ISOCTTRITASE, GLYCINE-ALANINE TRANSAMINASE AND DEVELOPMENT IN BLAS T0 CLADlELLA EMERSON 11 By Howard D. McCurdy J r . A THESIS S u b m i t t e d t o t h e S c h o o l f o r Advanced G r a d u a t e S t u d i e s o f M ic h i g a n S t a t e U n i v e r s i t y of A g r i c u l t u r e and A p p l i e d S c i e n c e i n p a r t i a l f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e d e g r e e of DOCTOR OF PHILOSOPHY D e p a r t m e n t o f M i c r o b i o l o g y and P u b l i c H e a l t h 19 59 P roQ uest Num ber: 10008648 All rights reserved IN FO RM ATIO N TO A LL USERS The quality of this reproduction is dependent upon the quality o f the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if m aterial had to be removed, a note will indicate the deletion. uest. ProQ uest 10008648 Published by ProQ uest LLC (2016). C opyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENTS The a u t h o r w i s h e s t o e x p r e s s h i s s i n c e r e t h a n k s and h e a r t f e l t a p p r e c i a t i o n t o D r . E . C. C a n t i n o f o r h i s a b l e g u i d a n c e and s u p p o r t d u r i n g t h e c o u r s e of th is study. He w i s h e s a l s o t o e x p r e s s h i s i n d e b t e d n e s s t o D r . W. L. Mallmann f o r h i s en c o u r a g e m e n t and i n t e r e s t and t o P a t r i c i a McCurdy f o r h e r g e n e r o u s h e l p i n t h e p r e p a ra tio n of t h i s t h e s i s . To Pat ill TSOCITRTTASE< GLYCINE-ALANINE TRANSAMINASE AND DEVELOPMENT IN B LAS TO CLAD TELLA EMERSON I I By Howard D. McCurdy J r . AN ABSTRACT S u b m i t t e d t o t h e S c h o o l f o r Advanced G r a d u a t e S t u d i e s o f M ic h ig an S t a t e U n i v e r s i t y o f A g r i c u l t u r e and A p p lie d S c i e n c e in p a r t i a l f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e d e g r e e of DOCTOR OF PHILOSOPHY D e p a r t m e n t o f M i c r o b i o l o g y and P u b l i c H e a l t h Year 1959 "7< .1 -'A \ „{ ' ' \ \ ■ \ \ \ . : Approved 3 0 t \ i ■ ] \<'k. \ A : \ \ y Qy, \ \ ^ ABSTRACT The enzym e i s o c i t r i t a s e , w h ic h h a d b e e n p o s t u l a t e d t o o c c u r i n B l a s t o c l a d i e l l a , h a s b e e n p u r i f i e d fro m e x t r a c t s fifty -fo ld and some o f i t s p r o p e r t i e s s t u d i e d . e x h i b i t s maximum a c t i v i t y a t pH 7 tra tio n s of th is o rg a n is m c a . The p u r i f i e d i n t h e p r e s e n c e o f 10 enzym e M concen­ o f b o t h c y s t e i n e and Mgf ' and h a s a M i c h a e lis c o n s t a n t o f _4 4 -d x 10 M fo r its s p e c i f i c s u b s t r a t e d - 'i s o c i t r i c a c id . .A s e c o n d enzym e g l y c i n e - a l a n i n e t r a n s a m i n a s e , n o t p r e v i o u s l y p u r i f i e d fro m an y s o u r c e , h a s b e e n p u r i f i e d _ca. e i g h t y - f o l d . S t o i c h i o m e t r i c d e t e r m i n a t i o n s show t h e f o l l o w i n g t o b e t h e r e a c t i o n c a t a l y z e d b y t h e enzym e g ly o x y la te a la n in e 1 ■ p y r u v a t e -s g l y c i n e T he r e a c t i o n w as fo u n d t o b e r e v e r s i b l e o n ly w i t h d i f f i c u l t y , a t r u e e q u i l i b r i u m w as n e v e r a t t a i n e d , b e c a u s e o f a n i n h i b i t i o n o f t h e enzym e b y p y r u v a t e . I n d i r e c t e v id e n c e i n d i c a t e d t h a t p y r i d o x a l - ^ - p h o s p h a t e i s t h e p r o s t h e t i c g ro u p . pH d . 5 . Maximum a c t i v i t y w as o b ta in e d a t None o f t h e s e v e n t e e n am ino a c i d s t r i e d re p la c e d a la n in e in s y s te m s w i t h g l y o x y l a t e . New m e th o d s w e re d e v e lo p e d w h ic h p e r m i t t e d e n z y m o lo g ic a l s t u d i e s o n c e l l f r e e p r e p a r a t i o n s fro m z o o s p o r e s a s w e l l a s p l a n t s a t v a r i o u s s t a g e s o f d e v e lo p m e n t- f r o m n e a r - s y n c h r o n o u s , s i n g l e g e n e r a t i o n c u l t u r e s o f o r d i n a r y ( O .G .) c o l o r l e s s p l a n t s . v The s y n t h e s e s o f i s o c i t r i t a s e and g l y c i n e - a l a n i n e t r a n s a m i n a s e , as r e f l e c t e d i n a c t i v i t y m e a s u r e m e n t s , w e r e f o l l o w e d d u r i n g t h e d e v e l o p m e n t of b o t h o r d i n a r y c o l o r l e s s and r e s i s t a n t s p o r a n g i a l ( R . S . ) p lan ts. I n O.C. c u l t u r e s t r a n s a m i n a s e s y n t h e s i s was a l i n e a r f u n c t i o n of p l a n t growth t h r o u g h o u t t h e g e n e r a t i o n t i m e , w h e r e a s i s o c i t r i t a s e e x h i b i t e d a pronounced la g ex ten d in g t o c a . o n e - th ir d of th e g e n e r a tio n tim e. T r a n s a m i n a s e and i s o c i t r i t a s e a c t i v i t i e s p a r a l l e l e d ea ch o t h e r d u r i n g R . S . d e v e l o p m e n t , r e a c h i n g maximum a c t i v i t i e s a t c a . t h r e e fifth s of t h e g e n e r a t i o n t i m e - - t h e p o i n t of no r e t u r n i n o r to g e n y - - m u c h h i g h e r t h a n a t any o t h e r s t a g e of t h e l i f e cycle. An a t t e m p t was made t o i n t e g r a t e t h e s e r e s u l t s i n t o an i n t e r p r e ­ t a t i o n of t h e f u n c t i o n of t h e s e enzymes i n m o rp h o g e n e s i s and l i g h t s t i m u l a t e d growth i n B l a s t o c l a d i e l l a . vi TABLE OF CONTENTS Page INTRODUCTION ........................................................................................................... 1 REVIEW OF THE LITERATURE................................................................................................ M o rp h o g e n e s is and L i g h t S t i m u l a t e d G row th ( L u m i s y n t h e s i s ) i n B l a s t o c j a d i e l l a e m e r s o n i i . . . . . ........................................................... I s o c i t r i t a s e . . . . . . . . . . . . . . ............................. G l y c i n e - a l a n i n e T r a n s a m in a s e ...................................................................... . The D e v e lo p m e n t o f S i n g l e G e n e r a t i o n , N e a r-S y n c h ro n o u s G u l t u r e s o f B l a s t o c l a d i e 1 1 a ................... GENERAL METHODS AND MATERIALS. . . EXPERIMENTAL WORK....................................................................................... 3 3 9 11 lU 16 18 PART I ............................. Is o c itrita s e 1 ) A n a l y t i c a l M e th o d s ............................... 2) P re lim in a r y O b s e rv a tio n s .......................... 3 ) P r e p a r a t i o n o f Enzyme fro m A c e to n e P o w d e r s ........................ U) P a r t i a l P u r i f i c a t i o n . . . .......................... 5») I d e n t i f i c a t i o n o f G l y o x y l a t e ...................................... 6 ) S t o i c h i o m e t r y and R e v e r s i b i l i t y . ................. 7 ) Enzyme P r o p e r t i e s ........................ 18 18 20 21 22 2\\ 26 29 PART I I G ly c in e - A la n in e T r a n s a m i n a s e ................................... ft 1 ) A n a l y t i c a l M e th o d s ............................................................ ...................... 2 ) P r e l i m i n a r y O b s e r v a t i o n s .......................... 3) P a r tia l P u r if io a tio n . . . . . ............ I4.) S t o i c h i o m e t r y , R e v e r s i b i l i t y and I d e n t i f i c a t i o n o f p ro d u c ts ........... 5 ) Enzyme P r o p e r t i e s ............................... 37 37 39 lj.0 I4.3 lj.6 PART I I I S t u d i e s on t h e P h y s i o l o g y o f D e v e l o p m e n t . . . . . . . . ......................... 1 ) P r e p a r a t i o n o f Z o o s p o re S u s p e n s i o n s ................. 2 ) C u l t i v a t i o n o f S y n c h ro n o u s , S i n g l e G e n e r a t i o n C u l t u r e s o f O .C . P l a n t s ............................. v ii 5U £6 TABLE OF CONTENTS - C o n tin u e d Page 3) R e l a t i o n s h i p Bet w e en Endogenous R e s p i r a t o r y R a t e and S t a g e o f Development of O r d i n a r y C olorlessP l a n t s . . . 38 h) R e l a t i o n s h i p o f I s o c i t r i t a s e and T r a n s a m i n a s e A c t i v i t i e s t o Development and M o r p h o g e n e s i s ...................... 61 AND CONCLUSIONS......................................................................................... Ik SUMMARY......................................................................................................................................... 83 LITERATURE................. CITED..................................................................................................... 86 APPENDIX....................................................................................................................................... 9h DISCUSSION v iii L IS T OF TABLES TABLE I. II. Page P a r t i a l P u r i f i c a t i o n o f I s o c i t r i t a s e ................................ . ................ 25 Rf J s o f 2 , U - D i n i t r o p h e n y l h y d r a z o n e s .............................. 27 I I I . I s o c i t r i t a s e - S t o i c h i o m e t r y ............................................................................ IV . I s o c i t r i t a s e : 28 C o f a c t o r R e q u i r e m e n t s .................................................... 31 V. I n V i t r o E f f e c t o f L i g h t on I s o c i t r i t a s e ........................................... 31 V I . R e s u l t s of T r a n s a m i n a s e P u r i f i c a t i o n .................................................... UU V T I . R f ’ s o f T r a n s a m i n a s e R e a c t a n t s .................................................................. U5? V I I I . S t o i c h i o m e t r y o f G l y o x y l a t e - A l a n i n e T r a n s a m i n a t i o n ................... U5 IX . I n h i b i t i o n o f T r a n s a m i n a s e ............................................................................ U8 X- Enzyme S p e c i f i c i t y of G l y c i n e - A l a n i n e T r a n s a m i n a s e .............. XI . I n V i t r o E f f e c t of L i g h t on T r a n s a m i n a s e . £0 ................................. 5>1 X I I - The E f f e c t o f I n h i b i t o r s on Zo os po re R e s p i r a t i o n ........................ 60 X I I I . Soluble P r o t e i n ............................................ XIV. D i s t r i b u t i o n o f I s o c i t r i t a s e a t V a r i o u s S t a g e s o f t h e L i f e Cycle ..................................................................................................... ix 62 62 LIST OF FIGURES FIGURE Page l a - I d . A d s o r p t i o n S p e c t r a o f t h e 2 ,U “ d i n i t r o p h e n y l h y d r a z o n e s o f P y r u v a t e , a - K e t o g l u t a r a t e , G ly o x y la te and t h e P r o d u c t o f t h e I s o c i t r i t a s e R e a c t i o n ....................................... ........................................ 2 . Is o c itrita s e : Enzyme A ssa y C urve ................. ........... . ............. 3 . Tim e C o u rse o f I s o c i t r i t a s e R e a c t i o n . . . . U- I s o c i t r i t a s e : 3h 35 .................. 35 pH O ptim um ...................................... 35 G o f a c to r R e q u i r e m e n t s . . . ..................... 35 6. Is o c itrita s e : E f f e c t o f S u b s t r a t e C o n c e n t r a t i o n .................. 36 7 . Is o c itrita s e : A c t i v a t i o n E n e r g y ............. 36 8 . T r a n s a m in a s e : Enzyme A ss a y C u r v e ............................................................ 53 9 - Tim e C o u rse o f G ly c in e - A l a n i n e T r a n s a m i n a t i o n ................................ 53 5 2 -5 b . I s o c i t r i t a s e : 1 0 . R e v e r s i b i l i t y o f G ly c i n e - A la n in e T r a n s a m i n a t i o n .................. 53 1 1 . T r a n s a m in a s e : E f f e c t o f H y d ro g e n I o n C o n c e n t r a t i o n . . . . . . . . 53 1 2 . Z o o s p o re s fro m O . C . p l a n t s . . . . . . . ............................................................. 68 1 3 . A g e r m lin g t h r e e h o u r s a f t e r i n i t i a t i o n o f g ro w th i n a n e a r s y n c h r o n o u s c u l t u r e ............... ....................... . ..................................................... 68 l i i ? A s i x h o u r o ld O. C. p l a n t fro m a s i n g l e - g e n e r a t i o n n e a r sy n ch ro n o u s c u l t u r e ............................. 68 1 5 - A n i n e h o u r o ld O. C. p l a n t . fro m a n e a r s y n c h r o n o u s c u l t u r e . 68 1 6 . A g ro u p o f s e v e r a l n i n e h o u r O. C. p l a n t s i l l u s t r a t i n g t h e u n i f o r m i t y e x h i b i t e d b y p l a n t s i n n e a r s y n c h ro n o u s c u ltu re s ................... 69 1 7 . T w elv e h o u r O . C . p l a n t s fro m a n e a r s y n c h r o n o u s s i n g l e ­ g e n e ra tio n c u l t u r e ................ 69 x L IS T OF FIGURES - C o n tin u e d FIGURE Page 1 8 . Growth c u r v e of a s i n g l e g e n e r a t i o n c u l t u r e o f B l a s t o c l a d i e l l a ...................................................................................................... 70 19* hog grow th c u r v e o f i n d i v i d u a l p l a n t ' of B l a s t o c l a d i e l l a . ■. . 70 2 0 . R e l a t i o n s h i p b e t w e e n c u l t u r e age and oxygen u p t a k e ................... 70 21. I s o c i t r i t a s e : r e l a t i o n s h i p t o d e v elopm ent of O.C. p l a n t s . . 71 22. I s o c i t r i t a s e : relatio nship t o O.C. c u l t u r e d e v e l o p m e n t . . . . 71 23* T r a n s a m i n a s e : relatio n sh ip t o O.C. c u l t u r e d e v e l o p m e n t . . . . 71 2U. T r a n s a m i n a s e : r e l a t i o n s h i p t o d e v elopm ent of O.C. p l a n t s . . 29* Enzyme S y n t h e s i s as a f u n c t i o n of p l a n t 71 g r o w t h ............................. 72 26. I s o c i t r i t a s e : relatio n sh ip t o R . S . d e v e l o p m e n t ........................... 73 27- T r a n s a m i n a s e : relatio n sh ip to R.S. c u l t u r e d e v e l o p m e n t .. .. 73 xi 1 INTRODUCTION The d i s c o v e r y ( C a n t i n o , 1951) t h a t t h e de v e l o p m e n t o f t h e p r i m i t i v e a q u a tic fu n g u s , B l a s t o c l a d i e l l a em ersonii , a l o n g two d i f f e r e n t morpho­ g e n e t i c p a th w a y s c o u ld be d e t e r m i n e d by a s i m p l e m a n i p u l a t i o n o f t h e e x t e r n a l e n v i r o n m e n t , h a s p r e s e n t e d an e x c e l l e n t o p p o r t u n i t y f o r s t u d y ­ i n g t h e r e l a t i o n s h i p bet w een m e t a b o l i s m and m o r p h o g e n e s i s . However, e v e n w i t h t h i s r e l a t i v e l y s i m p l e o r g a n i s m , a lm o st a model s y s t e m i n f a c t , t h e r e l a t i o n s h i p i s a complex one and t h e p r o g r e s s of o u r u n d e r ­ s ta n d i n g i n e v i t a b l y slow. But p r o g r e s s i s b e i n g made and each new e x p e r i m e n t and e v e r y new f i n d i n g h a s s e r v e d o n l y t o i n t e n s i f y t h e interest o f t h o s e engaged i n B l a s t o c l a d i e l l a r e s e a r c h . The c o m p a r a t i v e l y r e c e n t d i s c o v e r y t h a t l i g h t p l a y s a r o l e i n d ev elo p m en t h a s added a new a s p e c t t o an a l r e a d y f a s c i n a t i n g s t o r y w hich p e r h a p s w i l l h a v e b r o a d i m p l i c a t i o n i n our u n d e r s t a n d i n g of l i f e i t s e l f . In sev eral papers (see l i s t of r e f e r e n c e s ) , C an t in o and c o - w o r k e r s h a v e p o s t u l a t e d mechanisms f o r t h e m o r p h o g e n e t i c and l i g h t phenomena i n B lasto clad iella. An i n t e g r a l p a r t o f t h e s e h a s b e e n t h e k e y r o l e of an enzyme, i s o c i t r i t a s e , t h e p r e s e n c e of w h ic h was i m p l i e d b u t not e x p l i c i t l y dem onstrated. I n t h e work r e p o r t e d i n t h i s t h e s i s t h e p r e s e n c e o f i s o c i t r i t a s e i n B l a s t o c l a d i e l l a has b e e n c o n f i r m e d , t h e enzyme p a r t i a l l y p u r i f i e d and i t s p r o p e r t i e s and r e l a t i o n s h i p t o development s t u d i e d . A second enzyme, g l y c i n e - a l a n i n e t r a n s a m i n a s e , p e r h a p s i n v o l v e d In t h e _in v i v o r o l e of i s o c i t r i t a s e h a s b e e n s i m i l a r l y 2 e x a m in e d . T hese s t u d i e s ha ve a l s o i n v o l v e d t h e e l a b o r a t i o n of new t e c h n i q u e s f o r t h e c u l t u r e and s t u d y of t h e o r g a n is m w h ic h w i l l be of im portance in f u tu r e r e s e a r c h . 3 REVIEW OF THE LITERATURE M o rp h o g e n e s is and L i g h t S t i m u l a t e d Growth ( L u m i s y n t h e s i s " ) I n B l a s t o c l a d i e l l a em e r s o n i i The u n i f l a g e l l a t e z o o s p o r e s of B l a s t o c l a d i e l l a e m e r s o n i i ( C a n t i n o and H y a t t , 1953a.) a r e c a p a b l e of em barking on two a l t e r n a t i v e p a th w a y s l e a d i n g i n t o t h e d e v e l o p m e n t o f m a t u r e p l a n t s of q u i t e d i f f e r e n t _ m o r p h o lo g y . C a n t i n o (1 951) f oun d t h a t t h e i n c o r p o r a t i o n o f ca 10 p M sodium b i c a r b o n a t e i n growth m edia c o n t a i n i n g p e p t o n e al w ay s l e d t o t h e grow th o f e s s e n t i a l l y o n l y one of t h e two p l a n t t y p e s — t h e r e s i s t a n t s p o r a n g iu m ( R . S . } - - w h e r e a s i n t h e ab s e n c e of b i c a r b o n a t e o n l y o r d i n a r y co lo rless ( O . C . ) p lants * w ere p r o d u c e d i n t h e f i r s t g e n e r a t i o n . Both k i n d s o f t h a l l i c o n s i s t of a b a s a l s t a l k b e a r i n g a r h i z o i d a l s y s te m and an u p p e r g l o b o s e s a c - - t h e s p o r a n g i u m . In R.S. p l a n t s , ho w e v e r , t h e sp o ran g iu m h a s a v e r y t h i c k c h i t i n o u s w a l l ( C a n t i n o , L o v e t t , and H o r e n s t e i n , 195?) i m p r e g n a te d w i t h m e l a n i n ( C a n t i n o and H o r e n s t e i n , 1955) e n c l o s i n g a p r o t o p l a s m c o n t a i n i n g a p p r e c i a b l e q u a n t i ­ t i e s of l i p o i d a l p a r t i c l e s and gamma c a r o t e n e ( C a n t i n o and H o r e n s t e i n , 1956b) . O r d i n a r y c o l o r l e s s s p o r a n g i a on t h e o t h e r hand do n o t p r o d u c e m e l a n i n o r gamma c a r o t e n e , hav e c o m p a r a t i v e l y t h i n c h i t i n o u s w a l l s , and b e a r s e v e r a l p r o m i n e n t d i s c h a r g e p a p i l l a e t h r o u g h w hich z o o s p o r e s a r e released at m atu rity . " L i g h t s t i m u l a t e d growth i n t h e a b s e n c e of c h l o r o p h y l l . li Tn e x p e r i m e n t s d e s i g n e d t o d e t e r m i n e t h e mode o f a c t i o n o f b i ­ c a r b o n a t e ( C a n t i n o , 195>l) i t was found t h a t b i c a r b o n a t e a l o n e , i n c o n t r a s t t o i t s b e h a v i o r on p e p t o n e m e d i a , d i d n o t i n d u c e R . S . f o r m a t i o n on b a s a l m ed ia c o n t a i n i n g c a s e i n h y d r o l y s a t e as t h e n i t r o g e n s o u r c e . S u b s e q u e n t s t u d i e s i n which v a r i o u s i n t e r m e d i a t e s and i n h i b i t o r s w er e _2 i n c o r p o r a t e d w i t h 10 M b i c a r b o n a t e l e d t o t h e w o r k i n g h y p o t h e s i s on w h i c h , w i t h some m o d i f i c a t i o n s , a l l work s i n c e t h a t t i m e h a s b e e n b a s e d . Th us, i f a - k e t o g l u t a r a t e o r c i t r a t e w e r e p r o v i d e d or t h e r e s p i r a t o r y p o i s o n s a r s e n i t e o r s e m i c a r b a z i d e added t o t h e b a s a l medium w i t h b i c a r b o n a t e t h e a b i l i t y t o I n d u c e R . S . was r e g a i n e d . These and r e l a t e d o b s e r v a t i o n s , le d Cantino t o p o s t u l a t e t h a t th e in c r e a s e d c o n c e n tr a tio n s o f b i c a r b o n a t e t e n d e d t o d e c r e a s e t h e r a t e s of t h e d e c a r b o x y l a t i o n s c a t a l y z e d by i s o c i t r i c d e h y d r o g e n a s e and a - k e t o g l u t a r a t e d e h y d r o g e n a s e , t h e r e b y i n d u c i n g an a c c u m u l a t i o n of i n t e r m e d i a t e s b e t w e e n a - k e t o g l u t a r a t e and i t s p r e c u r s o r s i n t h e K r e b s ' c y c l e . Somehow t h i s was s u p p o se d t o i n i t i a t e v a r i o u s s h u n t mechanisms l e a d i n g t o t h e s y n t h e s i s of gamma c a r o t e n e , m e l a n i n and t h e o t h e r i n g r e d i e n t s r e l a t e d t o t h e g e n e s i s of a r e s i s t a n t sporangium. I n l a t e r s t u d i e s ( C a n t i n o , 1952) i t was found t h a t t h e b i c a r b o n a t e e f f e c t was r e l a t e d t o c r i t i c a l p e r i o d s i n t h e developm ent o f b o t h R . S . and O.C. p l a n t s . Beyond t h r e e - f i f t h s of t h e g e n e r a t i o n tim e of b o t h , t h e c o u r s e o f d e v e lo p m e n t c o u l d be a l t e r e d n e i t h e r by t h e a d d i t i o n n o r re m o v a l o f b i c a r b o n a t e . A change i n p e r m e a b i l i t y was t h o u g h t t o be a t l e a s t p a r t i a l l y r e s p o n s i b l e th o u g h t h e o p e r a t i o n o f more f u n d a m e n t a l p r o c e s s e s was n o t e x c l u d e d . 5 The d i s c o v e r y of a m u ta n t ( C a n t i n o and H y a t t , 1953b) b r i g h t o r a n g e i n c o l o r by v i r t u e o f i t s c o n t e n t of l a r g e amounts o f gamma c a r o t e n e and which d i d n o t p r o d u c e r e s i s t a n t s p o r a n g i a , offered f u r th e r opportunity f o r t h e s t u d y of t h e m e t a b o l i c b a s i s o f R . S . f o r m a t i o n . S tu d ie s of th e c o m p a r a t i v e p h y s i o l o g y of t h e mutan t and t h e w i l d t y p e were c a r r i e d out ( C a n t i n o and H y a t t , 1953c) . These r e v e a l e d t h e p r e s e n c e i n t h e l a t t e r of t h e e n z y m a t i c a c t i v i t i e s a s s o c i a t e d w i t h cy tochrom e o x i d a s e , a c o n i t a s e , a T P N - s p e c i f i c i s o c i t r i c d e h y d r o g e n a s e , a TPN ( a n d / o r DPN) d e p e n d e n t a - k e t o g l u t a r a t e d e h y d r o g e n a s e , s u c c i n o x i d a s e , f u m a r a s e and a D P N - s p e c i f i c m alic dehydrogenase. On t h e o t h e r h a n d , t h e m u ta n t e x h i b i t e d a l l o f t h e s e a c t i v i t i e s e x c e p t a c o n i t a s e and a - k e t o g l u t a r a t e d e h y d r o g e n a s e . Thus, t h e mutan t a p p e a r e d u n a b l e t o c a r r y out t h e two o x i d a t i v e d e c a r b o x y ­ l a t i o n s a s s o c i a t e d w i t h t h e presumed t r i g g e r mechanism o f R .S . morpho­ g e n e s i s as a f u n c t i o n a l u n i t . Moreover e n z y m a t i c a c t i v i t i e s of a Krebs* c y c l e , p r e v i o u s l y o n l y presum ed t o be p r e s e n t i n B l a s t o c l a d i e l l a on t h e b a s i s of i n d i r e c t e v i d e n c e , was c o n f i r m e d . Subsequent experim ents concerned w ith f i n d i n g a c a u s a t i v e r e l a t i o n ­ s h i p b e t w e e n t h e a c t i o n o f b i c a r b o n a t e and new b i o s y n t h e t i c p a t h w a y s , t h e n s u g g e s t e d a mechanism w her eb y b i c a r b o n a t e f i x a t i o n may be c o u p le d t o melanin p r o d u c tio n . F irst, i t was found t h a t m e l a n i n s y n t h e s i s by R . S . c ould be u n c o u p l e d _2n v i v o from t h e r e s t of t h e m o r p h o g e n e t i c p r o c e s s by means of i n c o r p o r a t i n g p h e n y l t h i o u r e a i n t o t h e growth medium w i t h o u t a f f e c t i n g t h e g e n e r a t i o n o f o t h e r w i s e s t r u c t u r a l l y norma l and v i a b l e R .S . (C a n tin o , 1953)- J h v i t r o s tu d ie s dem onstrated th a t th e a p p e a r a n c e of m e l a n i n f o r m a t i o n was a s s o c i a t e d w i t h a c e l l w a l l bound 6 p o l y p h e n o l o x i d a s e s y s t e m , n o t p r e s e n t i n O. C . p l a n t s , which was a l s o I n h i b i t e d b y p h e n y l t h i o u r e a ( G a n t i n o and H o r e n s t e i n , 1955)* The enzyme was found t o m e d i a t e e l e c t r o n t r a n s p o r t n o t o n l y b e t w e e n s u b s t r a t e and oxygen b u t TPN as w e l l ( b u t n o t w i t h DPN) . M ore over, i t a p p e a r e d t h a t a - k e t o g l u t a r a t e f u n c t i o n e d as an e s s e n t i a l f a c t o r f o r a c t i v i t y by v i r t u e of i t s r e d u c t i v e c a r b o x y l a t i o n and c o n c o m i t t a n t o x i d a t i o n of r e d u c e d TPN. Th us , i t a p p e a r e d t h a t an i m p o r t a n t l i n k had b e e n e s t a b ­ lished . O t h e r s t u d i e s ( c f . G a n t i n o , I 9 6 0 , f o r r e v i e w ) ha ve be en c o n c e r n e d w i t h e l a b o r a t i n g i n more d e t a i l t h e b i o s y n t h e t i c and m e t a b o l i s chan ge s acco mpan ying m o r p h o g e n e s i s . The r e l a t i o n s h i p b e tw e e n t h e f i n d i n g s d e r i v e d t h e r e f r o m and t h e b i c a r b o n a t e e f f e c t i s ho w ev er , i n most c a s e s , s t i l l obscure. I n s t u d i e s on n i t r o g e n m e t a b o l i s m ( G a n t i n o , L o v e t t and H o r e n s t e i n , 1957) i t h a s b e e n f ound t h a t w h e r e a s t h e t o t a l n i t r o g e n c o n t e n t o f O.C. and R . S . p l a n t s wer e c o m p a r a b l e , c e r t a i n s h i f t s i n d i s t r i b u t i o n d i d occur. The f o r m a t i o n of r e s i s t a n t s p o r a n g i a was accompanied by a s h a r p r e d u c t i o n i n t h e s o l u b l e amino a c i d p o o l and an a c i d s o l u b l e n i t r o g e n f r a c t i o n , t h e l o s s o f two e l e c t r o p h o r e t i c a l l y s e p a r a b l e p r o t e i n f r a c t i o n s and t h e a p p e a r a n c e o f a new one n o t p r e s e n t i n O.C. Furtherm ore, th e d e c r e a s e i n t h e a c i d s o l u b l e n i t r o g e n f r a c t i o n was e q u i v a l e n t t o t h e in c r e a s e in c h i t i n n i t r o g e n . A change i n t h e b a l a n c e o f two enzymes c o n c e r n e d w i t h c h i t i n s y n t h e s i s was a l s o d i s c o v e r e d which may be r e l a t e d to the net in c re a se in c h i t i n in R .S. Both R . S . and O.C. were found t o ha v e c o m p a r a b le e n z y m a t i c a c t i v i t y a s s o c i a t e d w i t h t h e a c e t y l a t i o n 7 of glucoseam ine, a r e a c t i o n involved i n t h e s y n t h e s i s of c h i t i n . But a c t i v i t y o f c h i t i n a s e , an enzyme c a t a l y z i n g t h e c o n v e r s i o n of c h i t i n ba c k t o a c e t y l g l u c o s a m i n e , was d e c r e a s e d g r e a t l y as compared t o O.C. S t u d i e s w ere a l s o c a r r i e d o u t w hich r e v e a l e d c e r t a i n chan ge s i n a e r o b ic metabolism a s s o c i a t e d w ith t h e gen esis of a r e s i s t a n t sporangium. C o r r e s p o n d i n g t o a d e c r e a s e i n endogenous r e s p i r a t o r y a c t i v i t y (Qo2 °£ O.C. = 90; Q0^ of R . S . = 0 . 1 ) (Brown and C a n t i n o , 1955 j C a n t i n o and H o r e n s t e i n , 1955) i s a p r o n o u n c e d d e c r e a s e i n e n z y m a t i c a c t i v i t y a s s o c ia te d w ith th e Krebs' c y c le . Thus t h e t e r m i n a l cy t o c h r o m e o x i d a s e i s l o s t and a c o n i t a s e and o t h e r enzymes of t h e c y c l e l e a d i n g from a - k e t o g l u t a r a t e t o m a l a t e e i t h e r d i s a p p e a r or a r e r e n d e r e d i n o p e r a t i v e . However, t h e i s o c i t r i c d e h y d r o g e n a s e needed f o r r e v e r s i n g t h e c y c l e by t h e r e d u c t i v e c a r b o x y l a t i o n of a - k e t o g l u t a r a t e r e m a i n s a c t i v e ( C a n t i n o and H o r e n s t e i n , 1955) * The s t u d i e s of t h e e f f e c t of b i c a r b o n a t e on m o r p h o g e n e s is were t e m p o r a r i l y i n t e r r u p t e d by t h e d i s c o v e r y of a n o t h e r f a c e t o f t h e r o l e o f C02 i n t h e l i f e h i s t o r y o f B l a s t o c l a d i e l l a e m e r s o n i i . I t was o b s e r v e d b y C a n ti n o and H o r e n s t e i n ( 1 9 5 6 a ) , t h a t c u l t u r e s of o r d i n a r y c o l o r l e s s p l a n t s grew more r a p i d l y and p r o d u c e d g r e a t e r y i e l d s o f p l a n t m a t e r i a l in the l ig h t th an in th e d a r k . concentrations (10 _4 M oreo ver, b i c a r b o n a t e , a l b e i t a t l o w e r M) t h a n f o r m o r p h o g e n e s i s , was found t o be i n v o l v e d . I n a s e r i e s of e x p e r i m e n t s w i t h l a b e l l e d and u n l a b e l l e d b i c a r b o n a t e i t was found t h a t i l l u m i n a t i o n induced a l a r g e i n c r e a s e i n C02 f i x a t i o n b y w hole p l a n t s o v e r d a r k l e v e l s and a c o r r e s p o n d i n g d e c r e a s e i n l a b e l l e d a - k e t o g l u t a r a t e and i n c r e a s e i n l a b e l l e d s u c c i n a t e w i t h i n t h e o r g a n i s m . 