i n e , 5 jumole o f a k e t o a c i d ( p y r u v i c , o x a l o a c e t i c , o r a - k e t o g l u t a r i c a c i d ) and 0.2 umole o f p y r i -d o x a l phosphate i n a f i n a l volume o f 0.9 ml o f 0.1 M-sodium phosphate b u f f e r , pH 7.6. The enzyme was p r e p a r e d as d e s c r i b e d under p h e n y l -a l a n i n e ammonla-lyase t h e n c e n t r i f u g e d a t 20,000 g f o r 20 m i n u t e s . The s u p e r n a t e n t was passed t h r o u g h a 2 cm x 20 cm column o f Sepadex G-25 ( P h a r m a c i a , U p p s a l a , Sweden),prepared w i t h t h e 0.1 M-sodium phosphate b u f f e r , pH 7.6, f o r r e m o v a l o f t h e l o w m o l e c u l a r weight m o l e c u l e s . 12,5 ml o f e l u a t e was c o l l e c t e d and 0.3 ml a l i q u o t s were u s e d i n t h e enzyme a s s a y . A l l p r o c e d u r e s were performed a t 0-5°C u n t i l t h e a s s a y was begun. o A f t e r a 2 hour I n c u b a t i o n a t 30 C, t h e r e a c t i o n was st o p p e d by a d d i t i o n o f 0.1 ml o f 10 N KOH w i t h c o n t i n u o u s a g i t a t i o n . The r e a c t i o n m i x t u r e s were l e f t a t room temp-e r a t u r e f o r 30 minutes and t h e n t h e absorbance was r e a d a g a i n s t a b l a n k a t 331 nm. The amount o f p r o d u c t formed was c a l c u l a t e d from a molar e x t i n c t i o n c o e f f i c i e n t o f 19,900 M 1 -1 cm •• C. p-Hydroxybenzoate h y d r o x y l a s e . A c r u d e enzyme p r e p a r a t i o n (0.2 ml o f s o n i c a t e d c e l l s ) was I n c u b a t e d w i t h : NADP + f 0.5 jumolej- g l u c o s e - 6 - p h o s p h a t e , 5.0 umolej glucose-6-phosphate dehydrogenase, 0.1 ml o f a s t o c k d i l u t e d 25 f o l d ; p - h y d r o x y b e n z o l c a c i d , 0.3 umole; i n a t o t a l volume o f 1.0 ml a t pH 7.2, I n c l u d i n g 0.17 ml o f 0.1 M-sodlum o phosphate b u f f e r . The r e a c t i o n m i x t u r e was i n c u b a t e d a t 30 C f o r one hour and t e r m i n a t e d by t h e a d d i t i o n o f 1 ml o f 0.5 N HC1, The d i h y d r o x y - p r o d u c t was measured by_ the method d e s c r i b e d by Arnow (1937) f o r 3 , 4 - d l h y d r o x y p h e n y l a l a n i n e (D0PA). To t h e 2 ml s o l u t i o n (1 ml r e a c t i o n m i x t u r e +1 ml 0.5 N HC1), 1 ml of n i t r i t e - m o l y b d a t e r e a g e n t (10 gm sodium n i t r i t e and 10 gm sodium molybdate i n 100 m l ) , and 1 ml 1 N sodium h y d r o x i d e s o l u t i o n was added, m i x i n g w e l l a f t e r each a d d i t i o n . The samples were r e a d a t 510 nm. D. P r o t e i n d e t e r m i n a t i o n . The method o f Lowry e t a l . (1951) was us e d f o r t h e d e t e r m i n a t i o n o f p r o t e i n . The s u p e r n a t a n t from s o n i c a t e d c e l l s grown on n i t r a t e o r p h e n y l a l a n i n e was u t i l i z e d a f t e r r emoval o f c e l l d e b r i s by c e n t r i f u g a t i o n . . B o v i n e serum a l b u m i n ( f r a c -t i o n V, Sigma C h e m i c a l Co., S t . L o u i s , Mo., U.S.A.) was used as t h e standard.. •*3. RESULTS I . C u l t u r i n g . . A.. The e f f e c t o f p h e n y l a l a n i n e and t y r o s i n e . Under o p t i m a l growth c o n d i t i o n s (mass c u l t u r i n g ) , t h e a d d i t i o n o f e i t h e r o f t h e s e amino a c i d s a t 1.0 mM had no e f f e c t on t h e growth of, N. l n c e r t a ( T a b l e 8),. w h i l e t h e s m a l l d i f f e r e n c e s o b s e r v e d f o r Ij. g a l b a n a were p r o b a b l y n o t s i g n i f i c a n t . Data from one l i t e r c u l t u r e s I n d i c a t e d t h a t t h e s e amino a c i d s reduced t h e c e l l y i e l d f o r 1^ g a l b a n a ( T a b l e 9) w i t h o n l y a s l i g h t d e c r e a s e i n t h e growth c o n s t a n t s . P h e n y l a l a n i n e n o t o n l y reduced t h e growth c o n s t a n t but a l s o t h e c e l l y i e l d f o r i , l n c e r t a ( T a b l e 10), w h i l e t y r o s i n e was s t i m u l a t o r y , I n c r e a s i n g b o t h t h e c e l l y i e l d and t h e growth c o n s t a n t . The f e e d i n g o f L - p h e n y l a l a n l n e o r L - t y r o s l n e o v e r t h e c o n c e n t r a t i o n range o f 0.025 t o 2.0 mM had no e f f e c t on t h e g r o w t h c o n s t a n t o r l a g p e r i o d o f N. l n c e r t a ( F i g u r e s 7 and 8 ). S i m i l a r i l y L - p h e n y l a l a n i n e had no e f f e c t on e i t h e r o f t h e s e growth parameters o f I j g a l b a n a ( F i g u r e 7 ) w h i l e L - t y r o s i n e was s t i m u l a t o r y a t l o w e r c o n c e n t r a t i o n s and i n -h i b i t o r y a t h i g h e r c o n c e n t r a t i o n s ( F i g u r e 8 ). G e n e r a l l y , l n t h e p r e s e n c e o f n i t r a t e , n e i t h e r amino a c i d e x h i b i t e d . any c o n s i s t e n t i n f l u e n c e o v e r t h e growth of e i t h e r a l g a l s p e c i e s . When n i t r a t e was. o m i t t e d from t h e c u l t u r e medium, TABLE § Growth Constants and Cell Yields from Mass Cultures Algal species and Aromatic addition .Growth constant (£(io)> - i hr Mean gener-; atIon time (tg) hr C e l l count at harvest 6 xlO /ml Dry c e l l yield mg/llter culture Size of culture l i t e r s Isochrysls galbana Nitrate 0 . 0 1 2 ( 1 ) 24. 5 ( 1 ) 1 3 . 3 ( i ) ' 178 ( 1 ) +• L-tyroslne^ 2^ 0.009 33.4 15.2 NB(3> 4x5 (?) + L-phenylalanlne v ' : 0.015 20.1 ND ND 2x5 Navlcula lncerta Nitrate 0.040 7.53 9.0 185 2x5 (2) + L-tyroslne v ' 0.039 7.72 8.6 l 6 l 2x5 (?) + L-phenylalanlne V A 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 Ef fec t of Aromatic Compounds on the Growth Constants and C e l l Y ie lds from One L i t e r Cultures of Isochrysls galbana Aromatic..« a d d i t i o n * i ; •Growth constant <K(10)> h r " 1 Mean gener-at ion time (tg) hr C e l l count at harvest x l 0 6 /m l Dry c e l l y i e l d mg/ l l ter cul ture Percentage y i e l d . • r e d u c t i o n * 6 ' N i t r a t e 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-obumarate 4 ^ 0.0141 21.4 1.50 68 42.0 (1) concentration .0.1.0 mM. (2) based on dry c e l l y ie ld and using n i t rate y ie ld as reference. (3) concentration 0.05 mM. TABLE 10 The Ef fec t of Aromatic Compounds on the Growth Constants•and C e l l Yie lds from One L i t e r Cultures of Navlcula lncerta Aromatic, . a d d i t i o n 1 1 ' Growth constant OL(io)> hr"1 Mean gener-at ion time (tg) hr C e l l count at harvest x l 0 6 / a l Dry c e l l y i e l d mg/llter cul ture Percentage y i e l d . . . r e d u c t i o n * " K l t r a t e 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 (2) based on dry c e l l mM. y ie ld and using n i t ra te y ie ld as reference. (3) percentage s t imu la t ion . (4) concentration 0.05 nd. •. Fig ure • o o P o <t 3 » 01 (X) O cr 1 P So 3 a P cr o h*- SO 3 3 o p. CI-m S-i M CD cr 0 P 9) CD !~» T3 -•> a 0> >i a cr O ca o> o o >-» "> tr1 H CO •o o 3-o Ct 3" 3 VJ *! r-> 01 P (-» io P 3 0 3 M CD 8" O 3 3 P cr P 3" 3 CD P. CR O E cr 3" I n Oi O O o >0 *. 1—1 cn 0 % o. £3 V o I N H I B I T O N o f g r o w t h c o n s t a n t - o cn o cn T—1—1—r / / I • x I \ ! GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) p p cn Isochrysis galbana (• ) o o /I o / I / \ I-l LAG PERIOD (days) —r cn - r~ ( Fig ure 00 • S3 0 0 p 0 h* < 3 P ca « 0 ct- •I rj- P l-i 3: ff P cr a t-^ - p. •-» 3- 3: O P^ cr CD 3" T : CD ct- P p cn CD • '. •"-*> x> CD CD •t O •* cr O ca P. 0 O "1 f h-i 1 01 cr O O 1 0 1-1 CO r* 01 3 H* CD CO 0 n 3 p H" cr O- 3-P CD 3 P CN •1 P O 3 a! P- cr '9*7 ro "cn x, SS O Oi o ^ 6 % m 1—' cn ° % fc> V . . I N H I B I T O N o f g r o w t h c o n s t a n t O D O O O GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) Isochrysis galbana (• ) o o o o CP .A \ 3 13-, 0 LAG PERIOD (days) 1 : o o 1 4 0.4 0.3 - 6" ~~o —-o • o o X~ x ^ ~~ ~~ — - * 0.2 h OPTICAL DENSITY N. lncerta G c NO3 (control) o--o L-Phe (0.50 mK) + NO3 X--X L-Phe (0.50 mM) - NO3 • • L-Phe (0.05 mM) - NO3 0.1 h 0.0 Figure 9. Growth curves of Navlcula lncerta on nitrate and L-phenylalanine with and without nitrate. / G 0.3 0.2 OPTICAL DENSITY N. l n c e r t a e — e NO-J (control) o — o L-Tyr ( 0 . 5 0 mM) + NO3 X x L-Tyr ( 0 . 5 0 mM) - NO3 —• L-Tyr ( 0 . 0 5 mM) - MO 3 0.1 0.0 J 1 1 I l _ - I I • ' 0 8 12 DAYS 16 20 24 Figure 1 0 . Growth curves of Navlcula l n c e r t a on n i t r a t e and L-tyroslne with and without n i t r a t e . 49. s e v e r a l d i s t i n c t p a t t e r n s were o b s e r v e d . N* l n c e r t a c o u l d u t i l i z e b o t h amino a c i d s as t h e s o l e N-source w i t h no change i n t h e l a g p e r i o d from n i t r a t e grown c e l l s ( F i g u r e s 9 and 10). The growth c o n s t a n t s were a p p r o x i m a t e l y one h a l f o f t h e c o n t r o l v a l u e ( F i g u r e s 11 and 12), i n d i c a t i n g t h a t b o t h amino a c i d s were n ot as e f f i c i e n t as a n i t r o g e n s o u r c e as was n i t r a t e . The l a g p e r i o d was t h e same on n i t r a t e as i t was: on b o t h amino acids.- When c e l l s were removed from t h e 0.5 mM tube and i n o c u l a t e d i n t o a n o t h e r tube w i t h t h e same c o n c e n t r a t i o n of amino a c i d , v e r y l i t t l e d i f f e r e n c e was measured i n t h e growth p a r a m e t e r s . Growth c o n s t a n t Lag p e r i o d ( h r " 1 ) (days) L - p h e n y l a l a n i n e 1st t r a n s f e r 0.085 3.6 2nd t r a n s f e r 0.080 3.8 L - t y r o s i n e 1st t r a n s f e r 0.067 3.6 2nd t r a n s f e r 0.05? 3.5 T h i s c o n f i r m e d t h a t t h e enzymes r e q u i r e d f o r t h e amino n i t r o g e n u t i l i z a t i o n were p r e s e n t and no enzymes were i n -duced. i s g a l b a n a . on the o t h e r hand, u t i l i z e d t h e h i g h e r c o n c e n t r a t i o n s o f p h e n y l a l a n i n e as e f f i c i e n t l y as n i t r a t e ( F i g u r e 11), a l t h o u g h as the c o n c e n t r a t i o n d e c r e a s e d , the gro w t h c o n s t a n t a l s o d e c r e a s e d . When t h e o p t i c a l d e n s i t y d a t a was p l o t t e d ( F i g u r e 13). i t became e v i d e n t t h a t t h e r e was a f o u r day l a g p e r i o d beyond t h a t o b s e r v e d f o r c e l l s grown on n i t r a t e . An i n o c u l u m from t h e 0.5. mM .tube ( o n l y L - p h e n y l a l a n i n e ) when used t o i n i t i a t e a s i m i l a r tube gave no change i n growth c o n s t a n t but t h e l a g p e r i o d was extended a o i—i UJ «n a- m < 1/5 8 ^ Q I O t < 3 fr o o ~ ~Z- 3 o „ *— c til o z £ 16 0.15 au 3.0 2.5 2.0 Q017 0.016 0.015 + 5 0 5 10 -X K ' *-control -Ji t—X: control control control V -V. ' K -2.5X105 5x105 10"'* 2.5x# 5x10*' 103 2.0x1a3 CONCENTRATION (M) Figure 7. Diagram of the effects of L-phenylalanlne on the growth constant and lag-period of Isochrysls galbana and Navlcula lncerta. O 1—4 rr UJ < 1/1 — 8 ^ o I o or o o -2: fi o ,o •—» <-/ 2 5 3A 0.125 ao 2.0 0.022 _ 0.021 ] 0.020 0.019 0.018 0.017 0.016 0.015 ro c •v £3 t\J cr Ul & u o + 20 + 10. 0 10 20 control control -# x x — v w *-con_t_rql_ * control • X x-2.5 xlO 5 5x105 10"'' 2.5X104 ExTO*1 10"3 2OX103 CONCENTRATION (M) Figure 8. Diagram of the effects of L-tyroslne on the growth constant and lag period of Isochrysls galbana and Navlcula lncerta. O 0.3 0.2 OPTICAL DENSITY 0.1 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 0.0 Figure 13. Growth curves of Isochrysls galbana on nitrate and L-phenylalanine with and without nitrate. 52. 0.3 0.2 OPTICAL DENSITY 0.1 I. galbana G C NO3 (control) o — o L-Tyr (0.50 Q M ) + NO3 X—-X L-Tyr (0.50 mM) - NO3 • L-Tyr (0.05 mM) - NO> a—-a L-Tyr (2.0 mM) - N0-0.0 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 a l . (1975) for growth on glycine. Growth of X i 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 c e l l yield or op-t i c a l density maximum also decreased. Also from the optical density data (Figure 14), i t 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 i n i t i a t e growth l n 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 i t ' 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 i n the presence of nitrate. 54. oris 0.10 OPTICAL DENSITY 0.05 0.00 G — G NO^ (control) X---X L-Tyr (0.50 mM)'- NO^  • ' L-Tyr (0.50 mM) + L-Phe (0.25 mM) - NO, -XL. \%~%^X-X-X< 0 12 _ i t 16 20 2U 0.15 0.10 OPTICAL DENSITY 0.05 aoo 1 / C G NO^ (control) X—-X L-Tyr (1.0 mM) - NO^  • • L-Tyr (1.0 mM) + L-Phe (0.5 mM) - NO3 J L QjQjfr-X-x-x 1 I- • I 1 1— 0 8 12 16 DAYS 20 24 Figure 15, The effect of L-tyroslne with and without L-phenyl-alanlne on the growth yields of Isochrysls galbana. N e i t h e r D - p h e n y l a l a n l n e nor D — t y r o s i n e were u t i l i z e d "by I. g a l b a n a as t h e s o l e N -source. No growth was d e t e c t e d o v e r t h e c o n c e n t r a t i o n range o f 0„025-2,0 mM f o r a p e r i o d :of a t l e a s t f o r t y d a y s . IJ. l n c e r t a . however was a b l e t o u t i l i z e b o t h D-amino a c i d s , but o n l y a f t e r a l o n g l a g p e r i o d " ( F i g u r e s l 6 and 17). A f t e r bO d a y s , growth on 0.5 mM D - p h e n y l a l a n l n e was 78% o f t h a t o b s e r v e d on L - p h e n y l a l a n i n e ( F i g u r e 9)* whereas on 0.5 mM D - t y r o s i n e , . growth was bQ% o f t h a t o b s e r v e d on L - t y r o s l n e ( F i g u r e 10) and was s t i l l i n c r e a s i n g ( F i g u r e 17). The l a g p e r i o d appeared t o be dependent on t h e c o n c e n -t r a t i o n o f t h e D-isomer, t h e g r e a t e r t h e c o n c e n t r a t i o n t h e s h o r t e r t h e l a g p e r i o d ( F i g u r e s 16, 1 7 f and 1 8 ) . The growth c o n s t a n t s were a l s o reduced from t h a t o b s e r v e d f o r n i t r a t e •and as t h e c o n c e n t r a t i o n d e c r e a s e d so d i d t h e : g r o w t h c o n s t a n t . I n a l l c a s e s , t h e c e l l s grown on t h e D-isomers were l i g h t brown i n p i g m e n t a t i o n u n l i k e c e l l s grown on n i t r a t e o r t h e L - i s o m e r s which were d a r k brown. The d i f f e r e n c e I n p i g m e n t a t i o n and d e c r e a s e d g r o w t h c o n s t a n t s i n d i c a t e d t h e D-amlno a c i d s were a poor s o u r c e o f n i t r o g e n f o r N. l n c e r t a . B o t h s p e c i e s when grown I n t h e p r e s e n c e o f L - t y r o s i n e r e l e a s e d an e t h e r i n s o l u b l e brown c o l o u r e d m a t e r i a l I n t o t h e medium. T h i s o c c u r r e d n o t o n l y w i t h mass c u l t u r e s and one l i t e r c u l t u r e s b u t a l s o I n t h e OD e x p e r i m e n t s . T h i s a p p e a r -ance o f browning o c c u r r e d a t t h e end o f t h e e x p o n e n t i a l phase, a p p r o x i m a t e l y a f t e r twenty-two days o f growth f o r I . g a l b a n a ( F i g u r e lb) and seven days f o r N. l n c e r t a ( F i g u r e 10}:. No 0.2 OPTICAL DENSITY 0.1 Figure 16". Growth curves of Navlcula lncerta on nitrate and.D-phenylalanine. £ £. control (nitrate) • —• 2©0 mM D-phenylalanlne + + 0.5 mM D-phenylalanlne X x 0.25 m M D-phenylalanine A A 0.1 mM D-phenylalanlne o- o .0.025 mM D-phenylalanlne / / •0.0 0 :-«4r-f-t>-4-+-^-x-'-o-<>-+-«rr:— 12 18 24 30 36 DAYS 0.2 OPTICAL DENSITY 0.1 Figure 17. Growth curves of Navlcula l n c e r t a on n i t r a t e and D-tyroslne. G £ co n t r o l ( n i t r a t e ) . — _ . 2.0 mM D-tyroslne + • + 0.5 mM D-tyroslne X- x 0,25 mM D-tyroslne A A 0.1 mM D-tyroslne o o 0.025 mM D-tyroslne 0.0 DAYS o o 1—4 CC — UJ T O < to — y >~ 8 ^ o I o or o IT) K I o or o 26.0 22.0 18.0 U.0 3.2 0.091 0.009 0.008 0.007 £ 0.006 e o.oo5 S 0.00/, 0.003 0.002 10 5 0 2U.0 20.0 16.0 12.0 3.2 0.091 ^ 0.017 o I 0016 c ^ 0.006 g 0.005 - C A 0.00/, 0.003 Q002 20 15 10 5 0 o . A— control (NCh) control (N03) o o -^ o Figure 2.5X105 5x105 10 4 2.5x# 5x10'* 10"3 2Dx1(? CONCENTRATION (M) 18. Diagram of the effects of D-phenylalanlne and D-tyroslne on the growth constant and lag period of Navlcula lncerta.. -59. c o l o u r a t i o n o c c u r r e d w i t h growth o f e i t h e r s p e c i e s on L-p h e n y l a l a n i n e . The i d e n t i t y o f t h i s c o l o u r e d s u b s t a n c e i s unknown. The v o l a t i l e p r o d u c t ( s ) r e p o r t e d by Vose e t a l . ( 1 9 7 1 ) were not d e t e c t e d i n i n v i v o e x p e r i m e n t s w i t h I , g a l b a n a mass c u l t u r e s . No a r o m a t i c compounds were t r a p p e d i n t h e KOH and no a l d e h y d e s o r k e t o n e s were d e t e c t e d i n t h e 2 , 4 - d i n i t r o -p h e n y l h y d r a z i n e . T h i s s u g g e s t s t h a t p o s s i b l y t h e v o l a t i l e p r o d u c t ( s ) was o n l y r e l e a s e d upon a c i d i f i c a t i o n o f t h e medium, thus i t would not. be r e l e a s e d under the c o n d i t i o n s of t h e s e two t r a p p i n g e x p e r i m e n t s . B. M e t a b o l i s m o f p h e n y l a l a n i n e and t y r o s i n e . When t h e medium, t o t a l l y d e v o i d o f c e l l s , was ex-t r a c t e d w i t h e t h e r and t h e e t h e r chromatographed, v e r y few a r o m a t l c s were d e t e c t e d i F o r b o t h s p e c i e s the o n l y p h e n o l i c :compound observed:.from b o t h t h e p h e n y l a l a n i n e and t y r o s i n e media was p - h y d r o x y b e n z o i c a c i d s An a c i d , p o s i t i v e a r e a f o r b e n z o l c - p h e n y l a c e t l c a c i d s was a l s o o b s e r v e d from the p h e n y l a l a n i n e medium. No o t h e r compounds were d e t e c t e d . E x t r a c t i o n o f t h e c e l l s from growth on b o t h amino a c i d s r e v e a l e d p - h y d r o x y b e n z o l c a c i d . From t h e t y r o s i n e grown c e l l s , p - h y d r o x y p h e n y l a c e t l c a c i d and t r a c e s o f b o t h p - h y d r o x y b e n z y l a l c o h o l and 3-bromo-p-hydroxybenzoic a c i d were a l s o d e t e c t e d f o r b o t h s p e c i e s . A c i d p o s i t i v e a r e a s f r o m p h e n y l a l a n i n e grown c e l l s were ob s e r v e d n o t o n l y f o r t h e b e n z o i c - p h e n y l a c e t i c a c i d a r e a , but a l s o f o r p h e n y l l a c t i c -p h e n y l p y r u v i c a c i d a r e a . F o r b o t h s p e c i e s , t h e major com-pound p r e s e n t from growth on t y r o s i n e was p - h y d r o x y b e n z o i c a c i d , and f o r p h e n y l a l a n i n e , b e n z o i c and/or p h e n y l a c e t i c a c i d . At no time was p-coumaric a c i d o r i n d i c a t i o n s o f t h e p r e s e n c e of c i n n a m i c a c i d e v e r o b s e r v e d . The a r o m a t i c s produced from p h e n y l a l a n i n e and t y r o -s i n e m e t a b o l i s m were i d e n t i f i e d m a i n l y by R f p o s i t i o n s and co-chromatography w i t h a u t h e n t i c samples. When p o s s i b l e , UV s p e c t r a were o b t a i n e d f o r c o m p a r i s o n w i t h known s p e c t r a . The c o l o u r r e a c t i o n of t h e p h e n o l i c a c i d s w i t h d i a z o t i z e d p - n l t r o a n l l i n e (PNA) a i d e d i n i d e n t i f i c a t i o n as d i d gas c h r o m a t o g r a p h i c a n a l y s i s o f t h e n o n - p h e n o l i c a c i d s . I n a l -most a l l c a s e s , so l i t t l e p r o d u c t was o b t a i n e d t h a t f u r t h e r c h e m i c a l c h a r a c t e r i z a t i o n was i m p o s s i b l e . The bromophenollc compound from I_. g a l b a n a was t e n t a t i v e l y i d e n t i f i e d as 3-bromo-p-hydroxybenzoic a c i d . I n s o l v e n t system C, p - h y d r o x y b e n z o l c a c i d had an R^ o f 0,36 w h i l e 3-bromo-p-hydroxybenzoic a c i d had an R f o f 0o.l5« S p r a y i n g w i t h PNA, 3-bromo-p-hydroxybenzoic a c i d t u r n e d s a l m o n - r e d i n c o l o u r w h i l e b o t h 3,5-dibromo-p-hydroxybenzolc a c i d ( R f o f 0.18) and p - h y d r o x y b e n z o l c a c i d r e a c t e d t o g i v e r e d c o l o u r e d s p o t s . The major peak i n t h e UV s p e c t r u m ( F i g u r e 19) o f t h e i s o l a t e d 3-bromo-p-hydroxybenzoic a c i d c o r r e s p o n d e d t o t h a t of p - h y d r o x y b e n z o i c a c i d w h i l e t h e f i n e s t r u c t u r e , a l s o i n t h e s p e c t r u m , was v e r y s i m i l a r to. 3,5-dlbromo-p-hydroxybenzoic a c i d ( F i g u r e 36J and t o t h e im-pure s y n t h e t i c sample of 3-bromo-p-hydroxybenzoic a c i d . The g r e a t I n s t a b i l i t y o f t h e I s o l a t e d 3-bromo-p-hydroxyben-z o i c a c i d r e s u l t e d i n f a i l u r e a t f u r t h e r a t t e m p t s t o f u l l y 1.6 F i g u r e 19. A b s o r p t i o n s p ectrum i n e t h a n o l o f t h e p h e n o l i c a c i d t e n t a t i v e l y i d e n t i f i e d as 3-bro.mo-p-hydroxybenzoic a c i d i s o l a t e d from I s o c h r y s i s g a l b a n a and N a v i c u l a i n c e r t a . 