8 I n a d d i t i o n a compound a p p e a r e d w h i c h , on t h e b a s i s of p a p e r chroma­ t o g r a p h y , was t h o u g h t t o be o x a l a t e . Both l a b e l l e d and u n l a b e l l e d g l u c o s e w ere a l s o consumed more r a p i d l y i n t h e l i g h t w h i l e d e p r e s s i n g bicarbonate f ix a ti o n . During j n v i t r o s t u d i e s , i t was found t h a t c e l l f r e e p r e p a r a t i o n s m e d i a t e d a r e d u c t i o n of TPN v i a i s o c i t r i c d e h y d r o g e n a s e wh ich was i n h i b i t e d by l i g h t . C o n v e r s e l y t h e same p r e p a r a t i o n s m e d i a t e d a l i g h t s t i m u l a t e d o x i d a t i o n of TPNH w hi ch was a c c e l e r a t e d b y a - k e t o g l u t a r a t e and b i c a r b o n a t e . Moreover s u c c i n a t e was foun d t o i n d u c e a d e t e c t a b l e amount o f C02 f i x a t i o n p r o v i d e d TPNH .and l i g h t w ere p r e s e n t and t h i s , t o o , a p p e a r e d t o be a T P N - s p e c i f i c r e a c t i o n . When u n l a b e l l e d a - k e t o g l u ­ t a r a t e and l a b e l l e d b i c a r b o n a t e w er e i n c u b a t e d w i t h c e l l - f r e e e x t r a c t s i n t h e p r e s e n c e of TPNH t h e r a d i o a c t i v i t y was r e c o v e r e d i n i s o c i t r a t e , s u c c i n a t e and a - k e t o g l u t a r a t e . Thus s t u d i e s on t h e l i g h t e f f e c t , i n t e r e s t i n g and of w ide i m p l i ­ c a t i o n i n t h e i r own r i g h t , had s e r v e d t o s u p p l y d i r e c t c o r r o b o r a t i v e e v i d e n c e f o r t h e r o l e of a - k e t o g l u t a r a t e as a l o c u s f o r t h e f i x a t i o n of b ic a r b o n a te in B l a s t o c l a d i e l l a . M ore over , i n an a t t e m p t t o i n t e g r a t e t h e o b s e r v e d f a c t s from t h e l i g h t e x p e r i m e n t s , G a n tin o and H o r e n s t e i n h a v e e x t e n d e d t h e o r i g i n a l c o n c e p t of t h e mechanism of b i c a r b o n a t e f i x a ­ t i o n t o i n c l u d e t h e o p e r a t i o n o f an S . K . T . ( s u c c i n a t e - k e t o g l u t a r a t e iso citrate) c y c l e w hich may p l a y a r o l e i n m o rp h o g e n e si s as w e l l . The S.K.T . c y c l e was t h o u g h t t o o p e r a t e as f o l l o w s : In the presence of b i c a r b o n a t e l i g h t e n e r g y h e l p s d r i v e t h e r e d u c t i v e c a r b o x y l a t i o n o f a - k e t o g l u t a r a t e t o i s o c i t r a t e and t h e n c e v i a an enzyme presumed t o be 9 i s o c i t r i t a s e t o s u c c i n a t e and a C2 f r a g m e n t . Some o f t h e s u c c i n a t e somehow y i e l d s more a - k e t o g l u t a r a t e (some o f which i s a p p a r e n t l y d e r i v e d from t h e n i t r o g e n p o o l ) t h u s c o m p l e t i n g t h e c y c l e , w h i l e t h e C2 f r a g ­ ment and e x c e s s s u c c i n a t e a r e i n t e g r a t e d i n t o t h e r e s t o f t h e m e t a b o l i c machinery t o y i e l d in c re a se d grow th. In morphogenesis, i f a s i m i l a r mechanism i s assumed t o o p e r a t e , t h e h i g h c o n c e n t r a t i o n s o f b i c a r b o n a t e _2 i n v o l v e d (10 M) must u p s e t a c r i t i c a l i n t e r n a l b a l a n c e so t h a t i n s t e a d of i n c r e a s e d g r o w t h , an e n t i r e l y new c o u r s e of d e v e lo p m e n t i s p u r s u e d c u lm in a tin g i n th e p r o d u c tio n of r e s i s t a n t sporangia,. That t h i s i s t h e c a s e , now seems a l l t h e more l i k e l y i n v ie w of t h e r e c e n t o b s e r v a ­ tions ( G a n t i n o , 195?) t h a t t h e R . S . t r i g g e r mechanism i s a l s o s t i m u l a t e d by l i g h t . Iso citritase One of t h e c r i t i c a l r e a c t i o n s i n t h e h y p o t h e t i c a l S . K . I . c y c l e f o r B l a s t o c l a d i e l l a was t h e C2-C4 c l e a v a g e of i s o c i t r i c a c i d t h o u g h t t o be m e d i a t e d b y t h e enzyme i s o c i t r i t a s e . E v id e n c e f o r su ch a d e v i a t i o n from t h e K r e b s 1 c y c l e i n v o l v i n g t h e f o r m a t i o n o f g l y o x y l a t e and s u c c i n a t e from t h e t r i c a r b o x y l i c a c i d s was f i r s t p r o v i d e d by Campbell e t a l . ( 1 953)* u s i n g e x t r a c t s of Pseudomonos a e r u g i n o s a . The s p e c i f i c s u b s t r a t e o f t h e r e a c t i o n was a t f i r s t o b s c u r e d by t h e f i n d i n g of t h o s e w o r k e r s t h a t , w h i l e c i t r a t e and a c o n i t a t e w e r e a c t i v e , i s o c i t r a t e was n o t . However, t h e r e s u l t s o f o t h e r s (S mith and G u n s a l u s , 195U; S aj and H i l l a r y , 1956; O l s o n , 1955) hav e s i n c e shown d - i s o c i t r a t e t o be t h e a c t u a l s u b s t r a t e f o r t h e r e a c t i o n c a t a l y z e d by t h e enzyme i s o c i t r i t a s e 195U)* ( S m it h and G u n s a l u s . The a c t i v i t y h a s b e e n i d e n t i f i e d i n a number o f a e r o b i c and 10 f a c u l t a t i v e b a c t e r i a ( S m i t h and G u n s a l u s , 1955', Wong and A j l , 1999; Madsen and H o c h s t e r , 1999) c e r t a i n f u n g i ( O l s o n , 1959; K o r n b e r g and C o l l i n s , 1 9 9 8 ) , and t h e c a s t o r b e a n ( K o r n b e r g and B e e v e r s , 1 9 9 7 ) . It h a s b e e n p a r t i a l l y p u r i f i e d f r o m p s e u d o m o n a s ( S m i t h and G u n s a l u s , 1991) an d B a k e r s 1 y e a s t (O lson, 1999). Growth c o n d i t i o n s ( S m i t h and G u n s a l u s , 1999; K o r n b e r g et_ ad_., 1998) f o r optimum enzyme p r o d u c t i o n b y b a c t e r i a h a v e b e e n shown t o b e a e r o b i c i n a c e t a t e and m i n e r a l s a l t s m e d i a . Glucose c o m p le te ly r e p r e s s e s i s o c i t r i t a s e f o r m a t i o n i n f a c u l t a t i v e b a c t e r i a ' and d e c r e a s e s a c t i v i t y somewhat i n a e r o b i c o r g a n i s m s } t h e r e f o r e , it a p p e a r s t o be a d a p t i v e l y formed in t h e s e o r g a n i s m s . S m i t h and G u n s a l u s (199U? 1 9 9 7 ) have s tu d ie d th e s p e c i f i c i t y , s t o i c h i o m e t r y , e q u i l i b r i u m and e n e r g e t i c s o f t h e r e a c t i o n c a t a l y z e d b y t h e p a r t i a l l y p u r i f i e d p s e u d o m o n a s en zyme. The enzyme c a t a l y z e s an a ld o l cleavage of d - i s o c i t r a t e : d -iso citrate — As i n d i c a t e d , succinate f- g l y o x y l a t e t h e r e a c t i o n i s r e v e r s i b l e w i t h an e q u i l i b r i u m c o n s t a n t i n t h e d i r e c t i o n o f t h e c o n d e n s a t i o n o f 3k (pH 7 - 6 , t o a f r e e e n e r g y change o f -2100 c a l o r i e s . however, t h a t , i n view of t h e f i r s t t h e second o r d e r k i n e t i c s 27°C) c o r r e s p o n d i n g The a u t h o r s p o i n t o u t , order k in e tic s o f t h e c l e a v a g e and of th e c o n d e n s a tio n , th e d i s t r i b u t i o n of r e a c t a n t s a t low s u b s t r a t e c o n c e n t r a t i o n s i s f a r t o w a r d s g l y o x y l a t e and su ccin ate. R e q u i r e m e n t s f o r a s u l f h y d y l compound and a d i v a l e n t m e t a l i o n h a v e b e e n d e m o n s t r a t e d by v a r i o u s i n v e s t i g a t o r s Smith and G u n s a l u s , 1997; O l s o n , 1999)* (Wong and A j l , C y s t e i n e and magnesium 1999? 11 r e s p e c t i v e l y ha ve b e e n f ound t o b e s t s a t i s f y t h e s e n e e d s f o r t h e Pseudomonas and y e a s t enzymes G ly c in e - a la n in e Transam inase P r e v i o u s w o r k e r s ( C a m p b e ll, S m it h and E a g l e s , 199U? Smith and G u n s a l u s , 1957; Wong and A j l , 1955? S a j and H i l l a r y , 1956) hav e d i s c u s s e d t h e p r o d u c t i o n of g l y c i n e as one of t h e p o s s i b l e f u n c t i o n s o f i s o c i t r i t a s e . S i n c e g l y c i n e i s a v e r y a c t i v e compound met a b o l i c a l l y , s uch a r e a c t i o n m ig h t c o n n e c t t h e t r i c a r b o x y l i c a c i d s t o a number of i m p o r t a n t i n t e r ­ m ediates in b i o l o g i c a l syntheses . of p r o te in , Thus b e s i d e s i t s r o l e as a component i t h a s b e e n i m p l i c a t e d i n t h e p r o d u c t i o n o f th y m i n e (Elwyn and S p r i n s o n , 195U) p u r i n e s (Elwyn and S p r i n s o n , 1 9 5 0 ) , c a r o t e n e ( MacKinney eit _ a l . , 1955) as w e l l as p u r i n e s y n t h e s i s and a c t i v e C-l u n i t m e t a b o l i s m (S h em in , 1 9 5 6 ) . G lycine i s r e a d i l y conv erted t o s e r i n e by many o r g a n i s m s ( S ak am i, 1955) t h r o u g h which i t m ig h t e n t e r c a r b o h y d r a t e s y n t h e s i s by way of h y d r o x y p y r u v a t e and 3“p h o s p h o g l y c e r i c a c i d ( S a l l a c h , 1956). A number o f s t u d i e s h a v e shown t h a t g ly o x y late is r a p i d l y converted t o g l y c i n e _in v i v o i n b o t h p l a n t s and a n i m a l s (W e in h o u se and F r i e d m a n n , 1951? W e i s s b a c h and S p r i n s o n , 1953? K o l e s n i k o v , 195U? T o l b e r t and Cohan, 1953) * The s o u r c e o f t h e amino g ro u p f o r t h e c o n v e r s i o n i s p r o b a b l y d e r i v e d f r o m t r a n s a m i n a t i o n r e a c t i o n s w i t h e n d o g e n o u s amino a c i d s . T r a n s a m i n a s e s c a t a l y z i n g am ino g r o u p t r a n s f e r f r o m a l l o f t h e n a t u r a l am ino a c i d s h a v e b e e n d e s c r i b e d few p u r i f i e d . (M eister, 1955) b u t r e l a t i v e l y A g r e a t d e a l of e f f o r t h as b e e n expended d u r i n g t h e p a s t two d e c a d e s i n a t t e m p t s t o p u r i f y t h e g l u t a m a t e - a l a n i n e and g l u t a m a t e - 12 a s p a r t a t e tra n s a m in a s e s (Green £ t a l . , O'Kane and G u n s a l u s , 195-7; L i s , 1 9 5 8 ) . 