12 OPTICAL DENSITY 0.8 OA 0.0 200 225 250 275 300 WAVELENGTH (nm) 325 350 c h a r a c t e r i z e and c o n f i r m i t ' s s t r u c t u r e . C. The e f f e c t and me t a b o l i s m o f o t h e r a r o m a t i c compounds. I n T a b l e 11, a summary of t h e e f f e c t o f t h e v a r i o u s a r o m a t i c compounds on t h e growth o f I. g a l b a n a and N. l n c e r t a i s p r e s e n t e d . The d e t a i l e d e f f e c t s o f each a r o m a t i c compound on t h e growth c o n s t a n t and l a g p e r i o d a r e p r e s e n t e d f o r r e f e r -ence i n Appendix E, The a r o m a t i c p r o d u c t s d e t e c t e d from t h e me t a b o l i s m o f each a r o m a t i c s u b s t r a t e a r e a l s o p r e s e n t e d i n T a b l e 11. A g e n e r a l c h r o m a t o g r a p h i c map o f t h e l o c a t i o n s o f the v a r i o u s a r o m a t i c compounds i s p r e s e n t e d f o r r e f e r e n c e i n Appendi B. F o r a l l b u t p-hydroxybenzaldehyde and p- h y d r o x y m a n d e l i c a c i d , t h e a r o m a t i c compounds which were added t o c e l l c u l t u r e s caused no r e d u c t i o n i n c e l l y i e l d . Where growth i n h i b i t i o n had o c c u r r e d , the maximum c e l l numbers were always o b t a i n e d a f t e r an extended i n c u b a t i o n p e r i o d . D. R e s u l t s o f r a d i o a c t i v e t r a c e r s . -Uptake o f p h e n y l a l a n i n e and t y r o s i n e . I n F i g u r e s 20 t o 27 t h e t i m e c o u r s e u p t a k e o f D L - t y r o -s i n e - 3 - - ^ C and D L - p h e n y l a l a n l n e - 3 - ^ C a t an i n i t i a l c o n c e n t r a -t i o n o f 10 and 100 juM a r e p r e s e n t e d . B o t h a l g a l s p e c i e s were c a p a b l e o f u p t a k e of b o t h amino a c i d s , but g r e a t v a r i a t i o n s e x i s t e d i n t h e i r a b i l i t y t o do so. The u p t a k e r a t e s f o r b o t h t h e s e amino a c i d s were c a l c u l a t e d ( T a b l e 12) and were based o on 10 c e l l s per ml. I n t h e s e c a l c u l a t i o n s , t h e D-isomer com-ponent o f e i t h e r amino a c i d was d i s r e g a r d e d . At t h e concen-t r a t i o n l e v e l s u t i l i z e d l n t h e s e s t u d i e s , n e i t h e r s p e c i e s was a b l e t o a c h i e v e more t h a n a t h i r t y p e r c e n t u p t a k e o f t h e TABLE 11 A summary of the Effect and Metabolism of Aromatic compound Effect on growth constant 1 Details l n 2 Phenylacetic acid p-Hydroxyphenyl-acetlc acid DL-Mandelic acid DL-p-Hydroxy-mandellc acid H.C. inhibitory L.C. stimulatory H.C. Inhibitory L.C. no effect H.C. mildly Inhibitory L.C. no effect H.C. > L.C. Inhibition decreased 41 Tables 9 and 10 42 43 44 Benzoic acid H.C. Inhibitory L.C. stimulatory 45 Tables 9 and 10 p-Hydroxybenzolc acid H.C. inhibitory L.C. no effect k6 Tables 9 and 10 p-Hydroxy-benzaldehyde H.C. Inhibitory L.C. no effect 47 3,5-Dlbromo-p-hydroxybenzolc acid 3,5-Dlbromo-p-hydroxybenz-aldehyde Cinnamic acid p-Coumarlc acid H.C. Inhibitory L.C. no effect H.C. very inhibitory L.C. no effect H.C. Inhibitory L.C. stimulatory H.C. no effect L.C. stimulatory US 49 50 51 m-Hyd roxybonzolc acid o-Hydroxybenzoic acid H.C. mildly inhibitory L.C. no effect H.C. Inhibitory L.C. stimulatory 53 54 (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 l i q u i d benzoic acldj m-OHBA = m-hydroxybenzolc acid; benzaldehyde? p-OHPAA = p-hydroxyphenylacetic acid) PHCA = phenylhydracryllo acid. TABLE 11 (oont.) Other Aromatic Compounds on Isochrysls galbana and Navlcula lncerta 63. Chromotographlc results -(compounds detected)"^ O t h e r c o m m e n t s TLC.GLC PAA,BA,p-OHBA TLC p-OHPAA,p-OHBA,p-OHBAlc TLC.GLC BA,p-OHBA TLC p-OHBA,p-OHBAlc TLC p-OHBA,m-OHBA 3-Br-p-OHBA, BA TLC p-OHBA.3-Br-p-OHBA p-OH BA,3-Br-p-OHBA p-OHBAlc -presence of PAA suggested that It was poorly metabolized. -presence of p-OHPAA suggested that i t was poorly metabolized. -no DL-mandelic acid was detected which suggested that both isomers were metabolized. -reduced c e l l yield for X» galbana at 1 & 2 mM. • -no DL-p-hydroxymandelic acid was detected-'which - suggested that both isomers were metabolized. -presence of BA suggested It was poorly metabolized, -no o-OHBA or any dlhydroxy phenolic acids were detected for either species. -UV-spectrum of 3-Br-p-OHBA identical to that i n Figure 19. -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. -no adaptation to this aldehyde when I. galbana was subcultured.' -medium turned brown (including a control with no cel l s ) therefore this aldehyde was photochemically unstable. -brown colour was not extractable into ether. TLC nothing detected -medium turned brown therefore photochemically unstable, -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 iden t i c a l to spectrum of synthetic PHCA.BA,p-OHBA sample (Figure 1*0). TLC -purple spot tentatively i d e n t i f i e d 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 2.500 2,000 DPM PER 107 CELLS 1,500 1J00O o—o non-adapted cells, Illuminated • — • non-adapted cells, no Illumination A—A pre-adapted cells, Illuminated pre-adapted cells, no Illumination / o 10 6 8 HOURS Figure 2 0 . Time course of uptake of DL-phenylalanlne-3-for Isochry3l3 galbana. The i n i t i a l phenylalanine concentration Has 1 0 ;iM, 12 1)500 1400 1,200 1,000 DPM PER 107 CELLS 800 600 400 200 o--o non-adapted cells, Illuminated non-adapted cells, no Illumination A—A pre-adapted cells. Illuminated A—A pre-adapted cells, no Illumination Figure 2 1 , Time course of uptake of DL-phenylalanlne-3- A^C for Isochrysls a;albana. The I n i t i a l phenylalanine concentration, was O.JQ mH. ON TABLE 12 Uptake Hates of Phenylalanine and Tyrosine by Isochrysls galbana and Navlcula l n c e r t a at Two Substrate Concentrations Uptake Hates (2) L-phenylalanlne I*. galbana Ex l n c e r t a 10 uM 100 uM ' 10 uM 100 uM Non-adapted c e l l s . Illuminated Non-adapted c e l l s , no Illumination Pre-adapted c e l l s , illuminated Pre-adapted c e l l s , no Illumination 0.020 0.010 0.003 0.003 0.07 0.05(^ 0.05 2.6 4.4 4.6 10.8 9.1 10.0 8.9 . (2) L-tyroslne 10 uM 100 uM 10 uM 100 uM Non-adapted c e l l s , Illuminated Non-adapted c e l l s , no Illumination Pre-adapted c e l l s . Illuminated Pre-adapted c e l l s , no Illumination 0.014 0.004 0.005 0.004 0.10 0.06 0.12 0.03 5.1 9.>> 34.0 31.5 13.5 8.1 (1) jumoles/hour/10'' c e l l s . (2) based on the assumption that the L-isomer was s e l e c t i v e l y assimilated p r i o r to the D-lsomer (see text). „ (3) averaged over 12 hours the rate was 0.11 umoles/hour/10' c e l l s . (4) i f averaged over 12 hours, the value was comparable to the others at t h i s concentration* _ ON 100000 8QQ00 60J300 DPM PER 107 CELLS 40000 20,000 0--0 non-adapted c e l l s , Illuminated • — • non-adapted c e l l s , no i l l u m i n a t i o n A—A pre-adapted o e l l s , Illuminated pre-adapted c e l l s , no Illumination _i i i_ 10 12 (HOURS Figure 22. Time course of uptake of DL-phenylalanlne-3-tTC f o r Navlcula l n c e r t a . The I n i t i a l phenylalanine concentration was 10 uJii 1 a v a i l a b l e L-amlno a c i d . P h e n y l a l a n i n e was a s s i m i l a t e d by JE. g a l b a n a a t b o t h 10 and 100 uM c o n c e n t r a t i o n s ( F i g u r e s 20 and 2 1 ) . A t b o t h t h e s e c o n c e n t r a t i o n s , i l l u m i n a t i o n enhanced t h e u p t a k e r a t e s ( F i g u r e s 20 and 21, T a b l e 12) r e g a r d l e s s whether o r n o t c e l l s had been p r e - e x p o s e d t o p h e n y l a l a n i n e . A t 10 uM s u b s t r a t e l e v e l , non-adapted c e l l s appeared t o a c c u m u l a t e p h e n y l a l a n i n e more r a p i d l y t h a n p r e - a d a p t e d c e l l s , but. t h i s d i d n o t appear t o be t r u e a t 100 uM. T h i s o b s e r v a t i o n may have been t h e r e s u l t of p r e - a d a p t i n g the c e l l s w i t h 100 uM o f L - p h e n y l a l a n i n e which r e s u l t e d i n an i n t e r n a l p o o l o f p h e n y l a l a n i n e . T h i s s u g g e s t e d t h a t I± g a l b a n a had t h e cap-a c i t y t o a c c u m u l a t e p h e n y l a l a n i n e a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . The t i m e c o u r s e u p t a k e of p h e n y l a l a n i n e by N. l n c e r t a I s p r e s e n t e d i n F i g u r e s 22 and 23. At l e a s t a 1 0 0 -f o l d i n c r e a s e l n u p t a k e r a t e s was e v i d e n t when compared t o I s o c h r y s l s - ( T a b l e 12).. At b o t h c o n c e n t r a t i o n l e v e l s , an i n i t i a l r a p i d u p t a k e o c c u r r e d f o l l o w e d by a d e c r e a s e i n r a t e T h i s was e s p e c i a l l y e v i d e n t a t t h e 10 uM c o n c e n t r a t i o n where t h e " l e v e l l i n g o f f " o c c u r r e d w i t h about 7 - 1 0 % o f t h e a v a i l -a b l e L - p h e n y l a l a n i n e a s s i m i l a t e d . The v a r i a t i o n between 2-12 hours may have been t h e r e s u l t o f N. l n c e r t a * s a b i l i t y t o a d h e r e t o t h e w a l l s o f t h e f l a s k w hich r e s u l t e d i n a l o w e r c e l l number t h a n e x p e c t e d ( t h u s a l o w e r number of DPM) ( o n l y i n t h e s e u p t a k e e x p e r i m e n t s were t h e a d h e r i n g c e l l s o f N a v l c u l a not s c r a p e d from t h e w a l l s o f t h e e x p e r i m e n t a l 67. v e s s e l ) . A t b o t h c o n c e n t r a t i o n s , e x c e p t f o r p r e - a d a p t e d c e l l s i n 10 uM p h e n y l a l a n i n e - C, l i g h t appeared t o enhance t h e u p t a k e r a t e s ( F i g u r e s 22 and 23, T a b l e 12). The u p t a k e of t y r o s i n e by I_. g a l b a n a was comparable t o t h a t o b s e r v e d f o r p h e n y l a l a n i n e ( T a b l e 12). I n F i g u r e s 2k and 25 t h e t i m e c o u r s e u p t a k e o f t y r o s i n e i s p r e s e n t e d . S i m i l a r t o t h e p h e n y l a l a n i n e r e s u l t s , l i g h t a l s o enhanced t h e u p t a k e of t y r o s i n e a t both c o n c e n t r a t i o n l e v e l s . Un-l i k e t h e r e s u l t s w i t h p h e n y l a l a n i n e , non-adapted c e l l s w i t h no i l l u m i n a t i o n a ppeared t o have d i f f i c u l t y a s s i m i l a t i n g t y r o s i n e a t t h e 10 ;uM l e v e l ( F i g u r e 2k, T a b l e 12). F o r b o t h t y r o s i n e and p h e n y l a l a n i n e p r e - a d a p t e d c e l l s o f I s o c h r y s l s appeared t o be a b l e t o a s s i m i l a t e b o t h t h e s e amino a c i d s more e f f i c i e n t l y a t 100 ;uM whereas non-adapted c e l l s were more e f f i c i e n t a t 10 uM, The a s s i m i l a t i o n o f t y r o s i n e by N_, l n c e r t a i s p r e s e n t e d "in F i g u r e s 26 and 27. The u p t a k e r a t e s were comparable t o p h e n y l a l a n i n e ( T a b l e 12), but non-adapted c e l l s a t 100 uM had a 3 - f o l d g r e a t e r r a t e . A 3 - f o l d d i f -f e r e n c e i n r a t e was a l s o o b s e r v e d between t h e p r e - a d a p t e d and non-adapted c e l l s on L - t y r o s l n e a t 100 uM. T h i s was p r o b a b l y t h e r e s u l t of p r e - a d a p t e d c e l l s c o n t a i n i n g a l a r g e i n t e r n a l p o o l o f t y r o s i n e . A t 100 uM l i g h t a ppeared t o s t i m u l a t e u p t a k e ( F i g u r e 27), b u t t h i s e f f e c t was o n l y ob-s e r v e d over the f i r s t e i g h t hours o f a s s i m i l a t i o n . L i g h t a l s o DPM PER 107 CELLS 1,800 1£00 1,400 1200 1000 800 600 400 200 o — o non-adapted c e l l s , I l l u m i n a t e d • — • non-adapted c e l l s , no I l l u m i n a t i o n A-—zV pre-adapted c e l l s , i l l u m i n a t e d /a s • A—A pre-adapted c e l l s , no I l l u m i n a t i o n / / Figure Zl*. time course of uptake of DL-tyroslne-S-^C f o r i s o c h r y a i s galbana. The I n i t i a l t y r o s i n e concen-t r a t i o n was 10 uM. o—o non-adapted c e l l s , I l l u m i n a t e d •—• non-adapted c e l l s , no i l l u m i n a t i o n A-—A pre-adapted c e l l s , I l l u m i n a t e d HOURS i t Figure 25. time cctirse of uptake of DL-tyrdslne-3- A C f o r I s o c h r y s l s galbana; The i n i t i a l t y r o s i n e concen-t r a t i o n » a 3 0;10 mM; HOURS Figure 26. Time course of uptake of DL-tyroslne - 3-^C f o r Navlcula l n c e r t a . The I n i t i a l tyrosine concen-t r a t i o n was 10 JJH. 180,000 160,000 uqooo 120,000 100,000 DPM PER 107 CELLS 80,000 .60,000 40,000 20,000 o — o non-adapted c e l l s , Illuminated non-adapted c e l l s , no i l l u m i n a t i o n A—A pre-adapted c e l l s , i l l u m i n a t e d ? A—A pre-adapted c e l l s , no I l l u m i n a t i o n 6 HOURS Figure 2 7 . Time course of uptake of D L - t y r o s l n e - 3 - 1 C f o r Navlcula l n c e r t a . The I n i t i a l tyrosine concen-t r a t i o n was 0.10 mK. stimulated tyrosine uptake at 10 uM but only for non-adapted c e l l s (Figure 2 6 ) . The pre-adapted ce l l s 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 "level l i n g off" observed i n 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 cel l s --• . adhering to the walls of the flask. In a l l these uptake studies no corrections were made lk for the metabolism of either amino acid. The loss of C0 2 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 C0 2 was minimized under illumination due to photosynthetic fixation of COg. It i s unknown i f the greater uptake rates In the light were related to this fixation of QQ^ o r t o the greater provision of energy for amino acid transport. In the uptake experiments, no account of the R e -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 cel l s than fqr pre-adapted cells of both species when c e l l 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 a b i l i t y o f b o t h a l g a l s p e c i e s t o c o n c e n t r a t e b o t h amino a c i d s and t h e s t i m u l a t o r y e f f e c t o f l i g h t , l e , i n c r e a s e d u p t a k e r a t e s and g e n e r a l l y l a r g e r c o n c e n t r a t i o n r a t i o s , was v e r y s u g g e s t i v e o f a c t i v e t r a n s p o r t . No i n -h i b i t o r s of p h o t o p h o s p h o r y l a t i o n and, m a i n l y , o x i d a t i v e p h o s p h o r y l a t i o n were examined i n r e l a t i o n t o amino a c i d u p t a k e t o c o n f i r m t h i s s u g g e s t i o n . No o t h e r parameters o f u p t a k e were examined. The c a t a b o l l c f i s s i o n o f t h e a r o m a t i c r i n g o f t y r o s i n e . When a x e n i c c u l t u r e s o f n i n e p l a n k t o n l c s p e c i e s , from t h r e e d i v i s i o n s , were I n c u b a t e d 2-weeks w i t h u n i f o r m l y C - r i n g - l a b e l l e d L - t y r o s i n e , a l l s p e c i e s were c a p a b l e o f r i n g c l e a v a g e t o produce "^CC^ ( T a b l e 13);. Whether i n c o n -t i n u o u s d a r k n e s s o r under c o n s t a n t i l l u m i n a t i o n each s p e c i e s r e t a i n e d t h i s c a p a b i l i t y . U n l i k e t h e r e s u l t s r e p o r t e d f o r t h e d e g r a d a t i o n o f p h e n y l a l a n i n e ( T a b l e 3)» U» l u t h e r l . C, h u x l e y l - . and S. c o s t a t u m were a b l e t o degrade t y r o s i n e t o COg. The p r e s e n c e o f a p - h y d r o x y l group on t h e a r o m a t i c r i n g appeared t o f a c i l i t a t e r i n g c l e a v a g e . A n a l y s i s o f t h e medium from t h e s e e x p e r i m e n t s sug-g e s t e d a p o s s i b l e r o u t e f o r d e g r a d a t i o n o f t h e c a r b o n s k e l -e t o n o f t y r o s i n e . . F o r each s p e c i e s , b o t h p - h y d r o x y p h e n y l -a c e t i c and p - h y d r o x y b e n z o i c a c i d s were d e t e c t e d ( T a b l e 1^), p-Coumaric a c i d was not d e t e c t e d i n any s p e c i e s examined. The p r e s e n c e o f p - h y d r o x y p h e n y l a c e t l c a c i d s u g g e s t e d a C^-fragment was i n i t i a l l y removed, but i f a C 2 - f r a g m e n t was 72. TABLE 13 T o t a l ^C02 Measured as P r o d u c t o f C a t a b o l i s m f r o m 2-weeks 1 14 I n c u b a t i o n o f A l g a e w i t h u n i f o r m l y R i n g - l a b e l l e d C - t y r o s i n e L i g h t Dark 14 _3 A l g a l s p e c i e s C02, dpm x 10 Haptophyta, I s o c h r y s l - s - g a l b a n a - 11.1 (0.50$)* 3.1 (0.14$) K o n o c h r y s i s l u t h e r l 4.0 (0.18$) 8.7. (0.3.9$) C o c c o l l t h u s h u x l e y i 56.0 (2.52$) 76.8 (3.46$) B a c 1 l l a r l o p h y t a Penr.ate d i a t o m N a v l c u l a I n c e r t a 82.1 (3.70$) 12.4 (0*56$) C e n t r i c d i a t o m Skeletonema c o s t a t u m 2.5 (0.11$) 5.6 (0.25$) C r y p t o p h y t a Rhodomonas l e n s 49.8 (2.24$) 71.1 (3.20$) • F i g u r e s i n p a r e n t h e s e s g i v e d a t a as $ o f added C - t y r o s i n e . TABLE 14 14 Radioactivity In Products Isolated from Uniformly Ring-labelled C-tyroslne Feedings p-OHB (1) P-OH0AO p-OHBA p-OH0CH2OH Algal species dpm x 10"3 Haotophyta  Isochrysis galbana Monochrysls lutherl Coocollthu3 huxleyl Bacill^rlophyta Pennate diatom Navlcula lncerta Centric Diatom  Skeletonema costatum Cryptophyta Rhodomonas lens Dark Light 180.2 137.8 ( 8.11)( 2 ) ( 6.21) 7.77 8.00 (0.35) (0.36) 7.3^  7.09 Dark Light 1.11 3.33 ( 0.05) (0.15) 1.11 2.89 (0.05) (0.13) 0.91 Dark Light 0.89 3.12 ( 0.04) ( 0.14) 1.11 .1.31 (0.05) (0.06) Dark Light 151.2 358.0 ( 6.81) (16.1 ) 14.2 22.9 (0.64) (1.03) 2.10 2.06 (0.09) (0.09) 8.46 16.0 Dark Light 8.22 2.4? ( 0.37) ( 0.12) 1.11 2.00 (0.05) (0.09) Dark Light 19.8 19.1 ( 0.89) ( 0.86) 3.78 0.89 (0.17) (0*04) (0.38) (0.72) (1) abbreviations aret p-OHB = p-hydroxybenzolc acid, p-OH0Ac = p-hydroxyphenylacetio abld, p-OHBA = p-hydroxybenzaldehyde, p-OH0CH2OH = p-hvdroxybehzylalcdtiol. (2) figures i n parentheses give data as % of - added l*C-tyrosine. i removed this, acid would not be detected. Similarly, i f 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 C 2-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 J J . lncerta the reduction product, p-hydroxybenzylalcohol was also detected (Table 14) but most of the radioactivity was observed i n the p-hydxoxy-benzoic acid,. Side chain degradation of phenylalanine and tyrosine. To confirm the loss of C0 2 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. t h e D-isomer content of both amino a c i d s t o be d i s r e g a r d e d . T h e r e f o r e , a l l c a l c u l a t i o n s f o r both s p e c i e s were based o n l y on the content of L-lsomer which was assumed to be 50# o f the added DL-mixture. The c e l l s u t i l i z e d i n these s t u d -i e s were pre-adapted on 0,1 mM L - p h e n y l a l a n i n e or L - t y r o s i n e because 1^ galbana had a l a g p e r i o d b e f o r e e i t h e r amino a c i d was u t i l i z e d as a n i t r o g e n source ( F i g u r e s 13 and 1*0. In Tables 15 and 16 the r e s u l t s of the l / 4 -C0 2 t r a p -pings f o r both a l g a l s p e c i e s and both amino a c i d s a r e p r e -sented. To enable a comparison between s p e c i e s , the v a l u e s i n both t a b l e s were based on 10 c e l l s . F o r l n c e r t a ( T able 16) t h i s was the a c t u a l r a d i o a c t i v e counts trapped, but twice t h i s number of r a d i o a c t i v e counts were trapped f o r I . galbana. The percentage v a l u e s should be used to compare the r a d i o a c t i v i t y trapped f o r each s e r i e s of amino a c i d s ( l e . - l - ^ C , - Z ^ C . and -3- l l 4 'c). lk The v a l u e s obtained f o r the CO,, i n the KOH t r a p 14 were confirmed by r e t r a p p i n g the CO2 i n a a - p h e n y l e t h y l -amine. Any d i f f e r e n c e was a s s o c i a t e d with a v o l a t i l e p r o -d u c t or products. In the f e e d i n g s of I . galbana with p h e n y l -a l a n i n e (Table 15)» a v o l a t i l e product was d e t e c t e d a t the Cg-Cg l e v e l which suggested the v o l a t i l e product of Vose et a l . (1971) was a Cg-C^ compound.- A v o l a t i l e product a l s o was d e t e c t e d f o r N. l n c e r t a (Table 16) but i t appeared t o lk be a C.-C compound. In the feedings of t y r o s i n e - C, a o 1 v o l a t i l e product a t the Cg-Cg l e v e l was d e t e c t e d f o r I s o c h r y s l s (Table 15) while no v o l a t i l e s were observed f o r 76. TABLE 15 T o t a l ^ C O j Measured a 3 a Product of Catabollsm from Incubation o f Pre-adapted C e l l s of Isochrysls galbana with L a b e l l e d Phenylalanine and Tyrosine Incubation Labe l l e d conditions juCl added Total conc-en t r a t i o n of Trapped l n KOH Re-trapped In V o l a t i l e pro-precursor (hours) L of -Isomer L-lsomer (uM) 14' COg. dpm x 10~ 3 /10 8cells dpm x 10"3/108 L-Phe-l- l UC L i g h t - 6 Llght-12 2.0 2.0 50.0 50.0 5.2 10.3 0.122)* 0.232) 5.4 10.3 (0.122) (0.232) 0 0 14 L-Phe-2- C Dark - 6 Dark -12 2.0 2.0 50.0 50.0 ' 21.2 50.0 0.482) 1.132) 21.3 50.3 (0.482) (1.132) 0 0 L i g h t - 6 Llght-12 1.0 1.0 1*6.9 46.9 4.3 10.2 0.202) 0.462) 0.29 2.2 (0.022) (o.io2) 4.04 (0.182) 7.96 (0.362) Dark - 6 Dark -12 1.0 1.0 U-6.9 •*6.9 17.0 16.1 0.772) 0.732) 5.32 15.0 (0.242) (0.682) 11.7 (0.532) 1.10 (0.052) L-Phe-S-^C L i g h t - 6 Llght-12 1.0 1.0 46.7 • 46.7 0.66 4.4 0.032) 0.202) 0.15 0.75 (o.oi2) (0.032) 0.51 (0.022) 3.61 (0.162) Dark - 6 Dark -12 1.0 1.0 1*6.7 06.7 9.2 14.1 0.422) 0.632) 1.3 7.6 (0.062) (0.342) 7.9 (0.362) 6.5 (0.292) L - T y r - l - l U C L i g h t - 6 Llght-12 2.0 2.0 50.0 50.0 24.1 52.4 (0.542) (1.182) 24.5 52.6 (0.552) (1.182) 0 0 . Dark - 6 Dark -12 2.0 2.0 50.0 50.0 79-3 169.0 (1.792) (3.762) 79.5 167.0 (1.792) (3.762) 0 0 L-Tyr-2- l UC L i g h t - 6 Llght-12 1.0 1.0 7^.0 47.0 9.0 18.3 (0.412) (0.822) 9.0 15.4 (0.412) (0.702) 0 2.9 (0.132) Dark - 6 Dark -12 1.0 1.0 47.0 47.0 • 11.8 22.2 (0.532) (i.oo2) 6.4 18.8 (0.292) (0.852) 5.3 (0.242) 10.0 (0.452) L-Tyr-3- 1 , fC L i g h t - 6 Llght-12 1.0 1.0 43.4 43.4 6.9 6.5 (0.312) (0.292) 2.1 1.5 (o.io2) (0.072) 4.8 (0.212) 5.0 (0.222) Dark - 6 Dark -12 1.0 1.0 43.4 43.4 6.1 12.4 (0.282) (0.562) 2.0 10.0 (0.092) (0.452) 4.2 (0.192) 2.4 (0.112) ' f i g u r e s l n parenthesis give data as 2 of 14„ C-precursor fed. 7 7 . 4 TABLE 16 T o t a l l^COj Measured as a Product of Catabollsm from Incubation of Pre-adapted C e l l s of Mavlcula l n c e r t a with Labelled Phenylalanine and T y r o s i n e Incubation p Cl Tota l cone-La b e l l e d conditions added e n t r a t l o n of precursor of L-lsomer (hours) L-lsomer (uM) Trapped In KOH . He-trapped In Vo l a t i l e - pro-g-phenyleChvlamlne ducts In- KOH: 1 ' 4 ' C 0 2 , dpm x 1 0 " 3 / 1 0 ° o e l l s dpm; x: 1 0 ~ ' 3 / 1 0 8 c e l l 3 L-Phe-l-^C L i g h t - 6 Llght-12 2.0 2.0 50.0 50.0 147.0 195.0 ( 3.32* ( .^39* *149.0 195.0 ( 3.35*) I 4.40*) 0: 0: Dark - 6 Dark -12 2.0 2.0 50.0 50.0 '+48.0 656.0 (11.1 * (14.8 * 450.0 659.0 (10.1 *) (14.8 *) 0 0: L-Phe-2-l!*C L i g h t - 6 Llght-12 1.0 1.0 46.9 46.9 25.3 14.8 ( 1.14* ( 0.67* 25.4 15.6 ( 1.14*) ( 0.70*) 0 0 Dark - 6 Dark -12 1.0 1.0 46.9 46.9 59.*+ 83.3 ( 2.68* ( 3.75* 5^.2 83.6 . ( 2.44*) ( 3.77*) (0.23*). 0. L-Phe-3- l UC L i g h t - 6 Llght-12 1.0 1.0 46.7 10.9 19. ^  ( 0.49* ( 0.87* 0.64 0.43 ( O.03*) ( 0.02*) 10.2 19.0 (0.46*) (0.85*) Dark - 6 Dark -12 1.0 1.0 1*6.7 1*6.7 13.6 21.7 ( 0.61* ( 0.98* 1.9 3.4 ( 0.09*) ( 0.15*) 11.7 18 ..4-(0.53*) (0.83*) L - T y r - l - l I f C L i g h t - 6 Llght-12 2.0 2.0 50.0 50.0 301.0 233.0 ( 6.78* ( 5,25* ) 304.0 ) 234.0 ( 6.84*), ( 5.26*) 0 0 Dark - 6 Dark -12 2.0 2.0 50.0 50.0 648.0 605.O (14.6 * (13.6 * ) 648.0 ) 606.0 (14.6 *) (13.7 *) 0 0 L-Tyr-2- 1 ) +C L i g h t - 6 Llght-12 1.0 1.0 47.0 47.0 32.1 41.1 ( 1.46* ( 1.85* 32.5 42.9 ( 1.46*) ( 1.92/0 0 0: Dark - 6 Dark -12 1.0 1.0 47.0 ' 47.0 '71.0 .. 173.0 ( 3.20* ( 7.80* 71.4 174.0 ( 3.22*) ( 7.84*) 0 0 L-Tyr-3- l 2 tC L i g h t - 6 Llght-12 1.0 1.0 t*3.i* 43.4 • 1.84 3.04 ( 0.08* ( 0.14* 1.85 3.12 ( 0.08*) ( 0.14*) 0 0 Dark - 6 Dark -12 1.0 1.0 i*3.i* i*3.i* 30.5 77.0 ( 1.38* ( 3.52* 31.0 77.7 ( 1.39*) ( 3,55*) 0 0 •figu r e s In parenthesis glye data as * of 1 C-precursor fed. 78 Navlcula (Table l 6 ) . The r e s u l t s l n Tables 15 and 16 confirm that C02 u n i t s were removed from the side chain of both amino acids.. l 4 1 k The detection of C0 2 when phenlalanlne or tyroslne-3- C were fed to both species indicated that carbon-3, next to 14 the aromatic r i n g , was removed as C0 2. When s p e c i f i c a l l y side chain l a b e l l e d phenylalanine and tyrosine was fed to both a l g a l species the r a d i o a c t i v i t i e s decreased i n the or-der C-l>C-2>C-3., The uptake rates of both phenylalanine .and tyrosine for pre-adapted c e l l s of each species were compar-able (Table 12), but tyrosine appeared to be metabolized 14 more rapidl y than phenylalanine based on the .amounts of C02 produced. The c e l l s 'Utilized f o r these studies were a c t i v e l y photosyntheslzlng ( l e . s t i l l growing anabollcally) ;. This was confirmed for both species by comparing the r a d i o a c t i v e v l t y f o r c e l l s under continuous i l l u m i n a t i o n with those i n continuous darkness. The values i n darkness were always^" greater than i n l i g h t . In almost a l l instances, the radio-a c t i v i t y obtained f o r a 6 hour incubation was l e s s than .ob-tained f o r the 12 hour incubation.; The outstanding example where t h i s was not the case was for N. Incerta tyroslne-1-14 C l i g h t and dark incubations (Table 16). This was p o s s i -bly related to the " l e v e l l i n g o f f observed i n the uptake of tyrosine (Figure 26), but the cause was unknown. The chromatographic analysis,of the medium from the (1) except i n Table 15 L-Tyr-3- 1^C-Light-6 vs Dark-6. 79. above e x p e r i m e n t s w i t h p h e n y l a l a n i n e and t y r o s i n e s p e c i f i c a l l y ^ C - l a b e l l e d i n t h e s i d e c h a i n r e v e a l e d s i m i l a r p a t t e r n s f o r b o t h s p e c i e s . Photographs o f t h e a u t o r a d i o g r a p h s a r e p r e s e n t e d i n F i g u r e s 28 and 29. The c o n t r o l s , F i g u r e 30, were i n -c l u d e d t o show t h e c o n t a m i n a n t s t h a t were p r e s e n t I n t h e 14 C-amino a c i d s t o be f e d . The DPM v a l u e s were d e d u c t e d f o r any c o n t a m i n a n t t h a t c o r r e s p o n d e d t o a m e t a b o l i c p r o -14 d u c t f rom t h e f e e d i n g s . B o t h p h e n y l a l a n i n e - 2 - C and -3-14 C f e e d i n g s c o n t a i n e d f a i n t hot a r e a s t h a t c o u l d p o s s i b l y be i n t e r p r e t e d as c i n n a m i c a c i d , but t h e s e were a l s o ob-s e r v e d I n the r e s p e c t i v e c o n t r o l s and were u n i d e n t i f i e d c o n t a m i n a n t s . B e f o r e any chromatograph was d e v e l o p e d , c o l d p - h y d r o x y b e n z o i c a c i d was s p o t t e d f o r r e f e r e n c e w i t h t h e 14 C - e t h e r e x t r a c t . The p o s i t i o n o f t h i s a c i d , s p o t 1, was o u t l i n e d w i t h a d o t t e d l i n e . I n T a b l e s 17 and 18 t h e DPM v a l u e s o b t a i n e d i n t h e v a r i o u s s p o t s o b s e r v e d i n t h e a u t o r a d i o g r a p h s o f I . g a l b a n a ( F i g u r e 28) and N. l n c e r t a ( F i g u r e 29) a r e p r e s e n t e d . A l l 8 t h e r a d i o a c t i v e v a l u e s I n b o t h t a b l e s were based on 10 c e l l s . Spot #9. d e t e c t e d f o r b o t h s p e c i e s , was a n u n -i d e n t i f i e d Cg-C^ compound. I t was c o n s i d e r e d t o be pos- ' s l b l y a N - m a l o n y l o r N - a c e t y l d e r i v a t i v e o f p h e n y l a l a n i n e . , b u t n e i t h e r o f t h e s e d e r i v a t i v e s have been r e p o r t e d i n a l g a e f o r any amino a c i d (see Pbkorny e t a l . . 1970), P h e n y l p y r u v i c - p h e n y l l a c t l c a c i d s as one s p o t were d e t e c t e d I n t h e e x t r a c t s o f b o t h s p e c i e s when p h e n y l a l a n -i n e was f e d . The p e r c e n t a g e v a l u e s f o r p h e n y l p y r u v i c -p h e n y l l a c t i c a c i d s were g r e a t e r when p h e n y l a l a n i n e - 2 - C\ gure 28. A u t o r a d i o g r a p h s o f chromatograms p r e p a r e d from t h e e t h e r e x t r a c t s o f C - s i d e c h a i n l a b e l l e d p h e n y l a l a n i n e and t y r o s i n e f e d t o I s o c h r y s l s g a l b a n a . D o t t e d l i n e i n -d i c a t e s t h e c h r o m a t o g r a p h i c p o s i t i o n o f a u t h e n t i c p-h y d r o x y b e n z o i c a c i d . A. 2% aqueous f o r m i c a c i d . B. Benzene : a c e t i c a c i d i w a t e r (10:7«3, V/V/V). 1. p.-Hydroxy b e n z o i c a c i d . 2. p-Hydroxybenzaldehyde. 3. p - H y d r o x y p h e n y l a c e t i c a c i d . h, p - H y d r o x y p h e n y l l a c t l c a c i d . 5. p - H y d r o x y p h e n y l p y r u v i c a c i d . 6. B e n z o i c a c i d . 7. P h e n y l a c e t i c a c i d . 8. P h e n y l l a c t i c and p h e n y l p y r u v i c a c i d s . 9. Unknown Cg-C-j compound (Unknown #1). 10. 3-hromo-p-hydroxybenzoic a c i d . 11. Unknown Cg-C 2 compound (Unknown #2). F i g u r e 29. A u t o r a d i o g r a p h s o f chromatograms p r e p a r e d f r o m t h e e t h e r lk e x t r a c t s o f C - s i d e c h a i n l a b e l l e d p h e n y l a l a n i n e and t y r o s i n e f e d t o N a v l c u l a l n c e r t a . D o t t e d l i n e I n d i c a t e s t h e c h r o m a t o g r a p h i c p o s i t i o n o f a u t h e n t i c p-hydroxy-b e n z o i c a c i d . A. 2% aqueous f o r m i c a c i d . B. Benzene » a c e t i c a c i d 1 w a t e r (10i7:3, V/V/V). 1 . p-Hydroxybenzoic a c i d . * 2. p-Hydroxybenzaldehyde. 3. p - H y d r o x y p h e n y l a c e t l c a c i d . k, p - H y d r o x y p h e n y l l a c t i c a c i d . 5. p - H y d r o x y p h e n y l p y r u v i c a c i d . 6. B e n z o i c a c i d . 7. P h e n y l a c e t i c a c i d , 8. P h e n y l l a c t i c and p h e n y l p y r u v i c a c i d s , 9. Unknown C,-C! compound (Unknown #1), 0 3 1 0 . 3-cromo-p-hydroxybenzoic a c i d . 1 1 . Unknown C £ - C 2 c o m P o u n d (Unknown #2). ' l g u r e 30. A u t o r a d i o g r a p h s o f chromatograms p r e p a r e d f r o m t h e e t h e r lk e x t r a c t s of C ^ s l d e c h a i n l a b e l l e d p h e n y l a l a n i n e and t y r o s i n e t o be f e d t o I s o c h r y s l s g a l b a n a and N a v l c u l a  l n c e r t a . D o t t e d l i n e i n d i c a t e s t h e c h r o m a t o g r a p h i c p o s i t i o n o f a u t h e n t i c p - h y d r o x y b e n z o i c a c i d . A. 2% aqueous f o r m i c a c i d . B. Benzene : a c e t i c a c i d 1 water ( 1 0 i 7 » 3 . V/V/V). 1, p - H y d r o x y b e n z o i c a c i d . 2, p-Hydroxybenzaldehyde. 3, p - H y d r o x y p h e n y l a c e t i c a c i d . k, p - H y d r o x y p h e n y l l a c t i c a c i d . 5. p - H y d r o x y p h e n y l p y r u v i c a c i d . 6. B e n z o i c a c i d . 7. P h e n y l a c e t i c a c i d . 8. P h e n y l l a c t i c and p h e n y l p y r u v l c a c i d s . 9. Unknown c g - c ^ compound (Unknown #1). 10. 3 - D r o m ° - P - n y d r o x y D e n z o i c a c i d . 11, Unknown Cg-C 2 compound (Unknown #2). Control LiM-IZ hr. Control Tr-3-mC Li9ht-12hr. TABLE 17 R a d i o a c t i v i t y i n P r o d u c t s I s o l a t e d from ^ C - l a b e l l e d F e e d i n g s from I n c u b a t i o n o f P r e - a d a p t e d C e l l s o f I s o c h r y s i s g a l b a n a . Labelled Incubation juCl Unknown #1 ( 1 ) 0Pyr-0Lact 0Ao Benz p-OHB p-OHBBr Total conditions £tdd ed of L-lsomer precursor (hours) dpm x 10~3 per 10 cells L-Phe-l-^C Light- 6 Llght-12 2.0 2.0 1.07 (0.022) o.48 (o.oi2) 0.48 (0.012) 0.33 (0.012) 1.55 0.81 (0.032) (0.022) Dark - 6 Dark -12 2.0 2.0 o.4o (o.oi;?) 0.27 (0.012) 0.09 (o.o 2) 0.05 (o.o 2) 0.48 0.3} (o.oi2) (o.oi2) L-Phe-2-1'*C Light- 6 Llght-12 1.0 1.0 1.98 (0.092) 2.15 (0.102) 1.52 (0.072) 2.53 (o. n2) 0.77 (0.032) 3.96 (0.182) 4.27 8.63 (0.192) (0.392) . Dark - 6 Dark -12 1.0 1.0 0.43 (0.022) 0.83 (0.042) o.io (o.o 2) 0.65 (0.032) 0.53 (0.02*) 0.38 (0.022) 1.06 I.85 (0.052) (0.082) L-Phe-3-lllC Light- 6 * Llght-12 1.0 1.0 5.12 ( 0 . 232) 1. 58 (0.072) 1.19 (0.052) 1.81 (0.082) 0.08 (0.0 %) 0.38 (0.022) 0.59 (0.032) 0.23 (o.oi2) 0.20 (0.012) 6.97 0.14 (0.012) 4.20 (0.312) (0.192) Dark - 6 Dark -12 1.0 1.0 0.84 (0.OU2) . l . i o (0.052) 0.70 (0.032) 0.48 (0.022) 2.31 (o.io2) 0.08 (0.0 2) 0.48 (0.022) 0.15 (0.012) 0.05 (0.0 2) 3.80 O.07 (0.0 2) 2.20 (0. 172) (o.io2) p-OH0Pyr p-OH0Lact 1 p-OH^ Ao Unknown #2(3) p-OHBA P-0H3 p-OHBBr Total dpn x 10"' per 108 cells L-Tyr-l-^C Light- 6 Llght-12 2.0 2.0 0.45 (0.012) 0.96 (0.022) 0.51 (0.012) 0.92 (0.02^ ) 0.97 1.88 (0.022) (0.042) Dark - 6 Dark -12 2.0 2.0 0.13 (o-o 2) 0.15 (o.o 2) 0.07 (0.0 2) 0.20 (0.0 2) 0.21 0.35 (0.0 %) (O.012) L-Tyr-2-^C Light- 6 Llght-12 1.0 1.0 0 0 5.62 (0.252) 2.86 (0.132) 1.3? 0.3' (o.o62) (o.oi2) (0.022) 0 7.14 3.20 (0.322) (o.i42) Dark - 6 Dark -12 1.0 1.0 0 0 5.62 (0 . 252) 9.52 (0 . 432) 1.39 (0.062) 0-32 (o.oi2) 0.91 (O.OW 0.48 (0.022) 7.34 10.9 (0.332) (0.492) L-Tyr-S-^C Light- 6 Llght-12 1.0 1.0 0 0 1.25 (0.062) . 3.51 (0. 162) 2.04 (0.092) 1.37 (0.062) 3.84 (0.172) 1.82 (0.052 ) 0.71 (0.032 ) 3.30 ( 0.152) 5.99 (0.272) 4.62 (0.212) 14.5 14.0 (0.652) (0.632) Dark - 6 Dark -12 1.0 1.0 0 0 3.54 (0 . 162) 5.70 (0 . Z6h) 4.10 (0.192) 2.77 (0.132) e.46 (0.382) 5.19 (0.232) 3.34 (0.172) 8.53 (0 . 382) 5.55 (0.252) 11.0 (0. 502) 24.4 34.3 (i.io2) d.552) (1) abbreviations a r e i 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 - p r e c u r 3 o r . V^j (iS unknown hao same Rf valuos as p-hydroxybcnzaldehydo. • TABLE 18 - R a d i o a c t i v i t y I n P r o d u c t s I s o l a t e d f r o m l i + C - i a b e l l e d F e e d i n g s from I n c u b a t i o n o f P r e - a d a p t e d o f N a v l c u l a l n c e r t a . Labelled precursor L-Phe-l-^C L-Phe-2-ltlC 14 L-Phe-3- C Incubation conditions (hours) J J C I add ed of L-lsomer Unknown #1 (1) 0Pyr-0Lact 0Ao Benz p-OHB p-OHBBr dpn x 10"3 per 10 8 c e l l s Light- 6 Llght-12 2.0 2.0 5 . 5 2 2 . 3 3 ( 0 . 1 2 * ) ( 0 . 0 5 * ) 3.74 2.40 (0.08*) (0.05*) Dark - 6 Dark -12 2.0 2 . 0 1 . 3 3 1 .23 ( 0 . 0 3 * ) ( 0 . 0 3 * ) 0.96 1.62 (0.02*) (0.04*) Light- 6 Llght-12 1.0 1 . 0 10 .7 8.94 (0.48*) (0.40*) 40.2 50.5 (1 .9 * ) (2.3 *) 21.5 11.7 (O .97*) (0.53*) Dark - 6 Dark -12 1 . 0 1 . 0 8 . 9 2 8.53 (0.40*) ( 0 . 3 3 * ) 38.6 48.1 (1.7 *) (2.2 *) 33.2 37.2 (1.5 *) (1.7 *) Light- 6 Llght-12 1 . 0 1 . 0 5.72 5.64 (0.26*) ( 0 . 3 0 * ) 54.5 45.3 (2.5 *) (2.0 *) 36.5 23.8 (1 . 6 * ) (1.1 *) Dark - 6 Dark -12 1 . 0 1 . 0 8 . 9 3 9 . 6 7 (0.40*) (0.44*) 45.7 46.7 (2.1 *) (2.1 *) 6 1 . 3 58 .3 (2.8 *) (2.6 *) 16.5 17.7 (0.75*) (0.80*) 9.40 (0.42*) 14.1 (O .63*) 1 .23 (0 . 0 6 * ) 1.49 (O .06*) 0.97 (0.04*) 2 . 1 3 (0.10*) 0 . 7 3 (0 . 0 3 * ) 0.77 (0.03*) 0.60 (0.03*) 0.89 (0.04*) Total 9 . 2 6 ( 0 . 2 1 * ) 4.73 ( 0 . 1 1 * ) 2 . 2 9 (0 . 0 5 * ) 2 . 8 5 (0.06*) 7 3 . 5 7 7 . 0 8O .5 9 3 . 8 1 15 .0 •95.8 1 2 7 . 0 1 3 2 . 0 ( 3 . 3 * ) ( 3 . 5 * ) ( 3 . 6 * ) ( 4 . 2 * ) ( 5 . 2 * ) ( 4 . 3 2 ) ( 5 . 7 * ) ( 5 . 9 * ) p-OH0Pyr p-OH0Lact p-0Hf$Ac Unknown #2^ c-OHBA „ r,tm p-UHdA p-OHB p-OHBBr Total dpm x 10" 3 per 10 8 c e l l s L - Tyr-l-^C Light- 6 2 . 0 0 2.24 (0.05*) ? , L l n Llght-12 2 . 0 0 1 . 0 3 (0 . 0 2 * ) 2.24 ( 0 . 0 5 * ) Dark - 6 2 . 0 0 1 .99 (0 . 0 5 * ) Dark -12 2 . 0 0 2 .78 (0.06*) 1$ jo.ogl L - T y r - 2 - 1 " ^ Light- 6 1 . 0 0 5 . 2 0 (0. 23*1 1 2.u ( 0 . 5 6 * ) 2 . 9 4 ( 0 . 1 3 * ) , n , , f t o 9 i , Llght-12 1 . 0 0 3 .19 (0. 1^*5 2 . 6 3 (0 . 12*) ^.33 (0.19*) ^0 .5 ( 0 . 9 g ) ^ r , k 'A H 0 9 . 9 0 (0. 45*) 4 . 6 4 ( 0 . 2 1 * ) 2^.0 ( 1.1*) - R ,. / -. n <f\ ter* ~ 1 2 0 10.8 (O .49*) 2 1 . 9 ( 0 . 9 9 * ) 0 .91 (0.04*) 3 8 . 5 |1.7 *) L-Tyr-3-' L UC " ^ " ^ }'° 2 1 0 < U , ( 0 » 4 ^ ) ' 25.7 ( 1 . 2 * ) 16.2 (0. 73*) 4 0 . 3 (1.8 *) 28.7 (1 . 3 * ) 1 2 1 0 (s s i) Llght-12 1 . 0 0 22.1 (1.0 *) 18.9 (0 . 8 5 * ) 1 0 . 1 (0.46*) 4l.O ( 1 . 9 *) 2 7 . 6 (1.2*) J g ^ (5*.4*) Dark - 6 1 . 0 0 1 1 .0 (0.49*) 5 3 ^ (2.4 *) 38.9 ( 1.8 *) 5 2 . 9 (2.4 *) 39.7 (1.8*) w n /„ „ *, r k " 1 2 1 ' ° 0 1*.5 ( 0 . 6 6 * ) 32 .5 ( 1 . 5 * ) 27.O ( 1 . 2 * ) 78 .5 ( 3 . 5 * ) 3 3 . 3 ( 1 - 5 * ) Ht'.o ( 0 . 4 * j (1) abbreviations aroi 0 P y r = phenylpyruvlc acid, 0 L a c t » 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 l n parentheses give data as * o f added C - p r e c u r s o r , 00 (3) unknown haa s a o Br values as p-hydroicybenzaldehyde. _-85. i 4 and -3— C were f e d , w h i c h s u g g e s t e d t h a t Gome C^-Cg com-pounds chromatographed w i t h t h e s e a c i d s . . I t was known t h a t b o t h m a n d e l i c and b e n z o y l f o r m i c a c i d s chromatographed i n t h i s r e g i o n (see Appendix B) and p r o b a b l y a c c o u n t f o r t h e i n c r e a s e i n r a d i o a c t i v i t y . The r e s u l t s f o r I s o c h r y s l s sug-g e s t e d t h a t m a n d e l i c and b e n z o y l f o r m i c a c i d s were p r e s e n t i n v e r y low amounts ( T a b l e 17), where w i t h N a v l c u l a t h e s e • two a r o m a t i c a c i d s c o m p r i s e d t h e major p o r t i o n o f t h i s 14 14 a r e a when p h e n y l a l a n l n e - 2 — C o r - 3— C were f e d . P h e n y l a c e t i c a c i d was the o n l y o t h e r s p o t d e t e c t e d 14 when p h e n y l a l a n i n e - 2 - - C was f e d t o bo t h s p e c i e s . The v a r i a t i o n s i n t h e p e r c e n t a g e v a l u e s f o r t h i s a c i d when f e d t o I s o c h r y s l s c o u l d be r e l a t e d t o the Cg-C^ v°l atlle p r o -d u c t t r a p p e d I n t h e KOH (T a b l e 15). I n CO t r a p p i n g ex-14 2 p e r l m e n t s , when C - p h e n y l a c e t i c a c i d was f e d t o b o t h s p e c i e s , 14 a v e r y l a r g e p e r c e n t a g e o f C - p h e n y l a c e t i c a c i d f r om t h e c o n t r o l was t r a p p e d i n t h e KOH. T h i s s u g g e s t e d t h a t X* g a l b a n a may e x c r e t e p h e n y l a c e t i c a c i d i n t o t h e medium and upon a c i d i f i c a t i o n t h i s a c i d c o u l d be v o l a t i l i z e d . The p e r c e n t a g e v a l u e s f o r N. l n c e r t a were r e l a t i v e l y c o n s t a n t and e s s e n t i a l l y no v o l a t i l e p r o d u c t was d e t e c t e d . T h i s s u g g e s t e d t h a t p h e n y l a c e t i c a c i d was not e x c r e t e d i n t o t h e medium by t h i s s p e c i e s . Two d i s t i n c t s p o t s were d e t e c t e d i n the b e n z o i c -p h e n y l a c e t i c a c i d r e g i o n f o r r^ . l n c e r t a when p h e n y l a l a n l n e -14 3- C was f e d ( F i g u r e 29). Only one s p o t was d e t e c t e d f o r 86. I. galbana (Figure 28) which corresponded to phenylacetic acid Indicating l i t t l e 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 ( s a l i c y l i c acid)* If s a l i c y l i c acid was present, Navlcula also possesses the capability to hydroxylate benzoic acid in the ortho-position. No indication of s a l i c y l i c acid was obtained for I. galbana. The C^-C^ vol a t i l e 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 l n 14 a very high number of DPM ln the KOH trap when ring- C-benzolc acid was u t i l i z e d . Benzoic acid is relativef' 9: ly v o latile and great care must be taken when i t i s -used in feeding experiments (B. E l l i s , personal communica^ tion).. This suggested that benzoic acid may be excreted i n -to the medium and volatilized upon acidification. In Figures 28 and 2 9 , 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 p h e n y l a l a n i n e ^ - 1 ^ ! and phenylacetaldehyde-benzaldehyde when p h e n y l a l a n i n e ^ - 1 * ^ were fed. _Both these aldehydes are known to chromatography i n t h i s region, but many other compounds, e s p e c i a l l y l i p i d s , chromatograph with these two aldehydes. Very s i m i l a r patterns were obtained i n the degrada-t i o n of tyrosine, but the degradation products a l l contain-ed a para-hydroxyl group. p-Hydroxyphenylpyruvic a c i d was not detected f o r N. incerta (Figure 2 9 , Table 18), but a very f a i n t spot (#5-Figure 28, Table 17) was observed f o r I . galbana when tyrosine-1- C was fed. This a c i d was not 14 14 observed f o r I . galbana when tyrosine-2- C and - 3 - C were fed. This being possibly due to only ' . l j i C i of these two amino acids being u t i l i z e d as opposed to 2 uCi f o r • tyrosine-' l - ^ C and the t a i l i n g of other spots over t h i s region. The 14 only other spot detected when tyrosine-1- C was fed cor-responded to p-hydroxyphenyllactic a c i d . When the percent-age values obtained f o r t h i s a c i d were compared to those obtained when t y r o s i n e ^ - 1 ^ was fed to both species (Tables 17 and 18), a d i s t i n c t Increase was observed. This sug-gested that a Cg-C 2 compound, p-hydroxymandelic a c i d , chromatographed with the p-hydroxyphenyllactic a c i d (see Appendix B). A s i m i l a r increase was noticed when the values of p-hydroxyphenyllactic a c i d were compared f o r the 14 14 tyroslne-2- C and - 3 - C feedings of N. lncerta (Table 18). This Indicated a Cg-C^ compound, probably p-hydroxybenzyl-a l c o h o l (see Appendix B) also chromatographed i n t h i s . 