1955; Camrnarata and Cohen, 1951; Only r e c e n t l y h a s a h i g h l y p u r i f i e d g l u t a m a t e - a s p a r t a t e - e n z y m e b e e n o b t a i n e d ( J e n k i n s and S i z e r , 1957; J e n k i n s _et _ a l . , 1 9 5 9 ) - O t h e r t r a n s a m i n a s e s f o r which p a r t i a l p u r i f i c a t i o n s have been r e p o r te d a r e ; gam m a-am inobutyric-glutam ate ( S c o t t and J a k o b y , 1 9 5 9 ) ; t y r o s i n e - g l u t a m a t e ( C a n e l l a k i s and Cohen, 1 9 5 6 ) ; s e r i n e - a l a n i n e ( S a l l a c h , 1955) and enzymes m e d i a t i n g s i m u l t a n e o u s t r a n s a m i n a t i o n - d e a m i n a t i o n r e a c t i o n s b e tw e e n a s p a r a g i n e and g l u t a m i n e and a number o f a - k e t o a c i d s ( M e i s t e r and T i c e , 1950; M e i s t e r , a t a l . , 1952; M e i s t e r , 1 95 1, 1955) i n c l u d i n g g l y o x y l a t e . R e p o r t s o f t r a n s a m i n a s e s c a t a l y z i n g t h e i n t e r c o n v e r s i o n of g l y o x y l a t e and g l y c i n e h a v e b e e n r a r e a n d , e x c e p t f o r t h e t r a n s a m i n ­ a t i o n r d e a m i d a t i o n enzymes, none h a s b e e n p u r i f i e d or t h o r o u g h l y s t u d i e d . Camrnarata and Cohen (1 950) and Awapara and S e a l e (1952) hav e r e p o r t e d an e n z y m a t i c t r a n s m i n a t i o n b e t w e e n g l y o x y l a t e and g l u t a m a t e c a t a l y z e d by r a t t i s s u e p re p a ra tio n s . W i l s o n e t a l . ( 1 955) d e m o n s t r a t e d s i m i l a r a c t i v i t y in various p la n t t i s s u e s . More r e c e n t l y a c e l l - f r e e e x t r a c t o f Pseudomonas h as b e e n o b s e r v e d by Campbell (1956) t o c a t a l y z e t h e t r a n s a m i n a t i o n o f g l y o x y l a t e t o g l y c i n e from g l u t a m a t e , a s p a r t a t e , a l a n i n e , a s p a r a g i n e and g l u t a m i n e . However, i t was n o t e s t a b l i s h e d w h e t h e r one o r s e v e r a l enzymes w er e i n v o l v e d and no f r a c t i o n a t i o n was attem pted . M e t z l e r _et aJ_. (1955) and Nakada and Weinhouse ( 1 953) ha v e r e p o r t e d c h e m i c a l t r a n s a m i n a t i o n s b e t w e e n g l y c x y l a t e and s e v e r a l amino a c i d s i n c l u d i n g a l a n i n e w i t h o u t t h e i n t e r m e d i a t e p a r t i c i p a t i o n of an o r g a n i c 13 catalyst . I t i s of i n t e r e s t t o n o te t h a t th e e q u i l i b r i u m of th e chem ical r e a c t i o n : L - A l a n i n e +- g l y o x y l a t e g l y c i n e -t- p y r u v a t e s tr o n g ly fa v o rs gly cin e form ation w ith a c a lc u la t e d n eg ativ e f r e e e n e r g y ch ange of two K c a l or mor e. M e t z l e r (1955) h a s p o i n t e d t h i s o u t as a p o s s i b l e e x p l a n a t i o n f o r t h e r a r i t y of r e p o r t s of e n z y m a t i c a l l y c a t a l y z e d t r a n s a m i n a t i o n s i n t h e d i r e c t i o n of g l y o x y l a t e f o r m a t i o n . Though a l a n i n e h a s b e e n r e p o r t e d t o p a r t i c i p a t e i n a number of e n z y m a t i c t r a n s a m i n a t i o n s ( S a l l a c h , 1955; Awapara and S e a l e , 1952; R o w s e l l , 1956; Rudman and M e i s t e r , 1953; A l t e n b e r n and H o u s e w r i g h t , 1955 )* t o t h e w r i t e r ' s knowledge t h e r e a r e no r e p o r t s , o t h e r t h a n t h a t o f Campbell ( 1 9 5 6 ) , o f a g l y o x y l a t e - a l a n i n e t r a n s a m i n a t i o n . Much of t h e s t u d y of t r a n s a m i n a s e s h a s b e e n co n c e r n e d w i t h t h e r o l e o f v i t a m i n B 6 and i t s a n a l o g u e s . P y r i d o x a l - 5 “p h o s p h a t e h a s b e e n e s t a b l i s h e d as a coenzyme f o r a l m o s t a l l of t h e t r a n s a m i n a s e s examined c r i t i c a l l y f o r a r e q u i r e m e n t ( C a m p b e l l , 1956; M e i s t e r and P e t e r s o n , 1955; B a x t e r and R o b e r t s , 1958$ Fincham and B o u l t e r , 1956; S c h l e n k and F i s h e r , 1957; J e n k i n s e t a l . , 1959)* a nd i t seems l i k e l y t h a t i t i s i n v o l v e d i n a l l t r a n s a m i n a s e r e a c t i o n s ( M e i s t e r , 1955)* That t h e coenzyme i s t i g h t l y b o u n d , may e x p l a i n t h e o b s e r v a t i o n t h a t c e r t a i n enzymes do n o t r e q u i r e t h e a d d i t i o n of p y r i d o x a l p h o s p h a t e f o r a c t i v i t y (Fincham and B o u l t e r , 1956; S c o t t and J a k o b y , 1956; c f . a l s o J e n k i n s et a l . , 1959* M e i s t e r , 1955)* Of p a r t i c u l a r i n t e r e s t i n our s t u d i e s o f g l y c i n e - a l a n i n e t r a n s ­ a m in a s e i s t h e f a c t t h a t M e i s t e r (1955) h a s p o i n t e d out t h e p o s s i b i l i t y iu of tra n sa m in a tio n s in v olving g ly o x y late not req u irin g the in te r ­ mediate p a r t i c i p a t i o n of t h i s c o f a c t o r . T h is p o s s i b i l i t y i s s u g g e s t e d b y t h e f i n d i n g s o f M e t z l e r _et _al. (195U) and Nakada and We i n h o u s e (19 53) w ith chemical t r a n s a m i n a t i o n s . The Dev elopmen t o f S i n g l e G e n e r a t i o n , N e a r -S y n c h r o n o u s C u l t u r e s of B l a s t o c l a d i e l l a In atte m p tin g to o b ta in s u ita b le m a te ria l f o r studying the p roper­ t i e s o f i s o c i t r i t a s e and g l y c i n e - a l a n i n e t r a n s a m i n a s e and e l u c i d a t i n g t h e i r p o s s i b l e f u n c t i o n s i n growth and d e v e l o p m e n t , i t was n e c e s s a r y t o d e v i s e new c u l t u r e t e c h n i q u e s . P r e v i o u s s t u d i e s of enzymes i n B. e m e r s o n i i have i n v o l v e d t h e u s e o f f i v e t o t e n d ay l i q u i d c u l t u r e s i n i t i a t e d b y means of r e l a t i v e l y l i g h t s u s p e n s i o n s of z o o s p o r e s from a g a r p l a t e c o l o n i e s o f r e s i s t a n t s p o r a n g i a ( C a n t i n o and H o r e n s t e i n , 1955) o r o r d i n a r y c o l o r l e s s p l a n t s ( C a n t i n o and H y a t t , 1 9 5 3 c ) . The h a r v e s t s from s uc h c u l t u r e s a r e a c t u a l l y t h e r e s u l t o f s e v e r a l g e n e r a t i o n s of p l a n t s and as su ch c o n t a i n v a r y i n g amounts of dead p l a n t m a t e r i a l and t h a l l i a t d i v e r s e s t a g e s of developm ent. I n t h e i n i t i a l e x p e r i m e n t s on i s o c i t r i t a s e i t was f ound t h a t r e s u l t s o b t a i n e d by t h e u s e of su ch c u l t u r e s wer e v a r i a b l e . sig n ifican t re su lts, More i t seemed, might be f o r t h c o m i n g i f means were d e v i s e d f o r o b t a i n i n g , as t h e r e s u l t of a s i n g l e g e n e r a t i o n o f p l a n t s , s u f f i c i e n t m a t e r i a l w i t h which t o c a r r y out p h y s i o l o g i c a l s t u d i e s . C l e a r l y , t h e p r o b le m of s i n g l e g e n e r a t i o n c u l t u r e s was one of d e v e l o p i n g a means of g e t t i n g s u f f i c i e n t l y h e a v y s u s p e n s i o n s o f z o o s p o r e s . B a r n e r and C a n t i n o (1 95 2) h a s a l r e a d y d e s c r i b e d means of i n d u c i n g 15 g e r m i n a t i o n o f m a t u r e o r d i n a r y c o l o r l e s s p l a n t s on a g a r and o b t a i n i n g zoospore su sp en sio n s th e re fro m . A lo g i c a l e x te n s io n of t h i s te c h n iq u e on a v e r y l a r g e s c a l e n o t o n l y p e r m i t t e d t h e g ro w th o f s i n g l e g e n e r a t i o n c u ltu re s o f o r d i n a r y c o l o r l e s s p l a n t s b u t i t was fo u n d t h a t s u c h c u l ­ t u r e s e x h i b i t e d e s s e n t i a l l y s y n c h r o n o u s g ro w th f o r m ost o f t h e g e n e ra tio n tim e . F u rth erm o re , in th e course of d e v e lo p in g th e s e c u ltu r e m e th o d s t h e l a r g e s c a l e p r e p a r a t i o n o f z o o s p o r e s u s p e n s i o n s p e r m i t t e d p h y sio lo g ic a l. s tu d ie s i n th e a q u a tic fu n g i. on z o o s p o r e s i n a way n o t p r e v i o u s l y a t t e m p t e d I t t h e r e f o r e becam e p o s s i b l e , f o r t h e f i r s t t i m e , t o s t u d y t h e e n z y m o lo g y o f d e v e l o p m e n t , u s i n g b o t h c e l l - f r e e p r e p a r a ­ tio n s and w h o le p l a n t s , a t e v e r y s t a g e o f t h e l i f e h i s t o r y o f B la st o c l a d i e l l a . 16 GENERAL METHODS AND MATERIALS The m e d ia u s e d f o r t h e c u l t i v a t i o n o f B . e m e r s o n i i w e re a s f o l l o w s : Medium PYG (now a v a i l a b l e a s D i f c o , C a n t i n o PYG B r o t h o r A g a r) c o n t a i n e d 3*0 gm g l u c o s e , 1 . 2 $ gm B a c t o P e p t o n e ( D i f c o ) , 1 . 2 5 gm y e a s t e x t r a c t ( D i f c o ) , 0 . 2 5 ml o f 0.01; p e r c e n t brom c r e s o l p u r p l e and 1000 m l d i s ­ t i l l e d w a te r. F o r c u l t i v a t i o n o f r e s i s t a n t s p o r a n g i a 0 . 7 5 gm s o d iu m b i c a r b o n a t e p e r l i t e r was a d d e d t o medium PYG. S o l i d medium f o r t h e m a i n t e n a n c e o f s t o c k c u l t u r e s and t h e p r o d u c t i o n o f z o o s p o r e s c o n t a i n e d 20 gm D i f c o B a c t o A g a r p e r l i t e r o f PYG. F o r t h e e a r l y s t u d i e s on i s o c i t r i t a s e , v a tio n of liq u id t h e m e th o d s f o r t h e c u l t i ­ c u l t u r e s w e r e a s d e s c r i b e d b y C a n t i n o and H y a t t ( 1 9 5 3 a ) . A l l o t h e r s t u d i e s w e r e made u s i n g c u l t u r e s grow n a c c o r d i n g t o m e th o d s d e v e lo p e d d u rin g th e c o u rs e of t h i s s t u d y and a r e d e s c r i b e d i n d e t a i l i n P a r t I I I o f t h e E x p e r i m e n t a l W o rk . c a tio n of i s o c i tr ita s e l i t e r fla sk s, M a te ria l fo r th e p a r t i a l p u r i f i ­ and t r a n s a m i n a s e w as o b t a i n e d b y i n o c u l a t i n g tw o - c o n t a i n i n g 1 . 