88. p o s i t i o n . No i n d i c a t i o n o f a Cg-C^ compound was o b t a i n e d f o r I. g a l b a n a . The o n l y o t h e r phenolic.compounds d e t e c t e d f o r b o t h 14 s p e c i e s when t y r o s l n e - 2 - C was f e d were p - h y d r o x y p h e n y l -a c e t l c a c i d and an unknown compound. T h i s unknown compound (#2) chromatographed i n t h e same r e g i o n as p-h y d r o x y b e n z a l d e h y d e . I t ' s i d e n t i t y was unknown, but t h e o n l y Cg-Cg compound i n t h e d e g r a d a t i v e sequence o f t y r o s i n e n o t a c c o u n t e d f o r was p - t i y d r o x y b e n z o y l f o r m i c a c i d . The c h r o m a t o g r a p h i c l o c a t i o n o f t h i s p h e n o l i c a c i d was n o t d e t e r m i n e d ( see M a t e r i a l s and Methods) and t h e unknown compound may be p - h y d r o x y b e n z o y l -f o r m i c a c i d . The Cg-Cg v o l a t i l e compound d e t e c t e d f o r g a l b a n a i n t h e ^ C O g s t u d i e s ( T a b l e 15) was p o s s i b l y p - h y d r o x y p h e n y l -a c e t l c a c i d . T h i s a c i d was found t o be q u i t e v o l a t i l e under a c i d i c c o n d i t i o n s and i t would be e x p e c t e d t o be t r a p p e d l n t h e KOH. -Chromatographic a n a l y s i s o f b o t h a l g a l s p e c i e s when 14 t y r o s l n e - 3 - C was f e d a l s o r e v e a l e d p - h y d r o x y b e n z a l d e h y d e , p - h y d r o x y b e n z o l c a c i d , and 3-bromo-p-hydroxybenzolc a c i d . When t h e r a d i o a c t i v i t y f o r unknown #2 ( T a b l e s 17 and 18) was s u b t r a c t e d from t h e combined v a l u e s f o r p-hydroxybenz-a l d e h y d e , v a l u e s were o b t a i n e d f o r p-hydroxybenzaldehyde. G e n e r a l l y t h e g r e a t e s t r a d i o a c t i v i t y o b s e r v e d i n any p r o d u c t 14 f r o m t h e m e t a b o l i s m o f t y r o s i n e - 3 - C by b o t h s p e c i e s was l n p - h y d r o x y b e n z o i c a c i d and/or 3-bromo-p-hydroxybenzoic a c i d . T h i s s u g g e s t e d t h a t t h e s e two compounds a p p e a r e d t o be t h e 89 , end p r o d u c t l n t h e d e g r a d a t i o n o f t y r o s i n e by b o t h a l g a l s p e c i e s . The r a d i o a c t i v i t y o b s e r v e d l n t h e l i g h t f o r t h e s e two a c i d s appeared t o be l e s s t h a n l n dark which s u g g e s t e d t h a t l i g h t promoted t h e i r d e g r a d a t i o n . At no time were any d l h y d r o x y p h e n o l l c compounds d e t e c t e d f o r e i t h e r a l g a l s p e c i e s l 4 when t y r o s l n e - 3 - C was f e d . F o r b o t h I±. g a l b a n a and N. l n c e r t a t h e p e r c e n t a g e s f o r t h e t o t a l r a d i o a c t i v i t y i n t h e i s o l a t e d p r o d u c t s f o r p h e n y l a l a n i n e and t y r o s i n e ( T a b l e 17 and 18) i n c r e a s e d as 14 t h e C-carbon moved c l o s e r t o t h e a r o m a t i c r i n g . I n t h e same e x p e r i m e n t s ( T a b l e s 15 and 1 6), t h e p e r c e n t a g e a c t i v i t y 14 / i n t h e COg d e c r e a s e d . When the t o t a l r a d i o a c t i v i t y ( i n t h e KOH and t o t a l e t h e r e x t r a c t ) o b t a i n e d i n the dark f o r 14 ^ 14 14 p h e n y l a l a n i n e - 1 - C, - 2 - C, and - 3 - C were compared, t h e v a l u e s were w i t h i n r e a s o n a b l e e r r o r o f each o t h e r . The l 4 14 l 4 same was t r u e f o r t y r o s l n e - 1 - C, - 2 - C, and - 3 - C. A co m p a r i s o n o f t h e d e g r a d a t i v e r a t e s ' f o r b o t h s p e c i e s I n d i -c a t e d t h a t N± l n c e r t a degraded p h e n y l a l a n i n e and t y r o s i n e t e n - f o l d f a s t e r t h a n £. g a l b a n a . T h i s t e n - f o l d d i f f e r e n c e c a nnot be e x p l a i n e d by uptake r a t e s as a 1 0 0 - f o l d d i f f e r e n c e was o b t a i n e d , but su g g e s t e d t h a t N a v l c u l a and p o s s i b l y I s o c h r y s l s a s s i m i l a t e d b o t h amino a c i d s i n t o a s t o r a g e p o o l . These amino a c i d s were t h e n m e t a b o l i z e d a t governed r a t e s , 14 When t h e chromatograms from t h e t y r o s i n e - 1 - C and 14 p h e n y l a l a n l n e - 1 - C f e e d i n g s were s p r a y e d w i t h PNA o r the s p r a y f o r a c i d s , o n l y p - h y d r o x y b e n z o i c a c i d was d e t e c t e d , . i 90. This was because of the addition of unlabelled p-hydroxy-benzoic aci d . The other aromatic and phenolic acids were l n such low concentrations that they were not detectable. At no time i n 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 a l g a l species, they were metabolized. The metabolic patterns (Figure 31) were t o t a l l y d i f f e r e n t from those ob-tained i n the metabolism of phenylalanine and tyrosine. This suggested that these two acids were not involved i n the degradation of either amino a c i d . The metabolism of cinnamic acid revealed mainly one other hot spot (spot #9) which corresponded to phenylhydracrylic a c i d . The i d e n t i t y of the spot to the upper l e f t of spot #9 i n X J galbana and N„: l n c e r t a (very f a i n t ) was unknown, It's lo c a t i o n suggest-ed that i t was benzoylacetlc acid, an oxidation product of phenylhydracrylic a c i d . (A s i m i l a r chromatographic r e l a -t ionship was observed f o r phenyllactic acid and phenyl-pyruvic acid, see Appendix B). The i d e n t i t y of the spot to the upper right of spot #8 i n N, Incerta was unknown. 14 It's l o c a t i o n and the po s i t i o n of the C-label i n c i n -namic acid suggested that i t was either a cg" c-j or Cg- C2 compound. No attempt was made to i d e n t i f y this spot, but possibly i t could have been phenylpropionic a c i d . No e v i -dence was obtained from either species that the aromatic nucleus of cinnamic acid was hydroxylated. F i g u r e 31. A u t o r a d i o g r a p h s o f chromatograms p r e p a r e d from t h e e t h e r e x t r a c t s o f c i n n a m i c a c l d - 2 - C and p-coumarlc a c l d - 2 - C fed t o I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . D o t t e d l i n e s I n d i c a t e t h e c h r o m a t o g r a p h i c p o s i t i o n s o f a u t h e n t i c s t a n d a r d compounds. A. 2% aqueous f o r m i c a c i d B. Benzene : a c e t i c a c i d : water (10*7:3, V/V/V). 1. p - H y d r o x y b e n z o i c a c i d . 2. p-Hydroxybenzaldehyde. 3. p - H y d r o x y p h e n y l a c e t i c a c i d . b. T r a n s - p - c o u m a r i c a c i d . 5. C i s - p - c o u m a r i c a c i d . 6. p - H y d r o x y p h e n y l h y d r a c r y l i c a c i d ( ? ) . 7. T r a n s - c i n n a m l c a c i d . 8. C l s - c l n n a m i c a c i d . 9 . P h e n y l h y d r a c r y l i c a c i d . A u t o r a d i o g r a p h s o f t h e e t h e r s o l u b l e m e t a b o l i c 14 p r o d u c t s from p-coumarlc a c i d - C r e v e a l e d o n l y one s p o t f o r b o t h s p e c i e s . T h i s c o r r e s p o n d e d l n R^ , t o t h e t e n t a -t i v e l y . I d e n t i f i e d p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d . As was ob s e r v e d l n t h e u n l a b e l l e d f e e d i n g s a p o r t i o n o f p-h y d r o x y p h e n y l h y d r a o r y l i c a c i d remained a t t h e o r i g i n . T h i s was a l s o a p p a r e n t i n t h e a u t o r a d i o g r a p h s . 14 D e g r a d a t i o n o f o t h e r C - l a b e l l e d s u b s t r a t e s . 14 14 The CO,, r e l e a s e d when p h e n y l a c e t i c a c i d - 1 - C 14 14 and -2- C and p - h y d r o x y p h e n y l a c e t i c a c i d - 1 - C and ^ 2 -14 C were f e d t o X« • g a l b a n a and N_. l n c e r t a a r e p r e s e n t e d ft l n T a b l e 19. The DPM v a l u e s were based on 10 c e l l s , , Twice t h e l i s t e d number of DPMs were t r a p p e d f o r 1^ g a l b a n a b u t o n l y 0.2 ti m e s t h e l i s t e d number f o r N_. l n c e r t a . The low v a l u e s were p o s s i b l y t h e r e s u l t o f (1) t h e c e l l s were no t p r e - a d a p t e d and (2) t h e c e l l s were n o t e x c e s s i v e l y p e r -meable t o e i t h e r of the s e a r o m a t i c a c i d s , .For b o t h s p e c i e s when t h e p e r c e n t a g e v a l u e s were compared, a g r e a t e r p e r c e n t -age was o b t a i n e d l n the CO^ when b o t h a c i d s were l a b e l l e d i n t h e C - l p o s i t i o n r a t h e r t h a n i n t h e C-2, S i m i l a r t o t h e r e -s u l t s o b t a i n e d l n t h e d e g r a d a t i o n o f b o t h p h e n y l a l a n i n e and t y r o s i n e , t h e c a r b o n n e x t t o t h e a r o m a t i c r i n g was removed as C 0 2 . A n a l y s e s o f t h e a u t o r a d i o g r a p h s , when p h e n y l a c e t i c 14 14 a c i d - 1 - C was f e d t o b o t h s p e c i e s showed mandelic a c i d - . C 14 and b e n z o y l f o r m i c a c i d - C. These two a c i d s and p-hydroxy-14 b e n z o i c a c i d were a l s o o b s e r v e d when p h e n y l a c e t i c a c i d - 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 C 0 2 , dpm. x 10 per, 10 cells Isochrysls galbana phenylacetic acld-l-^C 1 .00 0.05 1.5 (0.07%)^ 3 .5 ( 0 . l 6 # ) phenylacetic a c l d ^ - ^ C 0 .33 0 . 0 5 0.10 (0.01#) 0 .30 (0.04#) p-hydroxyphenylacetlc acld-l- l l fC 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 . 0 5 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- l 4C 0.054 0 .05 0.31 (0.26%) 1 .8 (l . 5 0 # ) 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 f e d . p-Hydroxymandellc a c i d was d e t e c t e d as t h e o n l y p r o d u c t from t h e m e t a b o l i s m of p - h y d r o x y p h e n y l a c e t i c . a c i d , w h i l e p -hydroxybenzaldehyde and p - h y d r o x y b e n z o i c a c i d were 14 a l s o d e t e c t e d when p - h y d r o x y p h e n y l a c e t i c a c l d - 2 - C was f e d t o b o t h s p e c i e s . These r e s u l t s a r e i n agreement w i t h t h e d a t a o b t a i n e d i n t h e d e g r a d a t i o n o f t h e s i d e c h a i n o f p h e n y l a l a n i n e and t y r o s i n e by b o t h a l g a l s p e c i e s . I n T a b l e 20 a r e p r e s e n t e d t h e r e s u l t s o f f e e d i n g 14 v a r i o u s C - l a b e l l e d Cg-C^ a r o m a t i c a c i d s . B o t h s p e c i e s were a b l e t o degrade t h e a r o m a t i c r i n g o f b e n z o i c a c i d - ( U ) -14 14 C - r i n g l a b e l l e d t o C0 2. I n e x a m i n i n g t h e a u t o r a d i o -g r a p h s , p - h y d r o x y b e n z o i c a c i d , m-hydroxybenzoic a c i d , and o - h y d r o x y b e n z o i c a c i d were o b s e r v e d as h y d r o x y l a t i o n p r o -d u c t s ' f o r N. l n c e r t a . Only o - h y d r o x y b e n z o i c a c i d was n o t d e t e c t e d f o r i x g a l b a n a and t h i s may have been t h e r e s u l t o f a v e r y r a p i d d e c a r b o x y l a t i o n o f t h i s a c i d as o b s e r v e d i n 14 T a b l e 20 (jje. a v e r y h i g h number o f c o u n t s I n t h e CO ? 14 f r o m s a l i c y l i c a c l d - 7 — C ) . The p r e s e n c e o f o-hydroxy-b e n z o i c a c i d was s p e c u l a t e d upon l n t h e N. l n c e r t a p h e n y l - " 14 a l a n l n e - 3 - C f e e d i n g e x p e r i m e n t . I t s p r e s e n c e was c o n - . f i r m e d i n t h i s e x p e r i m e n t . 3-Bromo-p-hydroxybenzolc a c i d was a l s o d e t e c t e d i n t h e a u t o r a d i o g r a p h s f r o m b e n z o i c a c i d -14 C f o r b o t h s p e c i e s . One o t h e r compound was d e t e c t e d l n t h e a u t o r a d l o -14 graphs f r o m s a l i c y l i c a c l d - 7 - C, b u t i t was not i d e n t i f i e d . S a l i c y l i c a c i d was d e c a r b o x y l a t e d -by b o t h s p e c i e s ( T a b l e 20). 9 5 . TABLE 20 T o t a l ^002 Measured-as a P r o d u c t o f C a t a b o l i s m f r om a 12-hour Dark I n c u b a t i o n o f Non-adapted C e l l s w i t h L a b e l l e d A r o m a t i c Compounds A l g a l s p e c i e s and L a b e l l e d p r e c u r s o r >uCi added T o t a l c o n c e n t r a t i o n f e d (mM.)(l) ^ C O g , dpm x 10"3 p e r 1 0 8 c e l l s I s o c h r y s l s g a l b a n a B e n z o i c a c i d - ( U ) -0.23 (0.01#) ( 2 ) ^ C - r l n g l a b e l l e d 1.0 0.05 s a l i c y l i c a c i d - 7-^C 1.0 0.05 20.5 (0.9W p - h y d r o x y b e n z o i c lk a c l d - 7 - C 1.0 0.05 2.k (0.11$) N a v l c u l a l n c e r t a b e n z o i c a c i d - ( U ) -^ C - r i n g l a b e l l e d * 1.0 0.05 0.61 (0 .03$) s a l i c y l i c a c i d - 7-^C 1.0 0.05 0.12 (0.01$) p - h y d r o x y b e n z o l c a c l d - ? ^ 1 ^ 1.0 0.05 2.5 (0.11$) (1) l a b e l l e d p l u s n o n - l a b e l l e d a r o m a t i c p r e c u r s o r . (2) f i g u r e s l n p a r e n t h e s i s g i v e d a t a as % o f t o t a l c o n c e n t r a t i o n f e d . p - H y d r o x y b e n z o i c a c i d was a l s o d e c a r b o x y l a t e d t o 14 y i e l d CO ( T a b l e 20) o r m e t a b o l i z e d t o produce 3-bromo-14 p - h y d r o x y b e n z o i c a c i d . The p r o d u c t i o n o f CO from p-14 h y d r o x y b e n z o i c a c i d - 1 - C was i n agreement w i t h t h e p r o -14 -\L d u c t i o n o f CO from t y r o s i n e - 3 - C and p - h y d r o x y p h e n y l -14 a c e t i c a c i d - 2 - C. At no t i m e was any d i h y d r o x y p h e n o l i c compound o b s e r v e d i n the a u t o r a d i o g r a p h s from the metabo-l i s m o f p h e n y l a c e t i c a c i d , p - h y d r o x y p h e n y l a c e t i c a c i d , b e n z o i c a c i d , p - h y d r o x y b e n z o i c a c i d , o r o - h y d r o x y b e n z o i c a c i d by e i t h e r a l g a l s p e c i e s . I n t h e KOH c e n t e r w e l l from t h e e x p e r i m e n t s w i t h t h e s e a r o m a t i c a c i d s , a v e r y h i g h background count was ob-t a i n e d . T h i s h i g h background was e l i m i n a t e d when t h e C 0 2 was r e t r a p p e d l n t h e a - p h e n y l e t h y l a m l n e . The v a l u e s i n T a b l e s 19 and 20 were r e s u l t s o b t a i n e d a f t e r r e t r a p p i n g t h e CO . 2 E. R e s u l t s o f enzyme a s s a y s . P h e n y l a l a n i n e ammonia l y a s e . From the r e s u l t s p r e s e n t e d i n T a b l e 21, t h i s enzyme d i d not appear t o be p r e s e n t l n a l g a e . When p o s i t i v e v a l u e s were o b t a i n e d i n t h i s a s s a y p r o c e d u r e , chromatography showed t h a t t h e r a d i o a c t i v i t y r e s i d e d i n compounds o t h e r t h a n c i n n a m i c a c i d . Transaminase Transaminase a c t i v i t y was obser v e d w i t h b o t h N„ l n c e r t a and I. g a l b a n a . but X. g a l b a n a showed t h i s a c t i v i t y o n l y a f t e r . p r i o r exposure t o e i t h e r p h e n y l a l a n i n e o r t y r o s i n e TABLE 21 P h e n y l a l a n i n e Aramonla-lyase A c t i v i t y I n Marine A l g a e A l g a l s p e c i e s . A c t i v i t y . A l g a l s p e c i e s ...  A c t i v i t y C h l o r o p h y t a D u n a l l e l l a t e r t i o l e c t a N a n n o c h l o r i s o c u l a t a Enteromorpha sp. Spongomorpha sp. U l v a so. Haptophyta : ) I s o c h r y s l s g a l b a n a M o n o c h r y s i s l u t h e r l B a c l l l a r i o p h y t a n i l •0 n i l n i l , n i l n i l n i l n i l P y r r o p h y t a Amphldlnium c a r t e r i Rhodophyta P o r p h y r i d i u m cruentum R h o d e l l a m a c u l a t a A n t i t h a m i n i o n s p . I r l d a e a s p . P o r p h y r a s p . Cyanophyta Agmenellum q u a d r u p l l c a t u m n i l n i l n i l n i l n i l n i l n i l C y c l o t e l l a nana N a v l c u l a l n c e r t a C r y p t o p h y t a Chroomonas s a l l n a " n i l n i l n i l Phaeophyta Fucus s p . L a m l n a r i a s p . ( b l a d e o n l y ) N e r e o o y s t l s sp. ( b l a d e o n l y ) n i l n i l n i l ( T a b l e 2 2 ) . T h i s d i f f e r e n c e may be r e l a t e d t o t h e f o u r day l a g p e r i o d shown by I s o c h r y s i s b e f o r e growth on L - p h e n y l -a l a n i n e o r L - t y r o s i n e as the s o l e n i t r o g e n s o u r c e u n l i k e N. l n c e r t a 0 When C - p h e n y l a l a n i n e o r t y r o s i n e was i n c u b a t e d w i t h an enzyme p r e p a r a t i o n from e i t h e r s p e c i e s , t h e major p a r t o f t h e r a d i o a c t i v i t y was not l o c a t e d i n t h e p h e n y l -p y r u v i c o r p - h y d r o x y p h e n y l p y r u v i c a c i d s ( F i g u r e 32) . There-f o r e , no t r u e e s t i m a t i o n o f t h e en z y m a t i c a c t i v i t y (based on t h e a l k a l i - c a t a l y z e d o x i d a t i o n of the s e two a r o m a t i c a c i d s t o t h e i r r e s p e c t i v e Cg-C-^ a l d e h y d e s ) c o u l d be o b t a i n e d and o n l y t h e o p t i c a l d e n s i t y r e a d i n g s from t h e enzyme a s s a y s a r e p r e s e n t e d i n T a b l e 22. Only I f t h e enzyme p r e p a r a t i o n s were f u r t h e r p u r i f i e d c o u l d a t r u e e s t i m a t i o n o f t h e t r a n s -aminase a c t i v i t y be o b t a i n e d . N. l n c e r t a showed a p p r o x i -m a t e l y a t e n - f o l d g r e a t e r t r a n s a m i n a s e a c t i v i t y t h a n I . galhana a t comparable t o t a l p r o t e i n c o n c e n t r a t i o n s ( T a b l e 22);. T h i s s u g g e s t e d t h a t t h e t e n - f o l d d i f f e r e n c e i n the d e g r a d a t i v e r o u t e s o f bo t h p h e n y l a l a n i n e and t y r o s i n e may be r e l a t e d t o t h e t r a n s a m i n a s e a c t i v i t y . I n c u b a t i o n o f non-adapted c e l l s o f I_. g a l b a n a w i t h lk C - p h e n y l a l a n i n e o r t y r o s i n e c o n f i r m e d t h a t no t r a n s a m i n -ase a c t i v i t y was p r e s e n t . Some r a d i o a c t i v e p r o d u c t s were e x t r a c t e d i n t o e t h e r ( F i g u r e 32) , but th e y d i d n ot c o r r e -spond t o t h e a r o m a t i c compounds e x p e c t e d from t h e de g r a d a -t i o n o f p h e n y l a l a n i n e o r t y r o s i n e . When p r e - a d a p t e d c e l l s o f I . g a l b a n a were us e d , a u t o r a d i o g r a p h i c p a t t e r n s TABLE 22 Transaminase A c t i v i t y i n C e l l E x t r a c t s o f N a v i c u l a i n c e r t a and I s o c h r y s l s g a l b a n a Transaminase a c t l v i t y ^ ^ Amino a c i d and N. i n c e r t a I . g a l b a n a a - k e t o a c i d s u b s t r a t e s Non-adapted c e l l s Non-adapted c e l l s P h e n y l a l a n i n e a d a p t e d c e l l s T y r o s i n e a d a p t e d c e l l s a - K e t o g l u t a r a t e + p h e n y l a l a n i n e a - K e t o g l u t a r a t e + t y r o s i n e 0,302 0,230 0.000 0.027 0.040 0.010 0.008 0.017 P y r u v a t e + p h e n y l a l a n i n e P y r u v a t e + t y r o s i n e 0.542 0.538 0.000 0.026 0.010 0.040 0,020 0.041 O x a l o a c e t a t e + p h e n y l a l a n i n e O x a l o a c e t a t e + t y r o s i n e 0.176 ' 0,388 0.000 0.024 0.010 0,020 0.030 0.030 C o n t r o l + p h e n y l a l a n i n e ^ ' C o n t r o l + t y r o s i n e ^ 2 ' 0.000 0,008 0.000 0.029 .0,000 0.000 ^ 0.010 0 P 0 2 0 (1) o p t i c a l d e n s i t y i n c r e a s e a f t e r 2 hours i n c u b a t i o n a t pH 7.6 (see t e x t ) . (2) no a - k e t o a c i d . 3d Fissure 32. A u t o r a d i o g r a p h s o f chromatograms p r e p a r e d from t h e e t h e r 14 14 e x t r a c t s o f C - p h e n y l a l a n l n e and C- t y r o s i n e t r a n s -aminase e x p e r i m e n t s w i t h e x t r a c t s o f I . g a l b a n a and N. l n c e r t a . O x a l o a c e t i c a c i d was u t i l i z e d as t h e a - k e t o a c i d l n each a s s a y , u lOOb DL-fVe-l-'K Noh-ad»p. cells + 101. c h a r a c t e r i s t i c o f t h e d e g r a d a t i v e r o u t e s f o r b o t h amino a c i d s were o b t a i n e d ( F i g u r e 32). A c o r r e c t i n t e r p r e t a t i o n o f t h e endogenous a - k e t o a c i d s u s e d by t h e s e a l g a e f o r t r a n s a m i n a s e a c t i v i t y ( T a b l e 22) i s n o t p o s s i b l e because of t h e impure enzymes u t i l i z e d l n t h e s e a s s a y s . O v e r a l l , a - k e t o g l u t a r i c a c i d , o x a l o a c e t i c a c i d , and p y r u v i c a c i d Were u t i l i z e d f o r t h e t r a n s a m i n a t i o n shown by b o t h s p e c i e s . P y r u v a t e r e s u l t e d i n t h e g r e a t e s t a c t i v i t y f o r N. i n c e r t a . b u t no a - k e t o a c i d c o n s i s t a n t l y r e s u l t e d i n g r e a t e r a c t i v i t y f o r I . g a l b a n a . The o n l y o t h e r e n z y m a t i c f a c t o r s t u d i e d was pH.' Only N. l n c e r t a e x t r a c t s were examined and t h e a c t i v i t y a t pH 7.6 was g r e a t e r t h a n a t e i t h e r 7.1 o r 8.0. The low v a l u e s of d o u b t f u l enzyme a c t i v i t y i n t h e .absence o f an a - k e t o a c i d s u g g e s t t h a t L-amino a c i d o x i d a s e may n o t be p r e s e n t i n e i t h e r X i g a l b a n a o r N± i n c e r t a . The c o r r e s p o n d i n g h i g h v a l u e s f o r g a l b a n a non-adapted c e l l s w i t h t y r o s i n e appear t o be due t o p r o d u c t s f r o m o t h e r un-i d e n t i f i e d r e a c t i o n s . I n a l l cases c i n n a m i c o r p-coumaric a c i d s were n e v e r d e t e c t e d as p r o d u c t s o f t h e s e enzyme, t e s t s , p - H y d r o x y b e n z o l c a c i d h y d r o x y l a s e . T h i s enzyme was o n l y a s s a y e d f o r i n N± l n c e r t a . I n two e x p e r i m e n t s , p h e n y l a l a n i n e , t y r o s i n e , and n i t r a t e grown c e l l s were examined, but no a c t i v i t y was d e t e c t a b l e . P r o t e i n d e t e r m i n a t i o n s . The Lowry method o f p r o t e i n e s t i m a t i o n gave a v a l u e o f 4 mg o f p r o t e i n p e r 20 mg o f N . , l n c e r t a f r e e z e d r i e d c e l l s and 5 mg o f p r o t e i n p e r 20 mg o f I. g a l b a n a f r e e z e d r i e d c e l l s . T h e - c e l l s examined were e i t h e r c u l t u r e d on n i t r a t e o r p h e n y l a l a n i n e . 1 0 3 . DISCUSSION The r e s u l t s I n d i c a t e t h a t I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a were c a p a b l e o f a s s i m i l a t i n g L - p h e n y l a l a n i n e and L - t y r o s i n e a g a i n s t a c o n c e n t r a t i o n g r a d i e n t ? " The u p t a k e f o r b o t h amino a c i d s was f a r s u p e r i o r f o r N. l n c e r t a . when compared t o I . g a l b a n a . I l l u m i n a t i o n g e n e r a l l y s t i m u l a t e d t h e u p t a k e r a t e s o f b o t h amino a c i d s by b o t h s p e c -l e s . T h i s e f f e c t o f l i g h t (an energy s o u r c e ) and t h e a s s i m -i l a t i o n a g a i n s t a c o n c e n t r a t i o n g r a d i e n t 1 s t r o n g l y s u g g e s t e d t h a t a c t i v e t r a n s p o r t was i n v o l v e d . To d a t e , o n l y t h e up-2 t a k e o f L - p h e n y l a l a n l n e i n a l g a e has been i n v e s t i g a t e d . I t was r e p o r t e d t h a t C h l o r e l l a f u s c a . C. v u l g a r i s , and Platymonas  s u bc o r d ' l f ormis a ss i ml l a t e d ' t h 1 s amino a c i d a g a i n s t r a concen-t r a t i o n g r a d i e n t ( P e d e r s e n e t a^., 1974} Van Sumere e t a l . . 1971i N o r t h e t a l . . 19^7) and l i g h t enhanced p h e n y l a l a n i n e u p t a k e i n v u l g a r i s "(Van Sumere e t a l . , 1971). I . g a l b a n a and N. l n c e r t a were a b l e t o • m e t a b o l i z e * L - p h e n y l a l a n i n e and L - t y r o s i n e when grown on t h o s e compounds, as t h e s o l e n i t r o g e n s o u r c e . I n d u c t i o n o f one o r more en-zymes was i n d i c a t e d f o r I . g a l b a n a by a f o u r day e x t e n s i o n o f t h e l a g p e r i o d o b s e r v e d f o r c e l l s p r e v i o u s l y c u l t u r e d on n i t r a t e a s t h e n i t r o g e n s o u r c e . No i n c r e a s e i n t h e l a g p e r i o d was ob-s e r v e d f o r N a v l c u l a which s u g g e s t e d t h a t t h e i n i t i a l enzyme o r enzymes were c o n s t i t u t i v e . These amino a c i d s were n o t t h e b e s t s o u r c e o f n i t r o g e n (1) see Addendum, p.159. (2) i n terms o f a r o m a t i c amino a c i d s . 104. f o r g rowth o f N a v l c u l a as t h e growth c o n s t a n t s were o n l y one h a l f t h e v a l u e o b t a i n e d f o r n i t r a t e ( b e s t N-source t e s t e d ) . I s o c h r y s l s u t i l i z e d L - p h e n y l a l a n i n e as e f f e c t i v e l y as n i t r a t e , b u t s e v e r e growth i n h i b i t i o n r e s u l t e d w i t h L - t y r o s l n e . A. s i m i l a r g r owth i n h i b i t i o n by L - t y r o s i n e was r e p o r t e d by Ingram and J e n s e n (1973) f o r A n a c y s t l s n i d u l a n s . but no a t t e m p t t o ex-p l a i n t h e cause o f t h e I n h i b i t i o n was u n d e r t a k e n . L - p h e n y l -a l a n l n e was r e p o r t e d t o i n h i b i t t h e growth o f Agmenellum  q u a d r u p l l c a t u m (Ingram and J e n s e n , 1973)• but t h e a d d i t i o n o f L - t y r o s i n e t o t h e medium c o m p l e t e l y r e v e r s e d t h i s i n h i -b i t i o n . They found t h a t L - p h e n y l a l a n i n e i n h i b i t e d 3-deoxy-D - a r a b l n o h e p t u l o s o n i c a c i d 7-phosphate s y n t h e t a s e (DAHP s y n t h e t a s e ) , t h e I n i t i a l enzyme i n t h e s h i k i m i c a c i d pathway ( F i g u r e . 1 ) . T h i s p r e v e n t e d t h e b i o s y n t h e s i s o f not o n l y L-t y r o s i n e but a l s o L - t r y p t o p h a n which i n h i b i t e d normal growth. I n an a t t e m p t t o e x p l a i n t h e e f f e c t o f L - t y r o s i n e on I. g a l b a n a . L - p h e n y l a l a n l n e was added t o t h e medium c o n t a i n i n g L - t y r o s i n e t o d e t e r m i n e i f feedback i n h i b i t i o n was t h e cause o f t h e reduced c e l l y i e l d . No change i n t h e c e l l y i e l d was o b t a i n e d . Normal growth o f I s o c h r y s l s was a l s o o b t a i n e d on n i t r a t e i n t h e p r e s e n c e o f L - t y r o s i n e which c o n f i r m e d t h a t f e e d b a c k i n h i b i t i o n was not i n v o l v e d as L - t y r o s l n e was a s s i m i l a t e d f rom t h e medium when n i t r a t e was p r e s e n t . Growth i n h i b i t i o n s h o u l d have r e s u l t e d i f L - t y r o s i n e was I n v o l v e d . These r e s u l t s s u g g e s t e d t h a t one o f t h e m e t a b o l i c p r o d u c t s from the degradation of L-tyrosine and not L-tyroslne i t s e l f 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 c e l l yields. The D-isomers of phenylalanine and tyrosine were also examined for their potential use as nitrogen sources. I.•galbana was unable to u t i l i z e either D-amino acid, but . N.•lncerta. after a lag period of about 11 days, u t i l i z e d 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 u t i l i z e D-phenylalanine, and one species to pos-sibly metabolize D-tyrosine. .. ' • Other reports indicated that D-phenylalanine was r not assimilated, i f assimilated at a l l , u n t i l a l l the L-phenylalanine was removed from the medium (Pedersen et  a l . , 1974; Van Sumere et a l . , 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 i n the DL-mixtures, the L-isomer was the i n i t i a l and possibly the only Isomer u t i l i z e d . 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 a l . , (1971) and Pokokny et a l . , (1970) respectively Both were metabolized, but when D-methlonlne was administered. 106. o n l y L - m e t h i o n i n e was found I n c o r p o r a t e d i n t h e a l g a l t i s -sues (24 s p e c i e s i n 8. d i v i s i o n s ) . T h i s s t r o n g l y I n d i c a t e d t h a t some of t h e D-isomer was r a c e m l z e d . They s u g g e s t e d t h i s c o n v e r s i o n proceeded v i a o x i d a t i v e d e a m i n a t l o n by D-amino a c i d o x i d a s e , f o l l o w e d by an L - s p e c i f i c r e a m i n a t i o n o r v i a e n z y m a t i c r a c e m i z a t i o n of t h e D-isomer. Which r o u t e was i n -v o l v e d was not d e t e r m i n e d . The extended l a g p e r i o d b e f o r e e i t h e r D - p h e n y l a l a n i n e or D - t y r o s i n e was u t i l i z e d as a n i t r o -gen s o u r c e by N_ l n c e r t a can be e x p l a i n e d by assuming t h a t t i m e i s r e q u i r e d f o r the i n d u c t i o n o f e i t h e r o f t h e s e en-z y m a t i c systems. i The i n i t i a l enzyme may be (A) n o n - o x i d a t i v e - a m m o n i a l y a s e s , (B) o x i d a t i v e - L - a m i n o a c i d o x i d a s e , o r (C) t r a n s a m i n a s e -amino t r a n s f e r a s e i n r e g a r d t o t h e m e t a b o l i c d e g r a d a t i o n o f L - p h e n y l a l a n i n e and L - t y r o s l n e " by d e a m i n a t l o n . I n no 14 x C-experiment was c i n n a m i c a c i d o r p-coumaric a c i d d e t e c t e d f o r I_j_ g a l b a n a o r N_j_ i n c e r t a which s u g g e s t e d t h a t t h e am-14 monia l y a s e s were not p r e s e n t . I n C-enzyme a s s a y s f o r p h e n y l a l a n i n e ammonia l y a s e on e i g h t e e n o t h e r a l g a l s p e c i e s , no a c t i v i t y was d e t e c t e d . Young et a l . ( 1 9 6 6 ) , who examined f o u r a l g a l s p e c i e s f o r p h e n y l a l a n i n e and t y r o s i n e ammonia l y a s e s a l s o found no a c t i v i t y . When n o n - l a b e l l e d L - t y r o s l n e was f e d t o n i n e t e e n s p e c i e s o f a l g a e , p-coumaric a c i d was n e v e r d e t e c t e d . So f a r as i s known, the ammonia l y a s e s ap-p e a r t o be s o l e l y r e s t r i c t e d t o v a s c u l a r p l a n t s , c e r t a i n f u n g i , and c e r t a i n s p e c i e s o f b a c t e r i a (see Towers and Subba Rao,, 1972) V 107. p I n enzyme a s s a y s f o r t h e amino t r a n s f e r a s e where no a - k e t o a c i d was p r e s e n t , no a c t i v i t y was o b t a i n e d f o r I» g a l b a n a and N . , l n c e r t a . w h i c h s u g g e s t e d t h a t t h e L-amino a c i d o x i d a s e was a l s o n o t a c t i v e i n e i t h e r s p e c i e s . But when an a - k e t o a c i d was p r e s e n t a c t i v i t y was o b t a i n e d f o r t h e amino t r a n s f e r a s e . E x c e l l e n t a c t i v i t y was o b t a i n e d f o r N a v l c u l a . but p r e - a d a p t e d c e l l s were r e q u i r e d b e f o r e a c t i v i t y was ob-t a i n e d f o r I s o c h r y s l s . T h i s s u g g e s t e d t h a t t h e f o u r day l a g p e r i o d o b s e r v e d b e f o r e growth of I s o c h r y s l s on L - p h e n y l -a l a n l n e o r L - t y r o s i n e as t h e s o l e n i t r o g e n s o u r c e was r e l a t -ed t o t h e i n d u c t i o n o f t h i s enzyme. No a c t i v i t y was o b t a i n -ed i f t h e c e l l s of I s o c h r y s l s were not p r e - a d a p t e d t o e i t h e r o f t h e s e amino a c i d s . Amino t r a n s f e r a s e a c t i v i t y has been p r e v i o u s l y de-t e c t e d i n a l g a e f o r L - p h e n y l a l a n i n e (Gassman e t a l . , 1968. J a c o b l , 1957 r Stenmark et a l . , 197*0 but no a c t i v i t y was r e p o r t e d f o r L - t y r o s i n e . The amino t r a n s f e r a s e f o r p h e n y l -a l a n i n e and t y r o s i n e has been demonstrated i n p l a n t s , f u n g i , b a c t e r i a , and a n i m a l s (see Towers and Subba Rao, 1 9 7 2 ) . The i m p o r t a n c e o f t h i s r e a c t i o n i n h i g h e r and l o w e r p l a n t s has no t been i n v e s t i g a t e d t h o r o u g h l y , p o s s i b l y because o f t h e i n t e r e s t i n t h e ammonia l y a s e s . . I n F i g u r e 33 a scheme e l u c i d a t e d by t h e s e s t u d i e s f o r t h e d e g r a d a t i o n of L - p h e n y l a l a n i n e f o r b o t h I, g a l b a n a and N. l n c e r t a I s p r e s e n t e d . T h i s scheme was based on t h e p h e n y l a l a n i n e - C s t u d i e s as w e l l as t h e m e t a b o l i s m o f non-r a d i o a c t i v e a r o m a t i c compounds. The compounds i n b r a c k e t s were not i d e n t i f i e d i n t h i s s t u d y but were e x p e c t e d . 108. L-phenylalanine phenylpyruvic acid <^>-CH 2 -^-C00H f \ CH 2 -CH-C00H RlH2 phenyllactic acid V ( f \ c H ? - C H - C 0 0 H ; ^ N H 3 ^ OH . 0 >^co2 phenylacetaldehyde < £ ^ - C H 2 - C H O phenylacetic acid < Q r - C H 2 - C O O H mandelic acid CH-COOH OH benzoylformic acid benzaldehyde ( ^ - C - C O O H ,S-co2 [ 0 C H Q ] ' I HO y benzoic acid C O O H m-hydroxy-benzoic acid [ C ? 2 ] n catechol CH J OH ;co 2 o-hydroxy-benzoic acid *- { ^ C O O H HO COOH H O - ^ ! p-hydroxybenzoic OHl acid U-dihydroxybenzene +COo 3-bromo-p-hydroxybenzoic yc-Tv acid H O - O - C O O H B'I P ^ ^ - 0 H ] »- BROWN ? semiquinone Figure 33. The degradative route of L-phenylalanlne in Isochrysls galbana and Navlcula lncerta. The compounds In brackets were not detected. 109. F o r b o t h s p e c i e s , t h e t r a n s a m i n a s e r e a c t i o n p r o d u c t p h e n y l -p y r u v i c a c i d and i t s r e d u c t i o n p r o d u c t , p h e n y l l a c t i c a c i d U \k 14 were d e t e c t e d when p h e n y l a l a n l n e - 1 - C , - 2 - C f and - 3 - C Ik were f e d . I n t h e d a r k f e e d i n g s w i t h p h e n y l a l a h i n e - 1 - Cf l e s s , t h a n 2,0% o f t h e r a d i o a c t i v i t y f o r I_. g a l b a n a and 10% o f t h e r a d i o a c t i v i t y f o r l n c e r t a were l o c a t e d i n t h e s e two a r o m a t i c a c i d s . The r e m a i n d e r o f t h e r a d i o a c t i v i t y f r o m Ik Ik t h e m e t a b o l i z e d p h e n y l a l a n l n e - 1 - C was i n t h e t r a p p e d C O g . The d e c a r b o x y l a t i o n o f p h e n y l p y r u v a t e would produce p h e n y l -a c e t a l d e h y d e , but t h i s a l d e h y d e was n o t p o s i t i v e l y i d e n t i -f i e d . A r a d i o a c t i v e s p o t was d e t e c t e d f o r b o t h s p e c i e s where i t s h o u l d chromatograph (see F i g u r e s 28 and 29 and Appendix Ik B f o r l o c a t i o n ) when p h e n y l a l a n i n e - 2 - C was f e d . T h i s a l d e -hyde ^ a s . s known t o be o x i d i z e d t o p h e n y l a c e t i c a c i d I n a i r and i t would be e x p e c t e d , i n v i v o , t o be o x i d i z e d t o p h e n y l a c e t i c a c i d . P h e n y l a c e t i c a c i d was d e t e c t e d f o r b o t h s p e c i e s when lk lk p h e n y l a l a n i n e - 2 - C and - 3 - C were f e d . T h i s a c i d was found t o be s t i m u l a t o r y t o growth i n c o n c e n t r a t i o n s a t o r below 0 .5 mM f o r I± g a l b a n a and below 0.1 mM f o r I n c e r t a . S i m i l a r l y A l g e u s (19^6) o b s e r v e d c o n c e n t r a t i o n s below 1.0 mM s t i m u l a t e d t h e growth o f Scenedesmus o b l i q u s a l t h o u g h he d i d n o t s p e c u l a t e on m e t a b o l i s m . I f I n h i b i t i o n above 1.0 mM o c c u r r e d i t was n o t s t a t e d . A t t h e a f o r e m e n t i o n e d c o n c e n t r a t i o n s f o r I s o c h r y s l s and N a v l c u l a . g r o w t h i n h i b i t i o n was o b s e r v e d . W i t h I s o c h r y s l s and N a v l c u l a . r e m a i n i n g p h e n y l a c e t i c a c i d 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 d i f f i c u l t y 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-t r o l l n the KOH. This suggested that phenylacetic acid was the v o l a t i l e product. Possibly some other Cg-C2 acid was i n -volved, but generally their concentrations were very low. The experiments of Vose et a l . (1971) indicated .that the .vol-a t i l e compound was not carbon monoxide or phenolic or olefinic l n nature. They suggested that an aromatic compound was i n -volved, which i s supported by the above results. Mandelic and benzoylformic acids were detected i n 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-f i n i t e increase. Which suggested that these two C £ - C 2 acids were p r e s e n t a l o n g w i t h p h e n y l p y r u v i c - p h e n y l a c e t i c a c i d s . I n t h e growth e x p e r i m e n t s , m a n d e l l c a c i d was f o u n d t o be m i l d l y i n h i b i t o r y f o r b o t h a l g a l s p e c i e s a t h i g h con-c e n t r a t i o n s , but a t low c o n c e n t r a t i o n s t h e growth o f I s o c h r y s l s was s t i m u l a t e d . When t h e medium f r o m t h e s e e x p e r i m e n t s was examined, m a n d e l i c a c i d was n o t d e t e c t e d but b e n z o i c a c i d was p r e s e n t . T h i s s u g g e s t e d t h a t t h e o b s e r v e d e f f e o t on g r o w t h may n o t be due t o m a n d e l i c a c i d b u t t o b e n z o i c a c i d . The e f f e c t on t h e growth o f many compounds may not i n f a c t be due t o t h e i n i t i a l s u b s t a n c e , but t o one o f i t s m o d i f i e d p r o d u c t s . B e n z o y l f o r m i c a c i d would be e x p e c t e d t o d e c a r b o x y -l a t e t o produce b e n z a l d e h y d e and CO^. When p h e n y l a l a n l n e -i l i 14 2- C and p h e n y l a c e t i c a c i d - 1 - C were f e d t o b o t h s p e c i e s , 14 CO;, was t r a p p e d i n t h e KOH w h i c h c o n f i r m e d t h i s d e c a r b o x y -l a t i o n . About 45/6 and J0% o f t h e m e t a b o l i z e d p h e n y l a l a n i n e -14 14 2- C i n t h e d a r k was I n t h e C0 2 f o r I s o c h r y s l s and N a v l c u l a r e s p e c t i v e l y . B e n z a l d e h y d e was not i d e n t i f i e d i n t h e s e two s p e c i e s , but i t s h o u l d chromatograph i n t h e same r e g i o n as p h e n y l a c e t a l d e h y d e . No r e p o r t s o f b enzaldehyde i n p h y t o -p l a n k t o n a r e known, but i t has been i d e n t i f i e d i n m a c r o s c o p i c m a r i n e a l g a e (Katayama, 1962). I t ' s b i o - o r g a n i c o r i g i n was unknown. Benza l d e h y d e was r e p o r t e d t o be i n h i b i t o r y t o t h e g r o w t h o f C h l o r e l l a v u l g a r i s a l t h o u g h i t s t i m u l a t e d c e l l u l a r r e s p i r a t i o n (Dedonder and Van Sumere, 1968, 1971). They a l s o o b s e r v e d t h a t i t was r a p i d l y o x i d i z e d by C h l o r e l l a t o b e n z o i c a c i d . T h i s a l d e h y d e undergoes f a i r l y r a p i d 112. o x i d a t i o n t o b e n z o i c a c i d i n a i r . B e n z o i c a c i d was d e t e c t e d as a p r o d u c t from t h e met-a b o l i s m o f DL-mandelic a c i d by b o t h a l g a l s p e c i e s . When - h 14 p h e n y l a l a n l n e - 3 - C was f e d , b e n z o i c a c l d - 1 - C was o n l y de-t e c t e d f o r N. l n c e r t a . T h i s s u g g e s t e d t h a t v e r y l i t t l e ben-z o i c a c i d was produced i n I . g a l b a n a o r i t was r a p i d l y met-a b o l i z e d . I n t h e d a r k about yi% o f t h e m e t a b o l i z e d p h e n y l -14 a l a n l n e - 3 - C was t r a p p e d as COg as compared t o 12$ f o r N. l n c e r t a . T h i s may have a c c o u n t e d f o r b e n z o i c a c i d not b e i n g d e t e c t e d . I n t h e m e t a b o l i s m o f b e n z o i c a c i d , t h e p a r a -and m e t a - h y d r o x y l a t e d d e r i v a t i v e s were d e t e c t e d f o r b o t h s p e c i e s , but s a l i c y l i c a c i d was o n l y d e t e c t e d from Nj, l n c e r t a . T h i s may p o s s i b l y be due t o a v e r y a c t i v e d e c a r b o x y l a s e i n I . g a l b a n a f o r s a l i c y l i c a c i d (see T a b l e 2 0 ) . I t was un-known i f t h e d e c a r b o x y l a t i o n of b e n z o i c a c i d o r i t ' s h y d r o x y -14 l a t i o