5 l i t e r s PYG and e q u ip p e d w i t h a e r a t i o n d e v ic e s , w ith heavy su sp en sio n s of z o o sp o re s . The c u l t u r e s w e re h a r v e s t e d a t _ca. t h i r t e e n h o u r s b y vacuum f i l t r a t i o n , w a sh e d w i t h c a . tw o l i t e r s d i s t i l l e d w a t e r and s u c k e d ,Td r y . " h o m o g e n iz a tio n e t c . , S p e c ific d e ta i ls about a r e t o be found i n t h e a p p r o p r i a t e s e c t i o n s . The b i o c h e m i c a l s u s e d d u r i n g t h e c o u r s e o f t h i s w ork w e r e o b t a i n e d as fo llo w s: so d iu m p y r u v a t e ( r e a g e n t g r a d e ) , am ino a c i d s , a - k e t o g l u t a r i c a c i d , and o x a l a c e t i c a.cid fro m N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n ; p h o sp h o p y rid in e n u c le o tid e (TPN), d i p h o s p h o p y r i d i n e n u c l e o t i d e (DPN), tri- 17 sodium g l y o x y l a t e m o n o h y d ra te , d, 1 - i s o c i t r i c a c i d l a c t o n e ( a l i o f r e e ) , and p y r i d o x a l - 5 ~ p h o s p h a t e from Sigma C hem ical Co.j p y r u v i c a c i d from C a l i f o r n i a C o r p o r a t i o n f o r B io c h e m ic a l R e s e a r c h . Iso c itric a c i d w as p r e p a r e d fro m t h e l a c t o n e b y h y d r o l y s i s w i t h a s l i g h t e x c e s s o f 2N p o t a s s i u m h y d r o x i d e i n a b o i l i n g w a t e r b a t h . F o r u s e , t h e s o l u t i o n was a d j u s t e d t o pH 7-U- Such p r e p a r a t i o n s c o n t a i n e d £0 p e r c e n t d - i s o c i t r i c a c id by a s s a y w i t h p i g h e a r t i s o c i t r i c d e h y d r o g e n a s e (O choa, 1 9 5 2 ) . Sodium p y r u v a t e was p r e p a r e d from t h e a c i d b y t h e m ethod o f R o b e r t s o n (19U 2) and r e c r y s t a l l i z e d fro m a w a t e r - a l c o h o l s o l u t i o n . A ll in o rg a n ic a v a ila b le , c h e m i c a l s and o r g a n i c s o l v e n t s w e re c o m m e r c i a l l y r e a g e n t g r a d e compounds . The g l a s s b e a d s u s e d f o r g r i n d i n g p l a n t t i s s u e i n t h e o m n im ix e r w e r e s i z e N o. 100 made b y t h e M i n n e s o t a M in in g and M a n u f a c t u r i n g C om pany. 18 EXPERIMENTAL WORK PART I Isocitritase 1) Analytical Methods C h e m ic a ls G ly o x y lic a c id w as a s s a y e d a s i t s 2 ,L |.- d i n i t r o p h e n y l - h y d r a z o n e (ENPH) c o l o r i m e t r i c a l l y b y t h e m e th o d s o f F rie d e m a n n and Haugen (19U 3) and s p e c t r o p h o t o m e t r i c a l l y b y an a d a p t a t i o n o f t h e m ethod o f S m ith and G u n sa lu s (1 9 5 7 )* K e to a c i d s w e re d e m o n s tr a t e d b y ch ro m a­ t o g r a p h y o f t h e i r DNPH d e r i v a t i v e s a c c o r d i n g t o t h e m ethod o f C a v a l l i n i ( 1 9 ^ 0 ) i n b u t a n o l - e t h a n o l - w a t e r ( 1+ : 1 ; 5 )> b u t a n o l - a c e t i c a c i d - w a t e r ( U : l : 5 ) j p r o p a n o l- a m m o n ia - w a te r ( 6 s 3 s i ) and tw o d i m e n s i o n a l l y i n p h e n o l - w a t e r ( 2 - 5 : 1 ) and b u t a n o l - p r o p i o n i c a c i d - w a t e r ( U . £ : 2 s'3 ) . P r o t e i n w as d e te r m in e d t u r b i d o m e t r i c a l l y b y t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n (S tg id tm an e t a l . « a lb u m in a s a s t a n d a r d . 1 9 5 1 ) u s i n g c r y s t a l l i n e b o v in e seru m R e a d in g s w e re made u s i n g a K le tt-S u m m e r s o n c o l o r i m e t e r w i t h a N o. 51+ f i l t e r . E s t i m a t e s on m ore h i g h l y p u r i f i e d enzym e p r e p a r a t i o n s w e re d o n e b y m eans o f U . V . a b s o r p t i o n m e a s u re m e n ts a t 215 and 225 Enzymatics a c c o r d i n g t o t h e m ethod o f W a d d e ll ( 1 9 5 6 ) . Succinic acid was determined monometrically using a succinoxidase preparation from pig heart according to the method of Krebs as described by Umbreit _et al. (1 9 5 7 )* The method was found to be specific for succinate, no other compound elicited oxygen uptake. 19 Iso c itric a c i d was d e t e r m i n e d e n z y m a t i c a l l y u s i n g t h e i s o c i t r i c d e h y d r o g e n a s e p r e p a r e d fr o m p i g h e a r t b y m e a s u r i n g TPN r e d u c t i o n s p e c t r o p h o t o m e t r i c a l l y a t 3 I4O mu (O c h o a , 1 9 5 2 ) . Iso c itrita se i n m ic ro m o le s: a c t i v i t y w as d e t e r m i n e d i n an a s s a y s y s t e m c o n t a i n i n g d ,l-is o c itra te , I4.O; c y s t e i n e h y d r o c h l o r i d e , c h l o r i d e , 1 0; p h o s p h a t e b u f f e r , pH m l, t e m p e r a t u r e , 30°C . 200. m agn esiu m The t o t a l v o lu m e was 3*1 The r e a c t i o n was i n i t i a t e d b y t h e a d d i t i o n o f s u b s t r a t e and s t o p p e d b y t h e a d d i t i o n o f 0 . 3 ml 2 N h y d r o c h l o r i c a c i d . "When n e c e s s a r y p r o t e i n was p r e c i p i t a t e d b y t h e a d d i t i o n o f 0 . 0 2 ml t e n p e r c e n t so d iu m t u n g s t a t . e and rem o v ed b y c e n t r i f u g a t i o n . A fte r b rin g in g t o 10 m l, 1 . 0 ml o f a 0 . 1 p e r c e n t o f 2 , 14- d i n i t r o p h e n y l h y d r a z i n e i n 2N h y d r o c h l o r i c a c i d w as ad ded t o 1 . 0 ml o f t h e r e a c t i o n m i x t u r e and a l l o w e d t o r e a c t s i x m i n u t e s a t room t e m p e r a t u r e . Two m l o f 95 p e r c e n t e t h a n o l was t h e n a d d e d , f o l l o w e d by U.O ml o f 1 . 5 N sodium h y d ro x id e . A f t e r 3 - 5 m i n u t e s t h e c o l o r was r e a d a t 1+90 and %k0 mu w i t h a g ly o x y la te s ta n d a rd . M ic ro m o le s o f g l y o x y l a t e p e r s a m p le = O.J435 x o p t i c a l d e n s i t y a t I4.9 O mi . U nder th e c o n d itio n s of th e a ss a y , th e o p t i c a l d e n s i t y r a t i o s , U90:5UC) o f t h e DNPH d e r i v a t i v e s of g ly o x y la te , p y r u v a t e and a - k e t o g l u t a r a t e a r e c a . 1 .5 * 1 * ° a nd 0 . 8 r e s p e c t i v e l y . The o p t i c a l d e n s i t y r a t i o , t h e r e f o r e , g a v e a n i n d i c a t i o n o f t h e p r e s e n c e o f k e to a c id s o th e r th a n g l y o x y la te . One u n i t o f i s o c i t r i t a s e a c t i v i t y = t h a t amount o f enzyme c a t a l y z i n g t h e f o r m a t i o n o f 1 . 0 uM o f g l y o x y l a t e p e r t e n m i n u t e s i n t h e p r o t o c o l g iv e n abo ve. S p e c i f i c a c t i v i t y = t h e num ber o f u n i t s o f a c t i v i t y p e r m i l l i g r a m p ro te in . 20 2) P r e li m in a r y O b s e rv a tio n s The i n c u b a t i o n o f c r u d e h o m o g e n a te s p r e p a r e d fr o m s i x d a y o l d c u ltu r e s of B l a s t o c l a d i e l l a w ith i s o c i t r i c a c id , le d to th e i d e n t i f i ­ c a t i o n o f g l y o x y l i c a c i d a s t h e p r o d u c t o f an e n z y m a t i c r e a c t i o n b y means o f p a p e r c h r o m a t o g r a p h y o f t h e 2 , 14- d I n i t r o p h e n y l h y d r a z o n e . H o w e v e r, a - k e t o g l u t a r a t e and p y r u v a t e w e re a lw a y s p r o d u c e d a s w e l l when l o n g e r i n c u b a t i o n p e r i o d s w ere u s e d . I n e x p e r i m e n t s i n w h ic h s u c c i n a t e , s u c c i n a t e and g l y o x y l a t e , s u c c i n a t e and sod ium b i c a r b o n a t e , and g l y ­ o x y l a t e w e r e i n c u b a t e d w i t h B l a s t o c l a d i e l l a h o m o g e n a t e s , i t was fo u n d t h a t o n l y g l y o x y l a t e gave r i s e t o t h e s e tw o o t h e r co m p o u n d s. T h i s was l a t e r shown t o b e due t o e n z y m a t i c and c h e m i c a l t r a n s a m i n a t i o n s b e tw e e n g l y o x y l a t e and e n d o g e n o u s am ino a c i d s . The d e m o n s t r a t i o n o f t h e s t o i c h i o m e t r y , and t h e s t u d y o f p r o p e r t i e s of th e i s o c i t r i t a s e p u rific a tio n . r e a c t i o n , w ere t h e r e f o r e d ep en d en t upon p r e l i m i n a r y I n a l l c a s e s w h e re a t t e m p t s w e re made t o o b t a i n a c t i v e , p a r t i c l e - f r e e p r e p a r a t i o n s b y means o f h i g h s p e e d c e n t r i f u g a t i o n o f h o m o g e n a te s p r e p a r e d u s i n g a m o to r d r i v e n g l a s s h o m o g e n i z e r , a c t i v i t y was lo w o r a b s e n t . I n e x p e r i m e n t s w h e re e i t h e r 2 2 ,0 0 0 x G s u p e r n a t a n t s , lo w s p e e d (£ 0 0 x G) s u p e r n a t a n t s , w ith i s o c i t r a t e , o r c r u d e h o m o g e n a te s w e re i n c u b a t e d i t w as fo u n d t h a t m o st o f t h e a c t i v i t y h a d b e e n r e t a i n e d i n th e h ig h speed s e d im e n t. H o w ev er, s o l u b l e p r e p a r a t i o n s fro m o r d i n a r y c o l o r l e s s p l a n t s su it­ a b l e f o r ammonium s u l f a t e f r a c t i o n a t i o n w e re f i n a l l y o b t a i n e d fro m e x t r a c t s of a c e to n e d r ie d pow ders. M ost o f t h e d e t e r m i n a t i o n s on s t o i c h i o m e t r y w e re d o n e u s i n g p r e p a r a t i o n s o b t a i n e d b y t h i s p r o c e d u r e . 21 I t was l a t e r f o u n d t h a t t h i s re sista n t m ethod was u n s u i t a b l e f o r u s e w i t h s p o r a n g i a b e c a u s e o f t h e i r v e r y t h i c k w a l l s w h ic h r e s i s t e d b re a k a g e u s in g t h e g la s s h o m o g e n iz er. The a c q u i s i t i o n o f a S e r v a l l O m n im ixer p e r m i t t e d t h e p r e p a r a t i o n o f s o l u b l e e x t r a c t s fr o m b o t h R . S . and O .G . w i t h o u t p r e l i m i n a r y t r e a t m e n t s o t h a t t h e u s e o f a c e t o n e pow der e x t r a c t s w ere th e n ab an d o n ed . 