n o r o d u c t s o r b o t h a c c o u n t e d f o r t h e CO t r a p p e d from 14 d p h e n y l a l a n i n e - 3 - C. The p r e s e n c e o f b e n z o i c a c i d i n t h e medium was i n -h i b i t o r y on growth of b o t h s p e c i e s a t h i g h c o n c e n t r a t i o n s . S i m i l a r r e s u l t s were o b s e r v e d f o r C h l o r e l l a v u l g a r i s (Dedonder and Van Sumere, 1968, 1971). They r e p o r t e d no g r o w t h s t i m -u l a t i o n a t l o w e r c o n c e n t r a t i o n s but t h e growth o f b o t h I . g a l b a n a and N . I n c e r t a was s t i m u l a t e d w i t h low c o n c e n t r a t i o n s o f b e n z o i c a c i d . Dedonder and Van Sumere (1968, 1971) a l s o o b s e r v e d t h a t c e l l r e s p i r a t i o n was i n c r e a s e d by b e n z o i c a c i d but t h e y d i d not examine t h e medium f o r any m e t a b o l i c p r o -d u c t s . B e n z o i c a c i d was o b s e r v e d t o g i v e a h i g h r a d i o a c t i v e count in the control when i t 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 ( s a l i c y l i c acid) had very l i t t l e 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 i n 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. E l l i s , personal communication). Whether ring cleavage was extra- or intra-diol was not known. This decarboxylation was also reported to occur In higher plants (see E l l i s , 1974), Very l i t t l e 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 C r a l g l e , 1966), but h i g h c o n c e n t r a t i o n s I n h i b i t e d t h e g r o w t h n o t o n l y o f I_. g a l b a n a b u t a l s o o f C h l o r e l l a v u l g a r i s (Dedonder and Van Sumere, 1971 )• E v i d e n c e s u g g e s t e d t h a t b o t h I . g a l b a n a and l n c e r t a (1) d e c a r b o x y l a t e d t h i s a c i d p r o b a b l y r e s u l t i n g l n 1,4-dlhydroxybenzene, (2) b r o m l n a t e d t h i s a c i d t o 3-bromo-p-hydroxybenzoic a c i d , and (3) e x c r e t e d t h i s a c i d I n t o t h e medium. When bo t h s p e c i e s were mass c u l -t u r e d on e i t h e r p h e n y l a l a n i n e o r t y r o s i n e , p - h y d r o x y b e n z o i c a c i d was o b s e r v e d i n t h e medium a f t e r a l l c e l l s were removed. I t was not d e t e c t e d l n t h e medium o f t h e c o n t r o l c u l t u r e s . I t must be k e p t i n mind t h a t p - h y d r o x y b e n z o l c a c i d , a p r e -c u r s o r o f u b i q u i n o n e b i o s y n t h e s i s , c a n be formed d i r e c t l y f r o m c h o r i s m i c a c i d (see F i g u r e 1) ( L u c k n e r , 1972) w h i c h may e x p l a i n I t s common, appearence i n a l l c e l l e x t r a c t s . D e c a r b o x y l a t i o n ^ o f p - h y d r o x y b e n z o l c a c i d a l s o o c c u r -r e d i n b o t h I j g a l b a n a and N. l n c e r t a . The p r o d u c t , 1,4-d l h y d r o x y b e n z e n e , was r e p o r t e d t o be i n h i b i t o r y on t h e g r o w t h o f C h l o r e l l a v u l g a r i s (Dedonder and Van Sumere, 1971)) p o s s i b -l y due t o t h e f o r m a t i o n o f p-benzoquinone which was r e p o r t e d t o be e x t r e m e l y t o x i c f o r e i g h t a l g a l s p e c i e s (Dedonder and Van Sumere, 1971.; McLachlan and C r a i g i e , 1966). I t was n o t known i f t h e a l g a l c u l t u r e medium promoted t h i s r e a c t i o n . The o x i d a t i o n o f a h ydroquinone t o a q u i n o n e , and t h e r e -v e r s e r e a c t i o n , p r oceeds t h r o u g h a semi quinone ( M l c h a e l i s , , I 9 6 l ) . These semlquinones a r e known t o be c o l o u r e d com-pounds and i t was o b s e r v e d t h a t l n t h e growth e x p e r i m e n t s u s i n g C^ v u l g a r i s w h i c h c o n t a i n e d p-benzoquinone, t h e medium (1) see Addendum. 115. showed a pronounced reddish colour formatlon(Dedonder and Van Sumere, 1971). . Whether th i s together with the photo-l y t i c degradation of 3-bromo-p-hydroxybenzoic ac i d caused the browning l n the cultures of I_, galbana and N± lncert a was not established. The CO^ trapped from t y r o s i n e - 3 - C was greater 14 than for phenylalanlne - 3 - C f o r both a l g a l species (Tables 14 l 4 17 and 18). The CO^ from t y r o s i n e - 3 - C was probably de-ri v e d from the decarboxylation of p-hydroxybenzoic ac i d while 14 f o r phenylalanine -3— C from benzoic ac i d 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 thi s acid was always less i n the l i g h t than i n the dark f o r the tyrosine feedings. This suggested that i t contributed to the browning. The content of bromine (65 mg/L,, Home, 1969) was s u f f i c i e n t to have allowed enough 3-bromo-p-hydroxybenzolc acid to be formed to account f o r the browning. I n F i g u r e a scheme e l u c i d a t e d by t h e s e s t u d i e s f o r t h e d e g r a d a t i o n of L - t y r o s i n e by b o t h I . g a l b a n a and N. l n c e r t a i s p r e s e n t e d . T h i s scheme was based on the t y r o s i n e - 1 * ^ s t u d i e s as w e l l as the m e t a b o l i s m o f n o n - r a d i o a c t i v e a r o m a t i c compounds. The compounds i n b r a c k e t s were n o t i d e n t i f i e d i n t h i s s t u d y but were e x p e c t e d . p - H y d r o x y p h e n y l p y r u v i c a c i d was o n l y d e t e c t e d f o r I s o c h r y s l s g a l b a n a . b u t the s i d e c h a i n r e d u c t i o n p r o d u c t , p - h y d r o x y p h e n y l l a c t i c a c i d was d e t e c t e d f o r b o t h L-tyrosine H O - c ^ - C H o - C H - C O O H IvJH 116. 2 H O - ^ C H 2 - C H - c o 0 h - sjf OH p-hydroxy- /f- p-hydroxy-phenylpyruvic acid HO'-^^-CH^f r-COOH phenyllactic acid J S N H 3 p-hydroxy 0 >co 2 phenylacetaldehyde H 0 - ^ J j ) - C H 2 - c h 0 p-hydroxy- ^ phenylacetic acid H O - ^ J ^ C H ^ C O O H p-hydroxy-mandelic acid p-hydroxy H O - ^ 2 V C H - C O O H OH y u i u x y - p benzoytformic acid H 0 - v _ v - C - c 0 0 h L | 6 J p-hydroxy- y-L ^2 benzaldehyde H O - ^ _ ^ C H O p-hydroxy-benzoic acid - H O - ^ ^ - C O O H 'HO-^ -CHJDH p-hydroxy benzyl-alcohol 3-bromo-p- hydroxy-benzoic ac id iand 1^-dihydroxybenzene [ H O ^ O H ] + C O 2 H C - ^ ) - C O O H Br ^ polymerization ^^[^O 0"] BROWN ? semiqumone Figure Jk. The degradatlve route of L-tyrosine i n Isochrysls galbana and Navlcula lncerta . The compounds In brackets were not detected. 117 e I . g a l b a n a and.N. l n c e r t a . The p r e s e n c e o f t h i s a c i d c o n -f i r m e d t h a t p - h y d r o x y p h e n y l p y r u v i c a c i d was produced by b o t h s p e c i e s . The e f f e c t o f p - h y d r o x y p h e n y l p y r u v a t e was examined on t h e growth of G o n l o t r l c h u m a l s l a l l ( F r i e s , 197*0 and i t was found t o s t i m u l a t e growth a t low c o n c e n t r a t i o n s . Ex-p e r i m e n t s w i t h t h i s a c i d and w i t h p h e n y l p y r u v a t e must be c a r e f u l l y I n t e r p r e t e d because t h e y a r e u n s t a b l e a t a l k a l i n e ,pH v a l u e s p r o d u c i n g t h e r e s p e c t i v e C -C a l d e h y d e and a C 0 o l f r a g m e n t . T h i s was not o b s e r v e d t o have o c c u r r e d i r i v i v o * lk as l e s s t h a n J% o f t h e m e t a b o l i z e d t y r o s i n e - 1 - C was i n t h e ether, phase w h i l e f o r I± g a l b a n a 75$ o f t y r o s i n e - 2 - 1 ^ C ^ lk and f o r N. I n c e r t a 31$ o f t y r o s l n e - 2 - C was i n t h e e t h e r phase. I f a C -fragment was l o s t , t h e s e p e r c e n t a g e s f o r t h e e t h e r phases would a l l be e x p e c t e d t o be around 3$» The C^-fragment a l s o s h o u l d be r a p i d l y m e t a b o l i z e d t o produce COg, 14 lk t h e r e f o r e t h e v a l u e s f o r C 0 2 t r a p p e d from t y r o s l n e - 1 - C and lk -2 - C s h o u l d be t h e same I f a Cg-fragment was removed. T h i s lk was not o b s e r v e d as 97$ o f the m e t a b o l i z e d t y r o s i n e - 1 - C was lk t r a p p e d i n CO^ f o r b o t h s p e c i e s w h i l e f o r I_. g a l b a n a 25$ and f o r l n c e r t a 69$ o f t h e m e t a b o l i z e d t y r o s i n e ^ - ^ C lk was i n the . CO,,. These f i n d i n g s c o n f i r m e d t h a t a Cg-fragment was n o t produced. D e c a r b o x y l a t i o n o f p - h y d r o x y p h e n y l p y r u v i c a c i d would r e s u l t i n p - h y d r o x y p h e n y l a c e t a l d e h y d e . T h i s a l d e h y d e was not lk lk d e t e c t e d i n t h e t y r o s i n e - 2 - C and -3 - C e x p e r i m e n t s because i t p r o b a b l y chromatographed w i t h p - h y d r o x y p h e n y l l a c t i c and p - h y d r o x y m a n d e l i c a c i d s . A v e r y f a i n t s p o t l n t h e c o r r e c t 118 o p o s i t i o n s was o b s e r v e d a f t e r a 2k hour f e e d i n g w i t h t y r o -s i n e - U - ^ C . Whether p - h y d r o x y p h e n y l a c e t a l d e h y d e s p o n t a n e o u s l y o x i d i z e d t o p - h y d r o x y p h e n y l a c e t i c a c i d , was not e s t a b l i s h e d , , p - H y d r o x y p h e n y l a c e t l c a c i d was d e t e c t e d i n the t y r o -ik lk s i n e - 2 - C and - 3 - C ' f e e d i n g e x p e r i m e n t s . I n t h e grow t h e x p e r i m e n t s , h i g h c o n c e n t r a t i o n s were i n h i b i t o r y on growth,, C h r o m a t o g r a p h i c a n a l y s i s from t h e growth e x p e r i m e n t s , r e -v e a l e d r e m a i n i n g p - h y d r o x y p h e n y l a c e t i c a c i d , p-hydroxyben-z o i c a c i d , and p - h y d r o x y b e n z y l a l c o h o l . I n the same t i m e p e r i o d , p - h y d r o x y m a n d e l i c a c i d was t o t a l l y m e t a b o l i z e d to p - h y d r o x y b e n z o i c a c i d and p - h y d r o x y b e n z y l a l c o h o l . T h i s sug-g e s t e d t h a t b o t h s p e c i e s had d i f f i c u l t y i n h y d r o x y l a t i n g the s i d e c h a i n of p - h y d r o x y p h e n y l a c e t i c a c i d t o produce p-hydroxy-m a n d e l l c a c i d . p -Hydroxymandelic a c i d was d e t e c t e d i n t h e p-hydroxy-lk lk p h e n y l a c e t i c a c l d - 1 - C and - 2 - C f e e d i n g s . I n t h e growth e x p e r i m e n t s a t h i g h c o n c e n t r a t i o n s I . g a l b a n a was a l m o s t t o t a l l y i n h i b i t e d w h i l e o n l y m i l d i n h i b i t i o n was o b s e r v e d f o r N. l n c e r t a . The o t h e r c g ~ C 2 p h e n o l i c compound, p-hydroxy-b e n z o y l f o r m i c a c i d was not d e t e c t e d . The unknown compound lk #2 i n T a b l e s 17 and 18 f o r the t y r o s i n e - 2 - C f e e d i n g may be t h i s compound. The c h r o m a t o g r a p h i c l o c a t i o n was a p p r o x i m a t e l y where i t t h e o r e t i c a l l y s h o u l d have chromatographed. T h i s a c i d when s y n t h e s i z e d , was found t o be u n s t a b l e and s p o n t a n e o u s l y produced p-hydroxybenzaldehyde and p r o b a b l y CO^o I n t h e lk t y r o s i n e - 2 - C f e e d i n g s , 59% o f t h e t y r o s i n e m e t a b o l i z e d by lb M lk N. i n c e r t a was i n t h e C 0 2 , w h i l e o n l y 2% was i n t h e CO2 119 c f o r I . g a l b a n a . p-Hydroxybenzaldehyde was d e t e c t e d when t y r o s i n e - 2 -\h 14 C and p - h y d r o x y p h e n y l a c e t i c a c i d - 2 - C were f e d t o b o t h __ g a l b a n a and N. l n c e r t a . T h i s a l d e h y d e was v e r y i n h i b i -t o r y on t h e growth o f f i v e o t h e r p h y t o p l a n k t o n (Dedonder and Van Sumere, 1971; McLachlan and C r a i g i e , 1966) but a t l o w c o n c e n t r a t i o n s one s p e c i e s , s i m i l a r t o N^ , l n c e r t a . was found t o be s t i m u l a t o r y t o growth ( F r i e s , 1974). The m e t a b o l i s m o f p- h y d r o x y b e n z a l d e h y d e produced m a i n l y p - h y d r o x y b e n z o i c a c i d and t r a c e s o f 3-"bromo-p-hydroxybenzoic a c i d and p-hydroxy-b e n z y l a l c o h o l f o r b o t h I s o c h r y s l s and N a v l c u l a . A t no t i m e f o r e i t h e r I s o c h r y s l s g a l b a n a o r N a v l c u l a  l n c e r t a were any d l h y d r o x y p h e n o l i c compounds d e t e c t e d . T h i s does not mean t h e y were not produced, but o n l y t h a t t h e y were not o b s e r v e d . T h i s s u g g e s t e d t h a t any h y d r o x y l a t i o n t o p r o -duce a d l h y d r o x y p h e n o l i c compound proceeded a t a r a t e s l o w e r t h a n t h e r a t e f o r the r i n g c l e a v a g e o f such a compound. When a h y d r o x y l a t i o n o f t h e s i d e c h a i n o f p h e n y l a c e t i c and p-h y d r o x y p h e n y l a c e t i c a c i d s o r t h e r i n g o f b e n z o i c a c i d was r e q u i r e d , t h e s e r e a c t i o n s were v e r y s l o w and were p o s s i b l y r a t e l i m i t i n g . Thus a d d i t i o n o f a second h y d r o x y l group i n t o t h e a r o m a t i c r i n g s h o u l d a l s o be r a t e l i m i t i n g , even though b o t h I s o c h r y s l s and N a v l c u l a p o s s e s s the p o t e n t i a l f o r h y d r o x y -l a t i o n o f t h e o r t h o - , meta-, and p a r a - p o s i t i o n s o f b e n z o i c a c i d . W ith t h e p r o d u c t i o n o f "^CC^ from b e n z o i c a c i d - ( U ) -14 C - r i n g l a b e l l e d , i t was s u g g e s t e d t h a t t h e r o u t e 120, f o r r i n g c l e a v a g e was t h r o u g h a Cg-C-^ compound. U n l i k e t h e 14 s i t u a t i o n i n h i g h e r p l a n t s , b e n z o i c a c i d - ( U ) - C - r l n g l a b e l l e d p r o d u c e d no ^ C O ( B e r l i n e t a l . , 1971J E l l i s e t a l . , 1 9 7 0 ) , I t was e s t a b l i s h e d t h a t h o r o o g e n t i s i c a c i d ( 2 , 5 - d i h y d r o x y -p h e n y l a c e t i c a c i d ) was an i n t e r m e d i a t e i n h i g h e r p l a n t s u n d e r -g o i n g r i n g c l e a v a g e i n t h e d e g r a d a t i o n o f t y r o s i n e t o C 0 2 ( E l l i s , 1 9 7 3 ) . T h i s was the same pathway o r i g i n a l l y t h ought t o be o n l y r e s t r i c t e d t o m i c r o b i a l and a n i m a l m e t a b o l i s m ( M e i s t e r , 1 9 6 5 ) . I n t h e d e g r a d a t i o n o f t h e a r o m a t i c r i n g o f p h e n y l a l a n i n e i n h i g h e r p l a n t s , 2 , 3 - d i h y d r o x y p h e n y l a c e t i c a c i d was b e l i e v e d t o undergo r i n g f i s s i o n (see Towers and Subba Rao, 1972). I n a s u r v e y o f t h e d e g r a d a t i o n o f u n l a b e l l e d t y r o -s i n e by 19 p l a n k t o n l c s p e c i e s ( T a b l e 2 3 ) , b o t h p-hydroxy-p h e n y l a c e t i c and p - h y d r o x y b e n z o l c a c i d s were d e t e c t e d . When 14 C - t y r o s i n e - ( U ) - r l n g l a b e l l e d was f e d t o seven s p e c i e s o f p h y t o p l a n k t o n , b o t h p - h y d r o x y p h e n y l a c e t i c a c i d and p-hydroxy- " b e n z o i c a c i d were a l s o r a d i o a c t i v e ( T a b l e 14). T h i s s u g g e s t e d t h a t t h e pathway o f t y r o s i n e d e g r a d a t i o n i n F i g u r e 34 was p r e s e n t i n most i f n o t a l l a l g a e . P h e n y l a l a n i n e , however, may n o t be degraded by a l l s p e c i e s . No p h e n o l i c compounds, o t h e r t h a n p - h y d r o x y b e n z o l c a c i d were d e t e c t e d . When t h i s 14 a c i d was d e t e c t e d , p h e n y l a l a n i n e - r l n g - 1 - C was a l s o degraded 14 t o C 0 2 < T h i s s u g g e s t e d t h a t the pathway i n F i g u r e 33 was t h e d e g r a d a t i v e r o u t e f o r p h e n y l a l a n i n e . The g r e a t number o f s p e c i e s t h a t d i d n o t degrade p h e n y l a l a n i n e t o p-hydroxyben-z o l c a c i d o r C 0 2 may be t h e r e s u l t o f poor o r no h y d r o x y l a s e (1) u n d e r t a k e n f o r p a r t o f t h i s d i s s e r t a t i o n . 121. TABLE 2 3 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). A l g a l species Control Tyrosine Phenylalanine Phenylalanine r l n g - l - 1 ^ 1 * Chlorophyta ( 2) Brachlmonas submarlna 3(+) Dur.allella t e r t l o l e c t a 1 ( ? ) Nannochlorls oculata Haotophyta  Coccollthus huxleyl Isochrysls galbana 1(+) Monochrysls l u t h e r l 1 ( ? ) B a c l l l a r o p h y t a Amphlprora paludosa 3(+) Navlcula l n c e r t a 1(+) Skeletonema costatum 3 ( ? ) T h a l a s s i o s i r a f l u v l a t l l l s 1(+) Cryptonhyta Chroompnas s a l l n a 1 ( + ) . 3 ( + ) Cryptomonas s t r . WHI 1 (+)J3(+) P r r r r o P h y t a ^ V O d l n l u m c a r t e r l ghodii5I^Hla_L f g i g g guadrupllcatum v ^ t h n p h v t a H e t e j ^ t h r l * SP> M HonaVlaritu s s a l l n a K+ ) . 2 ( + ).3( + ) K + ),2( + ) K + ) l ( + ) , 2 ( + ) K++).2( + ),3( + ) K?) K+) K 3 + ) . 2 ( 3 + ) , 3 ( + ) K + ) , 2 ( ? ) l ( + ) , 2 ( + ) K+ ) . 2 ( + y , 3 ( + > K + ) . 2 ( + ) . 3 ( + ) unknown K?) K++) K?) i(?) K + ) K + ) , 2 ( + ),3( + ) 1 ( ? ) l ( 4 + ) , 2 ( 3 + ) , 3 ( + ) l ( + ) , 2 ( + ) 3(++) l ( + ) . 2 ( ? ) 3 ( ? ) K+ + ) , 2 ( + ) , 3 ( ? ) 1 ( ? ) l ( + ) , 2 ( ? ) 1(+) l ( + ) . 2 ( + ) K + ) . 2 { + ) 1 (+) unknown +(light) 6 unknown +(light) 0 unknown unknown (1) from Table 3 . ( 2 ) n ^ i ^ ? m P r ^ S ^ t e c t e d In e * h e r extract!. 1 = p-hydroxybenzoic a c i d , 2 = p-hydroxy phenylacetic ac i d , 3 = P-hydroxybenzylalcohol. Intensity l n brackets. ? =* doubtful t + - trace, ++ = low concentration, 3+ = medium concentration, and k+ = hl?h concentra-t i o n as determined from colour Intensity on thi n layer chromatograms. ooncentra-122. a c t i v i t y . p - H y d r o x y b e n z o i c a c i d appeared t o be a common i n t e r m e d i a t e i n a pathway l e a d i n g t o r i n g f i s s i o n i n a l g a e . R i n g f i s s i o n i n a l g a e does n o t appear t o be i m p o r t a n t 14 as very, low r a d i o a c t i v e c o u n t s i n the COg were always ob-t a i n e d . The main d e g r a d a t i v e p r o d u c t , p - h y d r o x y b e n z o i c a c i d , was always d e t e c t e d . The e x c r e t i o n of t h i s compound i n t o the medium was o b s e r v e d i n mass c u l t u r i n g and. when open ocean s e a w a t e r was a n a l y z e d , t h i s p h e n o l i c a c i d was d e t e c t e d (Degens e t a l . , 1 9 6 4 ) . When the sediments (dead a l g a l c e l l s ) were a n a l y z e d , o - h y d r o x y b e n z o i c , m-hydroxybenzoic and p-hydroxy-p h e n y l a c e t l c a c i d s were a l s o d e t e c t e d . A pathway f o r t h e d e g r a d a t i o n of p h e n y l a l a n i n e t o b e n z o i c a c i d appears t o be p r e s e n t i n h i g h e r p l a n t s b u t no c l e a r e v i d e n c e has been o b t a i n e d . . I n h i g h e r p l a n t s , f u n g i , and b a c t e r i a o - h y d r o x y p h e n y l a c e t i c a c i d was formed from p h e n y l -p y r u v i c a c i d i n a s i m i l a r r e a c t i o n t o t h e f o r m a t i o n o f homo-g e n t i s l c a.cld from p - h y d r o x y p h e n y l p y r u v l c a c i d . The a r o m a t i c r i n g was f u r t h e r h y d r o x y l a t e d . t h e n r i n g f i s s i o n o c c u r r e d (see Towers and Subba Rao, 1972). G e n e r a l l y , i f p h e n y l a c e t i c a c i d was f e d , i t was h y d r o x y l a t e d i n the o r t h o - o r p a r a - p o s i t i o n s . These p r o d u c t s were e i t h e r (A) f u r t h e r h y d r o x y l a t e d f o l l o w e d by r i n g c l e a v a g e , (B) f u r t h e r h y d r o x y l a t e d f o l l o w e d by t h e r e d u c t i o n o f the s i d e c h a i n t o one c a r b o n b e f o r e r i n g c l e a v -age, o r (C) t h e s i d e c h a i n was reduced t o one c a r b o n f o l l o w e d by f u r t h e r h y d r o x y l a t i o n t h e n r i n g c l e a v a g e (see Towers and Subba Rao, 1972). I n a n i m a l s , p h e n y l a l a n i n e was h y d r o x y l a t e d t o form t y r o s i n e which was degraded v i a p - h y d r o x y p h e n y l p y r u v i c 123. c i n n a m i c acid < ^ C H = C H - C O O H phenylhydracrylic <fjVcH-CH2-COOH acid N— ' O H ^ benzoylacetic acid or benzaldehyde < Q - C H O CH3COOH ^ C - C H 2 C 0 0 H Y benzoic acid ^ > - C O O H p-hydroxy- , — v benzoic acid H O - < ^ ) - C O O H p - c o u m a r i c H 0 ^ 3 " C H = C H _ C 0 0 H a c i d N — ' p - h y d r o x y - . phenylhydracrylic H O v J ^ H - C H 2 ~ C O O H a c i d w OH ° r H O - ^ > - C H O + [ch^COOH p-hydroxy- r " - S * p-hydroxy-benzoylacel ic H0-f7-C-CH^C00H benzaldehyde a c i d L >=/ 5 2 J F i g u r e 35. The d e g r a d a t i v e r o u t e of c i n n a m i c and p-coumaric a c i d s i n I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . The compounds I n b r a c k e t s were not d e t e c t e d . 124 . a c i d t o h o m o g e n t i s l c a c i d . No e v i d e n c e f o r t h e h y d r o x y l a t l o n of p h e n y l a l a n i n e t o form t y r o s i n e was found f o r I± g a l b a n a o r N. I n c e r t a . The o t h e r p o s s i b l e d e g r a d a t i v e pathways a r e t h r o u g h p h e n y l a l a n i n e and t y r o s i n e ammonia l y a s e s . These enzymes have n ot been found l n a l g a e , but t h e i r p r o d u c t s a r e degraded l n a s i m i l a r pathway t o t h a t r e p o r t e d l n h i g h e r p l a n t s (see E l l i s , 1 9 7 4 ) . I n F i g u r e 35 t h e proposed scheme f o r t h e i r d e g r a d a t i o n i s p r e s e n t e d and i n v o l v e s a 0 - o x i d a t l o n of t h e s i d e c h a i n . I n t h e d e g r a d a t i o n of c i n n a m i c a c i d , p h e n y l -h y d r a c r y l i c a c i d and b e n z o i c a c i d s were d e t e c t e d and i n t h e d e g r a d a t i o n of p-coumaric a c i d , p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d , p - h y d r o x y b e n z a l d e h y d e , and p - h y d r o x y b e n z o l c a c i d were d e t e c t e d . Whether a c e t a t e was removed from the h y d r a c r y l i c a c i d o r i t ' s o x i d i z e d p r o d u c t I s unknown. A c e t a t e was r e -p o r t e d t o be l o s t d i r e c t l y from p - h y d r o x y - 3 - m e t h o x y p h e n y l -h y d r a c r y l i c a c i d (Toms e t a l . , 1 9 7 0 ) . The d e g r a d a t i v e r o u t e s f o r p h e n y l a l a n i n e and t y r o -s i n e i n a l g a e do n o t r e s u l t i n any energy y i e l d i n g r e a c t i o n s . 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R e g u l a t i o n o f t h e c h o r i s m i c a c i d b r a n c h - p o i n t i n a r o m a t i c a c i d s y n t h e s i s i n b l u e -g r e e n and g r e e n a l g a e . A r c h . M i k r o b i o l . , 66: 2 5 0 - 2 5 8 . Weber, H. L., and A Beck. 1970, C h o r l s m a t e mutase from E u g l e n a g r a c i l i s . P u r i f i c a t i o n and r e g u l a t o r y p r o p e r t i e s . E u r . J . Biochem., 16: 244-251. W r i g h t , D., S. A. Brown, and A. C. N e i s h . 1958. S t u d i e s o f l i g n l n b i o s y n t h e s i s u s i n g i s o t o p i c c a r b o n VI. F o r m a t i o n o f t h e s i d e c h a i n o f the o h e n y l o r o p a n e monomer. Can. J , Biochem. P h y s i o l . , 36: 1037-1045. Young, M. R., G. H. N. Towers, and A. C. N e i s h , 1966 p Taxonomic d i s t r i b u t i o n o f ammonia-lyases f o r L- p h e n y l -a l a n i n e and L - t v r o s l n e i n r e l a t i o n t o l i g n i f i c a t i o n . Can.. J . B o t . , 44: 341-349. APPENDIX A. 134 ROUTINE STERILITY CHECKS : The t e c h n i q u e o f e n r i c h m e n t o f p o s s i b l e m i c r o b i a l c o n t a m i -n a n t s ( b a c t e r i a and molds) by h e t e r o t r o p h i c growth I n a s e a w a t e r medium e n r i c h e d w i t h o r g a n i c m a t e r i a l was used f o r c h e c k i n g a l l a l g a l c u l t u r e s f o r such c o n t a m i n a t i o n . The STP and ST^ media o f T a t e w a k l and P r o v a s o l i (1964) were u s e d f o r s u c h c o n t a m i n a t i o n c h e c k s . One t o two drops o f t h e a l g a l c u l t u r e growth medium were added t o a tube o f each medium and I n c u b a t e d about t h r e e o weeks I n t h e d a r k a t room t e m p e r a t u r e (22-25 C) f o r a c o n t a m i -n a t i o n check. The appearance i n e i t h e r medium o f c l o u d i n e s s and t u r b i d i t y t y p i c a l o f b a c t e r i a l growth o r o f d i s c o l o u r a t i o n and t u f t - f o r m a t i o n t y p i c a l o f mold growth was t a k e n as e v i d e n c e o f p o s s i b l e c o n t a m i n a t i o n . From a p o s i t i v e t u b e , 1-2 d r o p s were s u b c u l t u r e d i n t h e same medium, f o r c o n f i r m a t i o n , and 1-2 drops were examined m i c r o s c o p i c a l l y . I n t h e absence o f any v i s i b l e changes i n t h e s t e r i l i t y check t u b e , t h e a l g a l c u l t u r e t e s t e d was c o n s i d e r e d t o be f r e e f r o m c o n t a m i n a t i o n . 135. APPENDIX B. SPRAY REAGENTS The s p r a y r e a g e n t s employed i n t h e d e t e c t i o n o f v a r i o u s a r o m a t i c compounds s e p a r a t e d by paper and t h i n l a y e r chroma-t o g r a p h y are« (A) P h e n o l l c s . D i a z o t i z e d p - n i t r o a n i l i n e r e a g e n t ( I b r a h i m and Towers, I960): 5 ml o f 0,J%p-nitroaniline, 1 ml o f 5% sodium n i t r i t e and 15 ml o f 20% sodium a c e t a t e were com-b i n e d , i n t h a t o r d e r , j u s t p r i o r t o u s e . A f t e r s p r a y -i n g w i t h t h i s s o l u t i o n t h e chromatogram was a l l o w e d t o d r y b r i e f l y and t h e n o v e r s p r a y e d w i t h a 5% aqueous NaOH s o l u t i o n . P h e n o l i c compounds appear as v a r i o u s -l y c o l o u r e d s p o t s . F e r r i c c h l o r i d e r e a g e n t J 2% f e r r i c c h l o r i d e i n 95$ e t h a n o l i d e t e c t s o r -t h o d i h y d r o x y p h e n o l i c compounds as b r o w n i s h o r r e d d i s h s p o t s , (B) A l d e h y d e s . 2 , 4 - d l n i t r o p h e n y l h y d r a z i n e r e a g e n t (Dawson e t al.« 1969. p. 517)* 0ek% 2 , 4 - d i n i t r o p h e n y l h y d r a z i h e i n 2N HC1; de-t e c t s a l d e h y d e s and k e t o n e s as y e l l o w s p o t s which t u r n red-brown when o v e r s p r a y e d w i t h 10$ aqueous NaOH s o l u t i o n . Appendix B (cont. ) 1 3 6 . (C) A c i d s . M e t h y l red-bromothymol b l u e r e a g e n t (Handbook o f Chromatography. V o l . 2 , 1 9 7 ^ , p. 1 3 2 ) : 0 . 2 gm m e t h y l r e d and 0 . 2 gm bromothymol b l u e l n a m i x t u r e o f 1 0 0 ml formaldehyde and kOO ml o f 96% _ e t h a n o l and a d j u s t pH t o 5 . 2 w i t h 0 . 1 N NaOH; d e t e c t s o r g a n i c a c i d s as y e l l o w s p o t s on a p i n k background. Upon exposure t o NH^ vapour, s p o t s become r e d - o r a n g e on a g r e e n background. Chromatographic p o s i t i o n s o f v a r i o u s 0^-0^ a r o m a t i c compounds i n s o l v e n t system C s i s o p r o p a n o l : c o n c . NH^OH: water ( 8 i l»l, V/V/V). s o l v e n t f r o n t p - h y d r o x y b e n z y l a l c o h o l O 3 , 5 - d i b r o r a o - p - h y d r o x y b e n z y l a l c o h o l b e n z o i c a c i d O 2 , 4 - d i h y d r o x y b e n z o l c a c i d m-hydroxybenzoic a c i d O 0~ p - h y d r o x y b e n z o i o a c i d C O O 3 . 4 - d i h y d r o x y b e n z o l c a c i d 3 . 5 - d i b r o m o - p - h y d r o x y b e n z o l c a c i d + + • 3 , 5 - d i b r o m o - p - h y d r o x y b e n z o i c a c i d mono-p o t a s s i u m s u l f a t e o r i g i n e s t e r Appendix B (contd.) Chromatographic positions of various aromatic compounds. 137. 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 P r e p a r a t i o n o f 3.5-dlbromo-p-bydroxybenzolo a c i d . 1.10 gm p - h y d r o x y b e n z o i c a c i d was d i s s o l v e d In. 2.5 .ml g l a c i a l a c e t i c a c i d . To t h i s , 1 ml B r 2 i n 1 ml g l a c i a l a c e t i c a c i d was c a r e f u l l y added. The r e a c t i o n m i x t u r e was hea t e d f o r one hour, t h e n t h e excess B r ^ . HBr, and a c e t i c a c i d were .d r i v e n o f f under a s t r e a m o f n i t r o g e n . The w h i t e p r o d u c t was r e c r y s t a l i z e d from d i l u t e a c e t i c a c i d . The y i e l d was 2.24 gm, (94$ o f t h e t h e o r e t i c a l y i e l d ) and t h e m e l t i n g p o i n t was 266-268°C ( L i t . 268°C, L e u l i e r e t a l . 1927). NMR spectrum i n dg a c e t o n e was 5.50 (S, -OH) and 7.10 (S, 2H), and t h e 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 i s p r e s e n t e d i n F i g u r e 6. I t was found t h a t t h i s b r o m o p h e n o l i c compound was u n s t a b l e o v e r a l o n g p e r i o d o f t i m e and degraded t o g i v e t h e spectrum shown l n F i g u r e 6. P r e p a r a t i o n o f ?.5-dlbromo-p-hydroxybenzaldehyde. 1.00 gm o f p-hydroxybenzaldehyde was d i s s o l v e d i n 1.5 ml g l a c i a l a c e t i c a c i d and t o t h i s 1 ml B r 2 i n 1 ml g l a c i a l a c e t i c a c i d was c a r e f u l l y added. T h i s r e a c t i o n m i x t u r e was hea t e d f o r one hour, t h e n under a s t r e a m o f n i t r o g e n , t h e ex-c e s s B r 2 , HBr, and a c e t i c a c i d were removed. The w h i t e p r o -d u c t was r e c r y s t a l i z e d from an a c e t i c a c i d w ater m i x t u r e and t h e f i n a l y i e l d was 2.21 gm, 96$ o f t h e t h e o r e t i c a l y i e l d and t h e m e l t i n g p o i n t was 181-183° C. The NMR ;spectrum i n dg Appendix C (oont.) 139. 140. Appendix C (cont.) acetone was 3.55 (S, -OH), 8 . 0 6 (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 i n 1 ml g l a c i a l a c e t i c a c i d . 0.5 ml B r 2 i n 0.5 ml g l a c i a l a c e t i c a c i d was added, and the reaction mixture heated f o r one hour. Under a stream of nitrogen, the excess B r 2 , HBr, and a c e t i c acid were removed, leaving a white c r y s t a l i n e product which was a tribromo substituted compound (see Auwers et a l . , 1899). This was dissolved Inacetohe arid water was added uht 11 a .faint t u r b i d i t y appeared which was redlssolved by adding more ace-tone. After 5-6 hours at room temperature, water was added and the p r e c i p i t a t e f i l t e r e d o f f . Following r e c r y s t a l l l z a t i o n from d i l u t e a c e t i c acid-, 0.023 gm of product (20$ y i e l d ) was obtained which had a melting point of 113-114° C ( L i t . ll6-H7°C). Method ( 2 ) Involved reduction of 3»5-dibromo-p-hydroxybenzaldehyde with l i t h i u m aluminum hydride. Some pro-duct was obtained but i n very poor y i e l d . Preparation of 3-bromo-p-hydroxybenzolc acid (Leuller et a l . , 1972). 3.5 S m p-hydroxybenzolc ac i d was added to 80 ml H202 so l u t i o n (ll.3-.ml-of 30$ H 20 2 d i l u t e d to 100 ml - 3.4 gm H 20 2/ 100 ml). To t h i s 4.15 ml HBr (48$ HBr i n HOAc) was added and A p p e n d i x C f c o n t . ) l 4 l . l e f t 24 h o u r s . The p r o d u c t was c o l l e c t e d by f i l t r a t i o n , and r e c r y s t a l l i z e d from b o i l i n g w a t e r . A y i e l d o f 4..91 gm o f a m i x t u r e was o b t a i n e d . A p p r o x i m a t e l y 60% was t h e monobromo,, 35% t h e 3,5-dlbromo, and 5% a t r l b r o m o d e r i v a t i v e ( p r o b a b l y 3,5-dlbromo-p-hydroxybenzylbromlde). The major MS peaks f o r t h e monobromo were m/e = 218 (83), 216 (89). 201 (95), and 199 (100). P r e p a r a t i o n o f p - h y d r o x y p h e n y l a c e t a l d e h y d e ( G r e e n s t e l n  e t a l . , I96D. To 100 mg t y r o s i n e d i s s o l v e d i n w a t e r , 200 mg n i n h y d r i n was added. T h i s was b o i l e d f o r 10 m i n u t e s , c o o l e d , and ex-t r a c t e d w i t h d l e t h y l e t h e r . The e t h e r e x t r a c t was e v a p o r a t e d t o d r y n e s s and p - h y d r o x y p h e n y l a c e t a l d e h y d e was p u r i f i e d by s u b l i m a t i o n a t 150* C I n vacuo; f o l l o w e d by chromatography i n s o l v e n t system A. The R f v a l u e i n s o l v e n t s y s t e m A and B a r e t S o l v e n t s y s t e m A S o l v e n t system B p - h y d r o x y p h e n y l a c e t a l d e h y d e 0.74 0.15 p - h y d r o x y p h e n y l a c e t i c a c i d 0.67 0.25 p - h y d r o x y b e n z a l d e h y d e 0.64 0.39 p - H y d r o x y p h e n y l a c e t a l d e h y d e when s p r a y e d w i t h d i a z o t i z e d p-n l t r o a n i l i n e produces a y e l l o w - r e d c o l o u r and when s p r a y e d w i t h 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e a y e l l o w c o l o u r . I n F i g u r e 8 t h e UV-spectrum i s p r e s e n t e d . Very l i t t l e p r o d u c t was ob-t a i n e d , t h u s f u r t h e r c h a r a c t e r i z a t i o n was i m p o s s i b l e . -The method o f L a n g h e l d (1907) u s i n g sodium h y p o c h l o r i t e t o s y n t h e s i z e p - h y d r o x y p h e n y l a c e t a l d e h y d e was t r i e d but a g a i n v e r y l i t t l e p r o d u c t was o b t a i n e d . P r e p a r a t i o n o f p - h y d r o x y b e n z o y l f o r m l c a c i d . The method d e s c r i b e d f o r t h e o x i d a t i o n o f m a n d e l i c 143. Appendix C ( c o n t . ) a c i d t o b e n z o y l f o r m i c a c i d was t r i e d ( O r g a n i c S y n t h e s i s , V o l . I., p , 2 4 l ) but t h e o n l y p r o d u c t r e c o v e r e d was p-hydroxyben-a l d e h y d e . I n p l a c e o f KMnO^ as t h e o x i d i z i n g r e a g e n t , a l -uminium i s o p r o p o x l d e was p r e p a r e d ( V o g e l , 19&7) and u s e d . 50 mg DL-p-Hydroxymandelic a c i d was d i s s o l v e d i n anhydrous a c e t o n e and 20 ml a l u m i n i u m i s o p r o p o x l d e was added. T h i s was a l l o w e d t o r e a c t f o r one hour, t h e n the r e a c t i o n was f i l -t e r e d and e v a p o r a t e d t o d r y n e s s . The r e s i d u e was d i s s o l v e d i n d i l u t e HC1 and e x t r a c t e d i n t o e t h e r . A v e r y poor y i e l d was o b t a i n e d . I n F i g u r e 9 t h e UV-spectrum o f t h e p r o d u c t i s p r e s e n t e d . I t was found t h a t t h i s p r o d u c t r a p i d l y d e c a r b o x y -l a t e s t o g i v e p - h y d r o x y b e n z a l d e h y d e , (see F i g u r e 9 ) . T h i s was a l s o c o n f i r m e d by chromatography. No o t h e r d a t a p e r t a i n -i n g t o t h i s compound was o b t a i n e d , but 'because o f i t s i n -s t a b i l i t y i t i s not l i k e l y t o be p r e s e n t i n e x t r a c t s from any m e t a b o l i c s t u d i e s . P r e p a r a t i o n o f p h e n y l h y d r a c r y l i c a c i d . -T h i s was p r e p a r e d u s i n g the method of Wrig h t e t a l . , 1958. 1 . 6 5 gm c i n n a m i c a c i d was d i s s o l v e d l n 3 ml of g l a c i a l a c e t i c a c i d c o n t a i n i n g 30$ HBr. A f t e r r e a c t i n g f o r t h r e e days t h e a c e t i c a c i d and HBr was removed under a st r e a m o f n i t r o g e n . The r e s i d u e was d i s s o l v e d and r e c r y s t a l i z e d from benzene. The c r u d e s o l i d was b o i l e d i n 8 ml water f o r 15 min, c o o l e d , t h e n e x t r a c t e d i n t o e t h e r . The e t h e r was e v a p o r a t e d and t h e r e -s i d u e was r e c r y s t a l i z e d t w i c e from c h l o r o f o r m t o g i v e a y i e l d o f 0 .29 gm o r 40$ of t h e t h e o r e c t l c a l y i e l d . The m e l t i n g p o i n t was 92-94°C ( L i t . 92-93°C) and the UV-spectrum I s p r e s e n t e d i n F i g u r e 10. The MS was found t o be m/e 166 (37), 148 ( 1 0 . 8 ) , 147 ( 1 2 1 ) , 107 (10 0 ) , 106 (325) , 105 (295) , 79 (61..5), 77 ( 5 0 ) , Appendix C ( c o n t . ) 144. F i g u r e 39. Absorption spectrum of p-hydroxybenzoylformic a c i d and I t s spontaneous degradation product - p-hydroxybenzaldehyde. Both spe c t r a are l n ethanol. p-hydroxybenzoylformic a c i d — p-hydroxybenzaldehyde OPTICAL DENSITY 250 275 300 WAVELENGTH (nm) 16 F i g u r e 4,0. Absorption spectrum of synthetic phenylhydracryllc a c i d l n ethanol. 1.2 I OPTICAL DENSITY Ci8| 0.0 r 200 225 250 275 300 WAVELENGTH (nm) 325 350 Appendix C ( c o n t . ) . — Is-5 • and 71 ( 2 3 . 5 ) , w h i l e t h e NMR l n dg a c e t o n e was 2 . 6 8 (d, 2H - C H 2 ~ , J = 7 H z ) , 5.11 ( t , 1H, -CH-, J = 7Hz), 6 .25 (m, I H , H of -OH and -COOH), and 7 .30 (m, 5H, a r o m a t i c ) . P r e p a r a t i o n of p - h y d r o x y p h e n y l h y d r a c r y l l c a c i d . The method used t o s y n t h e s i z e p h e n y l h y d r a c r y l i c a c i d was t r i e d and f o u n d t o be u n s u c c e s s f u l . The. same method was used w i t h p - a c e t o x y c o u m a r l c a c i d r e p l a c i n g p-coumaric a c i d . Some p r o d u c t was o b t a i n e d but h y d r o l y s i s o f the e s t e r t o t h e f r e e a c i d was u n s u c c e s s f u l because the p h e n o l i c hydrogen l n t h e p r e s e n c e of base forms a quinone w h i c h causes d e h y d r a t i o n back t o p-coumaric a c i d . A s i m i l a r problem was e n c o u n t e r e d l n t h e a t t e m p t e d s y n t h e s i s o f 4-hydroxy - 3-methoxyphenylhydra-c r y l i c a c i d (Toms e_t a l . , 1 9 7 0 ) . They b e l i e v e t h a t enzymatic s y n t h e s i s i s p r o b a b l y t h e o n l y way t o make t h e i r p r o d u c t and t h e same i s l i k e l y t r u e o f p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d . P r e p a r a t i o n o f s u l f a t e e s t e r s of p h e n o l i c compounds. The method o u t l i n e d by Hodgkin e t a l . , (1966) was used t o p r e p a r e the s u l f a t e e s t e r s o f p - h y d r o x y b e n z o i c a c i d , 3 i 5 -d i b r o m o - p - h y d r o x y b e n z o i c a c i d , 31 5-dibromo-p-hydroxybenzal-dehyde and p-hydroxybenzaldehyde. 1.2 umoles of t h e p h e n o l was d i s s o l v e d l n 10 ml water c o n t a i n i n g 0 . 5 gm o f p o t a s s i u m c a r -b o n a t e , 0.24 gm s u l f u r t r i o x i d e - p y r i d i n e complex ( A l d r i c h C h e m i c a l Co., S t . L o u i s , M i s s o u r i ) was s l o w l y added w i t h s t l r -r i n g . The s o l u t i o n s were c o o l e d t o 0 C f o r 24 hours and t h e r e s u l t i n g c r y s t a l s which were c o l l e c t e d were r e c r y s t a l i z e d f r o m water.. The y i e l d s were a p p r o x i m a t e l y 60% f o r b o t h a c i d s b u t f o r t h e a l d e h y d e s the y i e l d s were reduced t o 40$. T h i s was due t o d e c o m p o s i t i o n o r s i d e r e a c t i o n s which r e s u l t e d i n an I n i t i a l brown t o b l a c k c o l o u r e d p r o d u c t f o r b o t h a l d e h y d e s . APPENDIX D. 146. CHEMICAL PREPARATIONS OF RADIOACTIVE COMPOUNDS P r e p a r a t i o n o f L - t y r o s l n e - u n i f o r m l y r i n g - ^ C . The p r o c e d u r e used was based on t h a t o f E l l i s e t a l . (1973). One l i t e r of medium (0.2$ L - t y r o s i n e , 0,2$ KH 2P0^, 0.1$ MgS02^7H 20, 0.0001$ FeSO^«7H 20, 0.01$ p y r i d o x i n e , 0.6$ g l y c e r o l , 0.5$ s u c c i n i c a c i d , 0.1$ DL- m e t h l o n l n e , 0.2$ DL-a l a n i n e , 0.05$ g l y c i n e , 0.1$ L - p h e n y l a l a n i n e and 0.5$ h y d r o - . l y z e d soybean p r o t e i n i n d i s t i l l e d water w i t h pH a d j u s t e d t o 7.3) i n a 2,8 l i t e r E r l e n m y e r f l a s k was c o o l e d a f t e r a u t o -c l a v i n g and i n o c u l a t e d f rom a f r e s h s l a n t c u l t u r e o f E r w l n l a h e r b l c o l a (ATCC 21434). The f l a s k was t h e n p l a c e d on a r e -o c l p r o c a t l n g s h a k e r a t 30C f o r 14-16 h o u r s . The c e l l s were h a r v e s t e d , washed, and t h e n resuspended l n 5 ml b u f f e r 0.1 M KH 2P0ij, pH 6.0 ( w i t h 5 n>M m e r c a p t e o t h a n o l ) p e r gram o f c e l l s . The c e l l s were d i s r u p t e d by s o n i f i c a t i o n f o r 5 m i n u t e s , c e n -t r i f u g e d and the s u p e r n a t a n t was f r a c t i o n a t e d w i t h ammonium s u l f a t e . The p r o t e i n p r e c i p i t a t i n g between 30$ and 70$ s a t u r a t i o n was d i s s o l v e d l n t h e above b u f f e r , t h e n d i a l y z e d o v e r n i g h t a g a i n s t t h e same b u f f e r . 2 ml p o r t i o n s of t h i s o enzyme p r e p a r a t i o n were f r o z e n and s t o r e d a t -20 C u n t i l r e -q u i r e d . The a c t i v i t y o f t h e enzyme was measured by i n c u b -a t i o n o f 0.1 ml enzyme f o r 30 minutes a t 30°C w i t h 5 umoles S - m e t h y l - L - c y s t e l n e (SMC), 0.5 umoles p y r i d o x a l phosphate and 200 umoles o f pH 7.8 KH 2P0ij. b u f f e r i n a t o t a l volume o f 1 m l . Appendix D ( c o n t . ) L Z * 7 * A f t e r m e a s u r i n g t h e f o r m a t i o n o f p y r u v a t e (Friedemann, 196?), one u n i t (U) o f a c t i v i t y c o r r e s p o n d e d t o th e f o r m a t i o n o f 1 umole o f p y r u v a t e / m i n u t e . F o r t h e s y n t h e s i s o f L - t y r o s i n e - r i n g - ^ C , p h e n o l - U - ^ C (New England N u c l e a r ) w i t h a s p e c i f i c a c t i v i t y o f 9.3 mCi/mM was us e d . 