3 ) P r e p a r a t i o n o f Enzyme fro m A c e to n e P o w d e rs A f i v e d a y c u l t u r e was h a r v e s t e d i n t h e u s u a l m a n n er and h o m o g e n iz e d f o r e x a c t l y t e n m i n u t e s i n a m o to r d r i v e n h o m o g e n iz e r c o n s i s t i n g o f a T e flo n p e s t l e and g l a s s t u b e , u s i n g ^ .0 ml o f 0.02M p h o s p h a t e b u f f e r f o r e a c h gram (w e t w e i g h t ) o f p l a n t m a t e r i a l i n a n i c e b a t h . The h o m o g e n a te was t h e n c e n t r i f u g e d a t 2 2 ,0 0 0 x G f o r t h i r t y m i n u t e s , t h e s u p e r n a t a n t d i s c a r d e d and t h e s e d i m e n t r e s u s p e n d e d i n t h e same v o lu m e o f b u f f e r . The c e n t r i f u g a t i o n was r e p e a t e d and t h e s e d i m e n t r e s u s p e n d e d i n o n e - f i f t h v o lu m e o f b u f f e r . To t h e m i x t u r e was ad d ed q u i c k l y , w i t h s t i r r i n g , t e n v o lu m e s o f a c e to n e p r e v i o u s l y c h i l l e d t o -1 0 °G . The m i x t u r e was a l l o w e d t o s t a n d t e n m i n u t e s i n t h e c o l d , a f t e r w h ic h i t was f i l t e r e d b y s u c t i o n t h r o u g h a B u c h n e r f u n n e l , w a sh e d w i t h tw o v o lu m e s o f a c e t o n e and s u c k e d d r y . The m at w as t h e n d r i e d b y p a s s i n g a i r o v e r i t a t room t e m p e r a t u r e o r b y e v a c u a t i o n i n a vacuum d e s i c c a t o r o v e r p a r a f f i n . w as g ro u n d A f t e r d r y i n g , t h e m at t o a p o w d e r b y means o f a m o r t a r and p e s t l e and s t o r e d a t -1 8 ° G i n a vacuum d e s i c c a t o r u n t i l r e a d y f o r u s e . A c t i v e e x t r a c t s w e re p r e p a r e d a s f o l l o w s : One gram o f t h e a c e t o n e d r i e d p o w d e r was s t i r r e d v i g o r o u s l y w i t h 20 ml o f 0 .1M p h o s p h a t e 22 b u f f e r pH 7 -U c o n t a i n i n g 10 M c y s te in e f o r f i f t e e n m in u te s. The p r e p a r a t i o n was t h e n c e n t r i f u g e d a t 2 2 ,0 0 0 x G f o r 30 m i n u t e s and t h e sed im en t d is c a r d e d . The s u p e r n a t a n t c o n t a i n e d t h e enzym ej 3 -2 gm p o w d e re d ammonium s u l f a t e was t h e n a d ded s l o w l y , w i t h s t i r r i n g , e a c h 10 ml o f e x t r a c t i n t h e c o l d (ca. to p©^ c e n t s a t u r a t i o n ) . The p r e c i p i t a t e w h ic h fo r m e d was c e n t r i f h g e d and r e s u s p e n d e d i n o n e - f o u r t h t h e o r i g i n a l v o lu m e o f t h e same b u f f e r . U) P a r t i a l P u r i f i c a t i o n The r e s u l t s o f a t y p i c a l f r a c t i o n a t i o n a r e shown i n T a b l e I . A l l s t e p s w ere c a r r i e d S t e p O n e: o u t a t 0 -2 ° G and c o n t i n u o u s l y . P r e p a r a t i o n o f t h e Crude E x t r a c t . The p l a n t m a t e r i a l fro m s e v e r a l f o u r t e e n h o u r c u l t u r e s was h o m o g e n iz e d i n 0.1M p h o s p h a t e _3 b u f f e r , pH 7 -U* c o n t a i n i n g 10 _4 M c y s t e i n e and 10 M v ersen e (5 ml b u f f e r p e r gm w e t w e i g h t ) b y g r i n d i n g w i t h g l a s s b e a d s ( s e e G e n e r a l M etho d s and M a t e r i a l s ) i n a S e r v a l l O m nim ixer a t 1 6 , 0 0 0 rpm , f o r t h r e e m in u te s. The O m n im ixer was c o o l e d b y means o f a s a l t - i c e m i x t u r e a t c a . -5>°C. A f t e r d e c a n t i n g fr o m t h e b e a d s t h e h o m o g e n a te was c e n t r i f u g e d i n t h e c o l d a t 2 2 ,0 0 0 x G f o r t h i r t y m i n u t e s i n a S e r v a l l h i g h s p e e d c e n trifu g e . The somewhat s t r a w c o l o r e d , s l i g h t l y t u r b i d s u p e r n a t a n t w as u s e d f o r t h e s u b s e q u e n t f r a c t i o n a t i o n . S t e p Two: F i r s t Ammonium S u l f a t e F r a c t i o n a t i o n (Ammonium s u l f a t e f r a c tio n A in ta b le ) . T w e n ty -tw o gm o f f i n e l y p o w d e re d ammonium s u l f a t e was ad ded s l o w l y , w i t h m e c h a n i c a l s t i r r i n g , o f crude e x t r a c t . t o e a c h 100 ml The p r e c i p i t a t e w h ic h fo rm e d was rem oved b y 23 c e n t r i f u g a t i o n and s i n c e i t was a lw a y s fo u n d t o b e w i t h o u t a c t i v i t y , d isc a rd e d . The s u p e r n a t a n t was t h e n t r e a t e d w i t h an a d d i t i o n a l 10 gm p e r 100 m l. t h e p r e c i p i t a t e c o l l e c t e d b y c e n t r i f u g a t i o n and r e d i s s o l v e d i n o n e - f o u r t h t h e o r i g i n a l vo lu m e o f 0 .02M p h o s p h a t e b u f f e r pH 6 . 0 . S te p T h re e ; 1955) * P r o t a m i n e S u l f a t e T r e a t m e n t ( G olow ick and K a p la n , A s o l u t i o n o f p ro ta m in e s u l f a t e (20 mgm p e r ml o f 0.02M p h o s p h a t e b u f f e r , pH 6 . 0 ) w as ad d ed s l o w l y , d r o p w i s e , w i t h s t i r r i n g to t h e s o l u t i o n f r o m S t e p Two s u c h a s t o g i v e a c o n c e n t r a t i o n o f 0 . 2 7 mgm p ro ta m in e p e r m illig ra m p r o t e i n . rem o v ed b y c e n t r i f u g a t i o n . w as o b t a i n e d . ta in e d tie s The h e a v y , f l o c c u l e n t p r e c i p i t a t e was Most o f t e n a r e l a t i v e l y c l e a r s u p e r n a t a n t U n le ss th e s o l u t i o n does c l e a r , p oor r e s o l u t i o n i s in th e fo llo w in g s te p . o f p ro ta m in e s u l f a t e ob­ When i t d i d n o t , a d d i t i o n a l s m a l l q u a n t i ­ ( 0 . 1 ml p e r 10 m l) w e re a d d ed f o l l o w e d b y c e n trifu g a lio n u n til i t d id . S te p F o u r: Seco n d Ammonium S u l f a t e F r a c t i o n a t i o n s u lfa te B in ta b le ) . (Ammonium The p r o t a m i n e s u l f a t e s u p e r n a t a n t was b r o u g h t t o 27 p e r c e n t s a t u r a t i o n b y t h e a d d i t i o n o f 19*5 gm o f s o l i d ammonium s u l f a t e p e r 100 ml c e n t r i f u g e d and t h e i n a c t i v e s e d im e n t d i s c a r d e d . An a d d i t i o n a l 5 *U gm p e r 100 ml o f t h e o r i g i n a l , v olu m e was t h e n i n t r o d u c e d and t h e p r e c i p i t a t e c o l l e c t e d b y c e n t r i f u g a t i o n and r e d i s s o l v e d i n o n e - f i f t h v o lu m e o f 0 .02M p h o s p h a t e b u f f e r pH 5 * 6 . S te p F i v e * N e g a t i v e A d s o r p t i o n w i t h C alciu m P h o s p h a t e G e l . C a lc iu m p h o s p h a t e g e l w as p r e p a r e d a s d e s c r i b e d b y K e i l i n and H a r t r e e (1938) . S m a ll a l i q u o t s o f t h e s e c o n d ammonium s u l f a t e f r a c t i o n w e r e 2U f i r s t tr e a te d w ith v a rio u s q u a n titie s m l) t o d e t e r m i n e w h a t r a t i o o f t h e g e l s o l u t i o n ( 1 8 . 6 mgm/ o f g e l t o p r o t e i n w o u ld g i v e maximum a d so rp tio n of in a c tiv e p r o te in w ith o u t a p p re c ia b le a d so rp tio n of is o ­ c itrita se . I t was f o u n d t h a t , i n e v e r y t r i a l m ade, 1 . 0 mgm o f g e l p e r mgm p r o t e i n was o p t i m a l . The s t a n d a r d p r o c e d u r e t h e n , was t o add s l o w l y , w i t h s t i r r i n g , en o u g h o f a s o l u t i o n o f c a l c i u m p h o s p h a t e g e l t o g i v e a g e l t o p r o t e i n ra tio of one. The s u s p e n s i o n was t h e n e q u i l i b r a t e d b y s t i r r i n g by hand f o r f i f t e e n m in u te s . A t t h e end o f t h a t t i m e t h e g e l was rem oved b y c e n t r i f u g a t i o n and d i s c a r d e d . S te p S i x : T h i r d Ammonium S u l f a t e F r a c t i o n a t i o n (Ammonium s u l f a t e C ) . To t h e g e l s u p e r n a t a n t fro m S t e p F o u r was add ed 6 . h ml o f a s a t u r a t e d s o l u t i o n o f ammonium s u l f a t e e x tra c t. ( a d j u s t e d t o pH 7 . 5 ) t o e a c h 10 ml o f The s o l u t i o n was t h e n c e n t r i f u g e d and t h e s e d im e n t d i s c a r d e d . An a d d i t i o n a l 3*1 m l o f t h e ammonium s u l f a t e s o l u t i o n p e r 10 ml was t h e n ad ded and t h e s e d i m e n t c o n t a i n i n g t h e a c t i v i t y r e d i s s o l v e d i n 0.1M -3 _4 . p h o s p h a t e b u f f e r , pH 7 *b3 c o n t a i n i n g 10 M c y s t e i n e and 10 M v e r s e n e . 5) I d e n tif ic a tio n of G ly o x y la te G l y o x y l a t e was i d e n t i f i e d as t h e k e to a c id p ro d u c t of th e e n zy m a tic r e a c t i o n by c h ro m a to g ra p h y as i t s 2 , l i - d i n i t r o p h e n y l h y d r a z o n e (DNPH) a c c o r d i n g t o t h e m etho d o f C a v a l l i n i ( 1 9 5 0 ) , i n f o u r d i f f e r e n t s o l v e n t s , a s w e l l a s b y tw o d i m e n s i o n a l c o - c h r o m a t o g r a p h y i n p h e n o l and b u t a n o l p ro p io n ic a c id . G l y o x y l a t e 2 , U ~ d i n i t r o p h e n y l h y d r a z o n e g a v e tw o s p o t s i n b u t a n o l - e t h a n o l - w a t e r and i n p h e n o l and b u t a n o l - p r o p i o n i c a c i d . 25 TABLE I PARTIAL PURIFICATION OF ISOCITRITASE F ra c tio n Mgm P /m l S p e c ific A c tiv ity R ecov ery ' (p e r c e n t) P u rific a tio n (fo ld ) 1 Crude E x t r a c t H .i 0 .31 100 1 2 Arnm. S . (A) 31-U556 S a t . 7*6 0 .6 0 98 2 3 P r o ta m i n e S u p e r n a t a n t 2.U 1 .7 0 91 5 U Arnm . S . ( B) 27-3$% S a t . 3-0 U.oo 65 13 5 Gel S u p e r n a t a n t 1 .0 8 .30 kk 27 6 Arnm . S . ( C) 31-U056 0 .8 1 6 .2 0 26 52 C o n d itio n s — R e a c tio n m ix tu re c o n ta in e d : 5 uM c y s t e i n e HC1; 10 uM MgCl2; 200 uM p h o s p h a t e b u f f e r pH 7 -hi UO uM d , l - i s o c i t r a t e ; i s o c i t r i t a s e , c a . one u n i t . T o t a l v o lu m e 3*1 m l; T e m p e r a t u r e , 30 C. ' ' ( T o t a l u n i t s / t o t a l num ber o f u n i t s i n o r i g i n a l e x t r a c t ) x 1 0 0 . 