26 u C i were p l a c e d i n a tube and the e t h e r was r e -moved under a s t r e a m of n i t r o g e n . To t h i s tube 4 0 0 ;umoles SMC, 1,5 jumoles p y r i d o x a l phosphate and 8 0 0 mmoles o f pH 7.8 KR^POi), b u f f e r was added. A f t e r a d d i t i o n o f 160 roU o f t h e en-zyme p r e p a r a t i o n , t h e r e a c t i o n ( t o t a l volume 4.0 ml) was i n -c u b a t e d a t 30°C f o r t h r e e h o u r s . The r e a c t i o n m i x t u r e was s p o t t e d onto s e v e r a l s h e e t s o f Whatman 3MM chromatography paper and d e v e l o p e d i n s o l v e n t s y s t e m C. The t y r o s i n e - ^ C was l o c a t e d by a u t o r a d i o g r a p h y , and e l u t e d w i t h e t h a n o l . The e l u a t e was s p o t t e d on A v i c e l p l a t e s and chromatographed f u r t h e r i n p y r i d i n e : i s o a m y l a l c o h o l s g l a c i a l a c e t i c a c i d : w a t e r (8:4:1:1, V/V/V/V). The y i e l d o f L - t y r o s i n e - u n i f o r m l y r i n g - ^ C was 20 u C i o r 76% o f t h e o r e t i c a l v a l u e . P r e p a r a t i o n o f p - h y d r o x y p h e n y l a c e t l c a c l d - l - ^ C and -2-^C. The method i s based on t h a t o f K r i s t e n s e n (1973). 6 ml d l a l y z e d enzyme (3 ml L-amino a c i d o x i d a s e #12993 f r o m C a l -biochem c o n t a i n i n g 2 mg/ml p r o t e i n and 3 ml D-amlno a c i d o x i -dase #129852 from C a l b i o c h e m c o n t a i n i n g 0.5 mg/ml p r o t e i n ) i n T r i s - H C l b u f f e r (0 .4 M, pH 7.8) w i t h 12 ;uCl D L - t y r o s i n e ^ - ^ C and 0.1 mg of c o l d D L - t y r o s i n e i n 1 ml b u f f e r were i n c u b a t e d Appendix D ( c o n t . ) a t 30° C f o r 24 h o u r s . I n a s i m i l a r r e a c t i o n 12 u C i o f DL-14 t y r o s i n e - 3 — C was used t o p r e p a r e p - h y d r o x y p h e n y l a c e t i c 14 a c i d - 2 - C. B o t h r e a c t i o n s were stopped by a d d i n g cone. HC1, c e n -t r i f u g i n g , and e x t r a c t i n g t h r e e t i m e s w i t h ether.. The ex-t r a c t s were washed w i t h I N HC1, e v a p o r a t e d t o d r y n e s s , r e d l s -s o l v e d i n e t h e r , and chromatographed on A v l c e l p l a t e s l n s o l -v e n t system A. Each p r o d u c t was l o c a t e d by a u t o r a d i o g r a p h y and e l u t e d w i t h e t h a n o l . The y i e l d o f p - h y d r o x y p h e n y l a c e t i c a c l d - l - ^ C ( s p e c i f i c a c t i v i t y , 16.9 uCi/mmole) was 1.46 u C i and f o r p - h y d r o x y p h e n y l a c e t l c a c l d - 1 - C ( s p e c i f i c a c t i v i t y , 15.5 uCi/mmole) i t was 0.33 uCi.. U n l i k e t h e r e s u l t s i n 14 K r i s t e n s e n ' s p a p e r, o t h e r C - p r o d u c t s were o b s e r v e d i n t h e a u t o r a d i o g r a p h s . 14 P r e p a r a t i o n o f p-coumarlc a c i d - 2 - C„ T h i s p r e p a r a t i o n i s based on t h a t o f A u s t i n and Meyers (1965). I n t o 3 ml p y r i d i n e c o n t a i n i n g 3 d r o p s p i p e r -d i n e , 110 mg p-h y d r o x y b e n z a l d e h y d e , 100 mg m a l o n i c a c i d , and 14 , 100 u C i m a l o n i c a c i d - 2 - C (ICN C h e m i c a l and R a d i o i s o t o p e d i v i s i o n , 15 uCi/mmole) were d i s s o l v e d . The r e a c t i o n was he a t e d 2 hours on a steam b a t h , c o o l e d , a c i d i f i e d w i t h cone. HC1, made up t o 50 ml w i t h w a t e r and t h e n e x t r a c t e d w i t h e t h e r . The e t h e r was e x t r a c t e d w i t h 5% NaHCO^, whi c h was a c i d i f i e d and e x t r a c t e d w i t h e t h e r . T h i s e x t r a c t was p u r i f i e d by chroma-t o g r a p h y l n s o l v e n t system B y i e l d i n g 70 u C i p-coumaric a c i d -. 14_ APPENDIX E. THE EFFECT AND METABOLISM OF OTHER AROMATIC COMPOUNDS I n F i g u r e s 4 l t o 54 a r e p r e s e n t e d the e f f e c t o f v a r i o u s a r o m a t i c compounds on t h e growth c o n s t a n t and l a g p e r i o d o f I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . The a r o m a t i c com-pounds s t u d i e d were: p h e n y l a c e t i c a c i d F i g u r e 4 l Page 150 p - h y d r o x y p h e n y l a c e t i c a c i d 42 150 m a n d e l i c a c i d 43 151 p - h y d r o x y m a n d e l i c a c i d 44 151 b e n z o i c a c i d ^5 152 p - h y d r o x y b e n z o l c a c i d 4 6 152 p-hydroxybenzaldehyde 47 153 3,5-dibromo-p-hydroxybenzoic a c i d 4 8 153 3,5-dibromo-p-hydroxybenzaldehyde 49 154 c i n n a m i c a c i d 50 ' 154 p-coumaric a c i d ( b r i e f n o t e on p.: 155) 51 156 m-hydroxybenzoic a c i d 53 158 o - h y d r o x y b e n z o i c a c i d • 5^ 158 a % INHIBITON ol growth constant GROWTH CONSTANT (AOD/day) LAG PERIOD (days) Navicula incerta (x—x) 9 a 3 1 CL a s j q a 3-» •s < a o O o o c 3 f a B r t r t P 3" H" 3 CB 3 c r o t» CC P ' 3 r t 0, a B o • M c t P (K o •o CD •a 1 a-H* a o 3 p . O B >-» O a M r f 01 0 O o 3" 0 o H> ca P. ca o 3 » c t 3-a 1 a » 3 P o o IVJ — • — . O U l O o o g o O cn o cn o cn O O O O O O L P L n en o ro P o p o ro —• o cn Isochrysis galbana (• •i) t* <n c a> -p-r o • p> o a 0 3 r » >-> 0 rf . c t cn 0 S i 3 CO 0 0 s in 0 i o 3 o -l> P. c t r t S3 3" 0 CO <) O i-» 0 CO O 3 "> c ca "1 t-J -t CO 0 0 o 3 ct h* r t ca 3 O 0 o (0 3 "> •1 p . c t • o 0 1 0 3 CN VS p . d •1 CD o M v ; o •o p . CO o 3 t * vs 0 ca o o CO o c t 3 h~ •1 o < ca 0 t* o ca t-* P-VT O w ^ S. o £-m —I un • — I c. o >_ X , Vo INHI3IT0N of growth constant cn O GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) o o o o o o o o o o o o c n c n O L P O cn o cn p cn o Isochrysis galbana (• ) O p O O O p O o p o o o P, ° —* —* —. _ i ro ro ro cn oo co o * N J LAG PERIOD (days) o p — I I 1— o p - I 1 1 1— w e a V.. INHIBITON of growth constant GROWTH CONSTANT • • (AOD/day) LAG PERIOD (days) Navicula incerta (x—x) V <K a 3 n a. o EO <R » . T 1 P < S H- O O 0 o c 3 -<> u (D c t c t P H- 3 a> 3 c t O CD P 3 • c t p . CD P o H c t P u CR o xt ~> (9 :o t-1 O i p . 1! o 3 "1 p . a (H H CO • * Q o O 3" 0 •1 o •< H-CO (J-to o 3 n p c t CC 3 P NJ In o 0 1 i—i cn 2 o, CO CD 9 i ro ro — —» t cn o cjn o cn o un i i i i f I I a o o to o O o b s o o b b L n cn ro ro _* + Ul o cn O cn o Ln Isochrysis galbana (• ) o o b p b co b cn !•» in c 1 CO • e -*• • B 0 P 3 H io c t IH » J T 3 CD P B g (rt SO >~i o 3 O p . s. c t c t •3 3" so CD <» o o CD o 3 ~> 01 <-> c t CD SO SO O 3 c t c t 01 3 o SO o CO 3 •1 p . c t a SO H • P I TO • a •o rr (0 *<: 1 a •* •i o o p . X << o B "> P M 3 P. 01 CD o H o t* •3 O •1 P 01 o 01. P. en O o. O ^ Z o, O J > m 5 N 3o£ 5^ i—c cn 2 s, — ' O J r o fc> V. INHIBITON of growth constant • CO CO cn o ro ro _. cn o cn a GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) o o o b o b o b LAG PERIOD (days) cn — I — cn — l — Isochrysis galbana ( — --•) O O N I O I L I ^ W M - . O O O O O O O O O - i — i 1 — i 1 r-o o o p o p a p b b b b b o b b o o o ro ai oo o ro QD b C! o | § i f 3 2 V . INHIBITON of growth constant GROWTH CONSTANT (AOD/day) LAG PERIOD (days) Navicula incerta (x—x) r r 1 IP *-»• 3 o CO f «* to P '3 CK V-1 to n r x ) (0 • l - t* o p . . o . p cn • i . p s o + + • IS) o o + + C J o a o o P o o o c n p CD O O O CD CD o tn o o -r P o o - r -rsj °P Isochrysis galbana (• ) - P : 3 - P . cn . c t» CO - -p-O N - • p (R . a 3 - 1 - 1 * p . o P 55' E M c t >-> P P -4 a t* O o o o c - 3 '-•) - H* . CQ P - c t c t - p 3 CO 3 c t o • CO CO • P •1 r 3 ••"> c t - p . CD . p • O - f J c t • P - 01 cn O •a Vo INHIBITON of growth constant o p . o o p t-1 cn p in —I 1 1— o —r— GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) p p p p p p p p o -r— T o T-Isochrysis galbana (•— LAG PERIOD (days) o o o »—* CC ~ UJ <n °-< 8 ^ a i o tr o ° c o I— c gs o 4.0 3.0 2.0 0064 0.063 0.062 a061 0.060 0.059 0.058 0.057 0.056 OD55 0.05i, 0.053 0.052 +10 + 5 0 5 10 15 20 2.5x10s 5x105 10"4 2.5x1Cr" 5xl0/) 10~3 2Dx1(? CONCENTRATION (M) Figure l*7. Diagram of the e f f e c t s of p-hydroxybenzaldehyde on the growth constant and Jag period of Isochrysls galbana and Kavlcula l n c e r t a . 3.5 3.0 2.0 0.055 0.050 0.045 0.040 .0.035 0030 0.025 0.020 0.0 rX X— 3.5 3.0 0.018 / control . / / / / * — - — —x X •— _x ^ X control 0 0 10 10 20 20 30 30 40 40 50 50 100 - ' 10.0 2.5x10s 5x105 \6U 2.5x:6Z' 5x10'' 1fJ3 2J0X1O3 CONCENTRATION (M) Figure k8. Diagram of the e f f e c t s of 3,5-dlbromo-p-hydroxybenzolc ac i d on the growth constant and l a g period of Isochrysls  galbana and Kavlcula l n c e r t a . • S*' *0 •a V. INHIBITON of growth constant GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) LAG PERIOD (days) X 3- a » V4 h* r-l a P rt a CD So •1 B 3 P a 03 ct o 3 3" -» a a ct •z w P i CO <4 O CD o t "1 c •» H CD to •> O 3 ct •» 3 CO 3 CO o rt O a SO 3 ct cf P • P 3 1 P- a M o* P m o 5 rj o CD 1 •j TJ h- 1 O • •3 P. 'A a o •j !-•> o •A H «S CO cf o CD o 3 3" N •J P •< M CO a H- CD CO 1 co on CD O * ~ O J ro _> o o o o o o o o g o CD o b o b o b § on o —r-oo b ro "on x, O o zz o m zz\ 01 i—i on O & • o , ' oo ro 'a x, 9o Isochrysis galbana ( ) c n - F ~ o J r v j _ . o co o ° P p p p o o o o o o b o b b b o o g - t V — i 1 1 1 r-t* oq c •» CD On o • S3 CJ a P 0 y 3 P CO 0) O ct 1 p P H 3 B P ct O p 3 3 O a ct CD 3 1 H CD ct P P W CD XI CD CD •1 O I—" ct o 01 a o o "1 o H l-» CO 3 o 3 o p p- B r» '< o CO H- p 0) o t* )<1 a P H o if 3 3 ct P he an m a ro on on o, 3T ro i—i cn O £-Z -—\ 2 3, — oo t o b So V. INHIBITON of growth constant —• -» ro o o o o GROWTH CONSTANT (AOD/day) Navicula incerta (x—x) i I r + + + —* —• ro oo o o o o o p o O o o s b b b b on 071 cn on O cn o on Isochrysis galbana ( ) o b o b 1 — r o o p o p o o b b b b o b b ro ro ro ro ro CO O J cn cn ^o oo CO o —' LAG PERIOD (days) 6 ro on -J— cn 1 i Y I » i i i / i i ' i o —r-155. p-Coumarlc a c i d Chromatography o f the e t h e r e x t r a c t s of the medium from t h e OD tubes and t h e one l i t e r c u l t u r e s r e v e a l e d t h e c i s - and t r a n s - p - c o u m a r i c a c i d s , p - h y d r o x y b e n z a l d e h y d e , p - h y d r o x y b e n z o i c a c i d , and an u n i d e n t i f i e d spot which r e -a c t e d w i t h PNA t o g i v e a p u r p l e c o l o u r . T h i s compound was t e n t a t i v e l y i d e n t i f i e d as p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d and chromatographed d i r e c t l y below t h e c i s - p - c o u m a r i c a c i d (see Appendix <©). When t h i s compound was i s o l a t e d and a g a i n chromatographed, i t was ob s e r v e d t h a t a p o r t i o n chromatographed as b e f o r e , but a p o r t i o n remained a t t h e o r i g i n ( p u r p l e c o l o u r w i t h PNA), I n F i g u r e 52 t h e a b s o r p -t i o n spectrum o f p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d from b o t h c h r o m a t o g r a p h i c l o c a t i o n s i s p r e s e n t e d . B o t h s p e c t r a were i d e n t i c a l , but why a p o r t i o n remained a t t h e o r i g i n was unknown. When p - h y d r o x y p h e n y l l a c t i c a c i d and p-hydroxy-p h e n y l h y d r a c r y l i c a c i d were chromatographed i n s o l v e n t system A two d i s t i n c t s p o t s ( b o t h p u r p l e w i t h PNA) were o b t a i n e d . The ' l a c t i c a c i d ' chromatographed i n f r o n t o f th e ' h y d r a c r y l i c a c i d ' (see Appendix B ) , I n F i g u r e 52 t h e s pectrum of p - h y d r o x y p h e n y l l a c t i c a c i d i s p r e s e n t e d f o r c o m p a r a t i v e p u r p o s e s . No comparison of the p-hydroxy-p h e n y l h y d r a c r y l i c a c i d t o a s y n t h e t i c sample was p o s s i b l e because a l l . a t t e m p t s a t s y n t h e s i s were u n s u c c e s s f u l (see Appendix B ) . Q O i—• LU <{> < LO ~ y >-8 ^ Q X O cr co ° c 2 rt Q i— c - o O IS ° CT) 2.0 1.5 0.067 T 0.066 0.065 Oj (_) 0.064 C _ r t 0.063 o *> 0.062 ^* rt 0.061 +10 + 5 0 3.8 3.6 3A 3.2 0.028 0.027 _ 0.026 j 0.025 0.0 2U 0.023 0.022 0.021 0.020 0.019 0.018 +.10 0 10 20 30 rt c rt _Q rt o i l i/) >~ x : u o ^-control / - * — - — # K / control Figure 2.5X105 5x105 10 4 2.5x10^ 5x10^  10 3 2.0x103 - CONCENTRATION (M) 5 1 . Diagram of the effects of p-counarlc acid on the growth constant and lag.period of Isochrysls galbana and Navlcula lncerta. OPTICAL DENSITY F i g u r e 52. A b s o r p t i o n s p e c t r u m i n e t h a n o l o f t h e p h e n o l i c a c i d t e n t a t i v e l y i d e n t i f i e d as p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d f r om I s o c h r y s l s g a l b a n a and N a v l c u l a l n c e r t a . p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d (Rf=0.6) p - h y d r o x y p h e n y l h y d r a c r y l i c a c i d ( f r o m or i g i n ) , ! p - h y d r o x y p h e n y l l a c t i c a c i d 250 275 WAVELENGTH (nm) o o U in 0 2 LO • : o or o ° c 2 ro O ^  S S ~ $ . . . O 5.0 4.5 4.0 0.072 0.070 0.068 0.065 0.06/, 0.062 0060 O058 + 5 0 5 10 15 20 4.0 3.0 2.0 0.032 „ 0.030 i 0028 ro c ro JQ a, ai 0.026 0.024 0.022 0.020 0X118 0 10 20 30 40 control 2.5x16s 5x105 10* 2.5x10'' 5x10^  10~3 2.0X103 CONCENTRATION (M) Figure 53. Diagram of the e f f e c t s of m-hydroxybenzoic a c i d on the growth constant and l a g period of Iso c h r y s l s galbana and Kavlcula l n c e r t a . 2.5x10s 5x105 IO4 2.5x10* 5x10* 10~3 2J0X1CTB CONCENTRATION (M) Figure $k. Diagram of the e f f e c t s of o-hydroxybenzoic a c i d on the growth constant and l a g period of Iso c h r y s l s galbana' H and Navlcula l n c e r t a . v_n CO • 159. ; ADDENDUM Page 8. Table 1. Bac1liar1ophyta Phaeodactylum trlcornutum D-phenylalanine 2 mM Growth 88$ 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 activ i t y 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 a l . , 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, l n 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 a l . , 1956f Chantraine et a l . . 1973) and 2-hydroxy-3(3',5'-dibromo-4*-hydroxyphenyl)-acrylic acid l6o. ( C h a n t r a i n e e t a i . , 1973) have a l s o been i s o l a t e d . A l s o i n T a b l e 5» u n d e r Rhodophyta B r o n g n l a r t e l l a b y s s o i d e s #7,l6 ( F r i e s , 1973) Rhodomela l a r l x #16 ( W e i n s t e i n e t a l . . 1976) Page 103. I n t e r n a l and e x t e r n a l amino a c i d c o n c e n t r a t i o n s f o r t h e u p t a k e o f L - p h e n y l a l a n l n e and L - t y r o s l n e . Amino a c i d E x t e r n a l I n t e r n a l p o o l cone. (mM)^ - and c e l l s p o o l cone. I . g a l b a n a N. i n c e r t a u t i l i z e d (mM) Phe non-adap c e l l s Phe pre-adap c e l l s T y r non-adap c e l l s T y r pre-adap c e l l s (1) a f t e r one h o u r ' s i n c u b a t i o n . (2) L t = l i g h t t Dk = d a r k j and adapt - a d a p t e d . The above i n t e r n a l c o n c e n t r a t i o n s c o m p r i s e t h e s o l u b l e and i n s o l u b l e p o o l s . I n each c a s e t h e i n t e r n a l c e l l u l a r c o n c e -n t r a t i o n s o f amino a c i d was g r e a t e r t h a n t h e e x t e r n a l c o n c -e n t r a t i o n . T h i s s u g g e s t e d b o t h s p e c i e s c a n a s s i m i l a t e t h e s e amino a c i d s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . The d e g r a d a t -i v e r a t e s f o r b o t h L - p h e n y l a l a n i n e and L - t y r o s i n e f o r b o t h s p e c i e s was l e s s . t h a n t h e i r u p t a k e r a t e s . 2 L t 0.1 0.7 59. Dk 0.1 0.5 49. L t 0.01 0.2 18. Dk 0.01 0.1 14. L t 0.1 1.1 54. Dk 0.1 0.5 48. L t 0.01 0.03 8. Dk 0.01 0.02 3. L t 0.1 1.0 183. Dk 0.1 0.6 161. L t . 0.01 0.1 38. Dk 0.01 . 0.04 36. L t 0.1 1.2 73. Dk 0.1 0.3 43. L t 0.01 0.05 3. Dk 0.01 0.04 3. 161. Page 114. D e c a r b o x y l a t i o n o f p - h y d r o x y b e n z o l c a c i d may be accom-p l i s h e d by an o x i d a t i v e o r a n o n - o x i d a t i v e mechanism. I n the schemes ( F i g u r e s 33, and 34), t h e p r o d u c t i o n o f 1 , 4 -d l h y d r o x y b e n z e n e would be o b t a i n e d as a r e s u l t o f o x i d a t i v e d e c a r b o x y l a t i o n w h i l e p h e n o l would be produced by n o n - o x i d a -t i v e d e c a r b o x y l a t i o n . No e v i d e n c e was o b t a i n e d as t o w h i c h r o u t e was used by b o t h s p e c i e s . Page H 5 t L i n e 5« " C e l l s o f b o t h s p e c i e s produce a brown e t h e r -i n s o l u b l e compound". T h i s may a l s o be a r e s u l t o f p e r o x i d a -ses a c t i n g on s i m p l e p h e n o l s t o g i v e p o l y m e r i c compounds. Page 124, L i n e 5« The p r e s e n c e o f p h e n y l a l a n i n e ammonia l y a s e was d e t e c t e d i n D u n a l l e l l a marina ( L o f f e l h a r t e t a l . . 1973) and t h e p r e s e n c e of l u n u l a r i c a c i d i n algae, i n c l u d i n g a N a v l c u l a sp. ( P r y c e , 1972) s u g g e s t s t h i s enzyme may be w i d e l y d i s t r i b u t e d i n a l g a e . The b i o s y n t h e s i s o f l u n u l a r i c a c i d i s t h ought t o be t h r o u g h a C v - C _ a c i d ( c i n n a m i c o r p-coumaric 6 3 a c i d ) w i t h 3 m a l o n i c a c i d u n i t s . U n l i k e i n h i g h e r p l a n t s , where t h e ammonia l y a s e s a r e t h e major enzymes and t h e t r a n s a m i n a s e s a r e mino r , t h e r e v e r s e i s p r o b a b l y t r u e f o r a l g a e . I n t i m e , f r o m c e l l s c u l t u r e d under t h e c o r r e c t p h y s i o l o g i c a l c o n d i t i o n s , t h e ammonia l y a s e s w i l l be d e t -e c t e d i n a l g a e . Page 131. Ragan and C r a i g l e , 1975 see Ragan and C r a g l e , 1976. ADDENDUM LITERATURE CITED Abe, HY, M. Uchlyama, and R. S a t o . 1974. I s o l a t i o n o f p h e n y l -a c e t i c a c i d and i t s p-hydroxy d e r i v a t i v e as a u x i n - l i k e s u b s t a n c e s from U n d a r l a p i n n a t l f I d a . A g r . B i o l . Chem., \ 381 897-898 . 16.2. A u g l e r , J . , and P. M a s t a g l i . 1956. S u r un compose phenol!que brome e x t r a i t de I ' a l g u e rouge H a l o p l t y s i n c u r v u s . Comptes Rendus., Acad. S c i , P a r i s , s e r . D, 242 :190-192. C h a n t r a i n e , J . , G. Combaut, and J . T e s t e . 1973. P h e n o l s bromes d'une rouge, H a l o p y t l s i n c u r v u s : a c i d e s c a r b o x y l i q u e s . P h y t o c h e m l s t r y , 12s 1793-1795. F o r s b e r g , C. 1965. N u t r i t i o n a l s t u d i e s o f C h a r a i n a x e n i c c u l t u r e s . P h y s i o l o g i a P l a n t a r u m , 18J 2 7 5 - 2 9 0 . F r i e s , L. 1973. Growth s t i m u l a t i n g e f f e c t s o f t h e bromophenol, - l a n o s o l , on r e d a l g a e i n a x e n i c c u l t u r e . E x p e r i e n t i a , 29« 1436-1437. Hayward, H. 1965. S t u d i e s on t h e growth o f Phaeodactylum t r l c o r n u t u m ( B o h l i n ) I . The e f f e c t o f c e r t a i n o r g a n i c n i t r o g e n o u s s u b s t a n c e s on growth. P h y s i o l o g i a P l a n t a r u m , 18« 2 0 1 - 2 0 7 . L o f f e l h a r d t , W., B. Ludwlg, and H. K l n d l . 1973. T h y l a k o i d - g e b u n d -ene L - P h e n y l a l a n i n - A m m o n i a k - L y a s e . H o p p e - S e y l e r ' s Z. P h y s i o l . Chem., 354: 1006-1012. P r y c e , R. J . 1972. The o c c u r r e n c e o f l u n u l a r i c and a b s c s i c a c i d s i n p l a n t s . P h y t o c h e m l s t r y , 11: 1759-1761. Ragan, M. A., and J . S. C r a i g i e . 1976. Physodes and t h e p h e n o l i c compounds o f brown a l g a e . I s o l a t i o n and c h a r a c t e r i z a t i o n o f p h l o r o g l u c i n o l polymers from Fucus v e s l c u l o s u s (L.).-. Can. J . Biochem.,54: 6 6 - 7 3 . S c o t t , R. 1954. O b s e r v a t i o n s on t h e iodoamino a c i d s o f marine a l g a e u s i n g l o d i n e - 1 3 1 . N a t u r e , 173: 1 0 9 8 - 1 0 9 9 . W e i n s t e i n , B., T. L. R o l d , C. E. H a r r e l l , J r . , M. W. Burns I I I , and J . R. Waaland. 1975. R e e x a m i n a t i o n o f t h e bromophenols i n t h e r e d a l g a Rhodomella l a r l x . P h y t o c h e m l s t r y , 14: 266?-2670. 

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