26 It g e n e r a l l y g a v e o n l y one s p o t i n b u t a n o l - a c e t i c a c i d b u t o c c a s i o n a l l y gave e v id e n c e of tw o . The m o b i l i t i e s o f t h e 2 , U - d i n i t r o p h e n y l h y d r a z o n e s o f a - k e t o g l u t a r a t e , g l y o x y l a t e , p y r u v a t e and o x a l a c e t a t e a r e l i s t e d in T a b le I I . G l y o x y l a t e was a l s o i d e n t i f i e d a b s o r p t i o n sp ec tru m o f i t s a s t h e p r o d u c t b y means o f t h e 2 , Ij-d in itro p h e n y lh y d ra z o n e as w e ll as th e a l k a l i i n s t a b i l i t y of th e l a t t e r . The s p e c t r u m o f t h e g l y o x y l a t e d e r i v a t i v e e x h i b i t s a n a b s o r p t i o n maximum a t U55 mu a ^d i s r e a d i l y d i s ­ t i n g u i s h a b l e fro m t h o s e o f t h e d i n i t r o p h e n y l h y d r a z o n e s o f p y r u v a t e and a - k e t o g l u t a r a t e w h ic h do n o t e x h i b i t an y m arked a l k a l i i n s t a b i l i t y . The a b s o r p t i o n s p e c t r a o f t h e DNPH d e r i v a t i v e s o f t h e unknow n, g l y o x y l a t e , a - k e t o g l u t a r a t e and p y r u v a t e a r e shown i n F i g u r e s l a , l b , l c and I d . 6) S t o i c h i o m e t r y and R e v e r s i b i l i t y D e t e r m i n a t i o n s on s t o i c h i o m e t r y w e re c a r r i e d s u l f a t e p r e p a r a t i o n fro m a c e t o n e e x t r a c t s . o u t u s i n g t h e ammonium The r e s u l t s a r e g i v e n i n T a b l e I I I and a r e i n a g r e e m e n t w i t h t h e e s t a b l i s h e d r e a c t i o n : d -iso c itra te y —— g ly o x y la te A ls o i n t h e same t a b l e a r e t h e r e s u l t s ^ su c c in a te of d e te rm in a tio n s of is o - c i t r a t e d i s a p p e a r a n c e and g l y o x y l a t e p r o d u c t i o n o b t a i n e d i n r u n s u s i n g ammonium s u l f a t e f r a c t i o n s The r e v e r s i b i l i t y o f e x t r a c t s fro m f r e s h m a t e r i a l . o f t h e r e a c t i o n was c o n f ir m e d b y i n c u b a t i n g 20 uM e a c h o f g l y o x y l a t e and s u c c i n a t e f o r one h o u r i n t h e u s u a l i n c u b a t i o n m i x t u r e c o n t a i n i n g t h e a c e t o n e enzy m e. m ic r o m o le s o f i s o c i t r a t e w e r e p r o d u c e d . Two and o n e - h a l f 27 TABLE I I R F’S OF 2 , 1'- DINITROPHENYLHYDRAZONES K eto Acid Phenol G ly o x y la te S o lv e n t B u ta n o lE th an o l 1 .6 5 •3 9 •5o 2 •7 3 •U3 •5 7 1 2 O x alacetate .6 1 B u tan o lA c e tic .7 7 P ro p a n o lAmmonia .7 6 -83 .8 8 .8 0 •9 5 .8 5 j CVI a-K eto g lu tu ra te oo P y ru v a te B u tan o lP ro p io n ic .6 1 •3 0 .5 8 P h e n y l h y d r a z o n e s w e r e p r e p a r e d and c h r o m a to g r a p h e d a c c o r d i n g t o t h e m ethod o f C a v a l l i n i ( 1 9 5 0 ) . The i s o c i t r i t a s e in a l l so lv e n ts. r e a c t i o n p r o d u c t had t h e same m o b i l i t i e s a s g l y o x y l a t e 2b TABLE ITT ISOCITRITASE-STOICHIOMETRY Enzyme d -Iso c itra te G ly oxy3.at e Su c c i n a t e k- 9 U.o 5*0 5-U 5-3 U -i 3 .8 5*2 U .o — U *9 — Ammonium S u lfa te -A c e to n e 1 2 Ammonium S u lfa te A C o n d itio n s: As f o r i s o c i t r i t a s e a s s a y e x c e p t t h a t i n c u b a t i o n was f o r one h o u r ; enzyme c a . 2 u n i t s . 29 7 ) Enzyme P r o p e r t i e s S ta b ility : F r o z e n m a ts and a c e t o n e p o w d e rs o f B l a s t o c l a d i e l l a may b e s t o r e d f o r s e v e r a l w e ek s w i t h o u t a p p r e c i a b l e l o s s o f a c t i v i t y . Ciu.de e x t r a c t s s t o r e d a t U°G l o s e a c t i v i t y w i t h v a r i a b l e r a p i d i t y ( o f t e n a s much a s 30-U-0 p e r c e n t i n f o u r h o u r s ) . p ro te o ly tic T h i s may b e d u e t o a c t i v i t y i n t h e e x t r a c t s f o r t h e r e i s some a c c u m u l a t i o n o f n in h y d rin p o s itiv e su b sta n c e s. P a r tia lly p u rifie d enzyme s o l u t i o n s may b e s t o r e d f o r some w eek s a t - l 8 °C w i t h r e l a t i v e l y l i t t l e 50 p e r c e n t lo ss in a c ti v ity . H o w ever, a b o u t i s l o s t on o v e r n i g h t s t o r a g e a t U°C. R e a c tio n R a te: As d e l i n e a t e d i n F i g u r e 2 t h e r a t e o f g l y o x y l a t e f o r m a t i o n i s d i r e c t l y p r o p o r t i o n a l t o enzyme c o n c e r r t r a t i o n up t o a b o u t tw o u n i t s . C o n se q u e n tly , a l l a s s a y s o f en zy m atic a c t i v i t y w ere k e p t i n t h e r a n g e o f a b o u t one u n i t . re a c tio n is The t i m e c o u r s e o f t h e i s o c i t r i t a s e show n i n F i g u r e 3• E f f e c t o f pH : The i s o c i t r i t a s e of B l a s t o c l a d i e l l a has a r a t h e r s h a r p pH optimum a t pH 1 •h a s shown i n F i g u r e I4 . w e re c a r r i e d A ll e n zy m a tic a s s a y s o u t a t t h i s pH. S p e c ific ity : Though n e i t h e r p u r e d - i s o c i t r a t e n o r 1 - i s o c i t r a t e w e r e a v i l a b l e f o r t e s t i n g t h e s p e c i f i c i t y o f t h e p u r i f i e d e nzy m e, s p e c i f i c i t y f o r th e d -is o m e r is im p lie d b y th e s to ic h io m e tr y d a t a . T h is f o l l o w s fro m t h e f a c t t h a t t h e enzyme p r e p a r a t i o n u s e d f o r t h e d e t e r m i n ­ a tio n of i s o c i t r a t e i s s p e c i f i c f o r th e d -iso m e r w h ile b o th iso m ers w e r e a lw a y s p r e s e n t i n t h e r e a c t i o n m i x t u r e i n e q u a l c o n c e n t r a t i o n s . 30 Crude e x t r a c t s from R .S . and O.C. p l a n t s p r e p a r e d u s i n g t h e Qmnimixer d id n o t p r o d u c e any 2 , L j - d i n i t r o p h e n y l h y d r a z o n e - f o r m i n g compound when i n c u b a t e d w i t h c i t r a t e o r c i s ^ a c o n i t a t e . T h is i s of p a r t i c u l a r i n t e r e s t s i n c e v a r i o u s w o rk e rs (D a g le y and Dawes, 1955$ S m ith e t a l . , 1956; and Wheat and A j l , 1955) h a v e r e p o r t e d an enzyme from b a c t e r i a , c i t r i t a s e , w hich c l e a v e s c i t r a t e t o o x a l a c e t a t e and a c e ta te . M o reo v er, o x a l a c e t a t e was n e v e r d e t e c t e d as a p r o d u c t o f i s o c i t r a t e u t i l i z a t i o n by c ru d e e x t r a c t s p r e p a r e d u s i n g t h e g l a s s homo­ g e n i z e r o r from a c e t o n e pow ders o f o r d i n a r y c o l o r l e s s p l a n t s th o u g h our c o n d i t i o n s w ere such t h a t i t would have b e e n d e t e c t e d and C a n tin o (1 9 5 3 c ) d e m o n s tr a t e d t h e p r e s e n c e o f a c o n i t a s e a c t i v i t y i n su ch p r e p a r a t i o n s . N e i t h e r d i d t h e y p r o d u c e o x a l a c e t a t e from c i t r a t e as d e t e r m in e d by l i g h t a b s o r p t i o n m easu rem ents a t 280 mu (Greenwood and Greenbaum, 1953)* C o - f a c t o r R e q u i r e m e n ts : I s o c i t r i t a s e r e q u ire s a m e ta llic io n f o r maximum a c t i v i t y as w e l l as a s u l f h y d r y l g ro u p . G l u t a t h i o n e was found t o b e o n ly c a . 60 p e r c e n t as e f f e c t i v e as c y s t e i n e a t t h e c o n c e n t r a t i o n g e n e r a l l y u s e d (T a b le TV) . Though Mg was r o u t i n e l y employed a s t h e m e t a l l i c c o f a c t o r th r o u g h o u t t h e s e s t u d i e s Mn' was found e q u a l l y e f f e c t i v e when u s e d w i t h t r i s b u f f e r a t t h e same c o n c e n t r a t i o n . The c o f a c t o r s a t u r a t i o n c u r v e s f o r b o t h Mg and c y s t e i n e a r e _3 shown i n F i g u r e s 5^- and 5 b . B o th gave maximum a c t i v i t y a t jca. 10 M c o n c e n tra tio n . E f f e c t of S u b s tr a te C o n c e n tra tio n : When i s o c i t r a t e c o n c e n t r a t i o n ( a s t h e d - is o m e r ) was p l o t t e d a g a i n s t r e a c t i o n v e l o c i t y a t y p i c a l s u b s t r a t e c o n c e n t r a t i o n c u rv e was o b t a i n e d . R earran g em en t o f t h e d a t a 31 TABLE TV ISOCITRITASE: S u I f h y d r y 1 Comp ound uM G ly o x y la te p e r 10 m i n u t e s C o n d itio n s: COFACTOR REQUIREMENTS A C y ste in e B G lu ta th io n e 1.2.3 0.77 As f o r i s o c i t r i t a s e a s s a y ; en zy m e, a c e t o n e e x t r a c t d i a l y z e d U h o u r s a g a i n s t 0 .1M p h o s p h a t e b u f f e r , pH 7*U* TABLE V IN VITRO EFFECT OF LIGHT ON ISOCITRITASE L ig h t uM G l y o x y l a t e p ro d u c e d i n 10 m in u te s '' 1 .2 6 * Dark 1 .2 7 "A vg. o f tw o d e t e r m i n a t i o n s . C o n d itio n s: As f o r i s o c i t r i t a s e a s s a y ; e x c e p t t h a t t h e l i g h t t u b e s w e re s u b j e c t e d t o l i g h t o f i n t e n s i t y 300 f o o t c a n d l e s . D ark w e r e s h i e l d e d w i t h alum inum f o i l . Enzym e, Ammonium S u l f a t e C. 32 i n t h e f o l l o w i n g fo rm o f t h e M l c h a e l i s e q u a t i o n ( L i n e w e a v e r and B u r k , I 9 3 I4 ) p e r m i t t e d a g r a p h i c a l e s t i m a t i o n o f t h e M i c h a e l i s c o n s t a n t a s U*8 x 10 M ( F i g u r e 6) . [ S ] _ ^ E f f e c t of T e m p e ra tu re : . [ S ] * Km_ v \/ D e term in atio n s o f th e r e a c t i o n r a t e s a t s e v e r a l te m p e ratu res p e rm itte d a g ra p h ic a l e s tim a tio n of th e a c ti v a tio n e n e r g y o f t h e enzyme c a t a l y z e d r e a c t i o n a c c o r d i n g t o t h e A r r h e n i u s e q u a t i o n ( N e i l a n d s and S tu m p f , 1958; A r r h e n i u s , 1 9 1 5 ) !°g K = E = 10 .,700 c a l o r i e s p e r m o le . E ffe c t of L ig h t: ft ( j ) L C ( F i g u r e 7) The Q10 o f t h e enzyme r e a c t i o n i s 1 . 8 . The i n t e n s i t y o f t h e i n c i d e n t l i g h t on t h e i l l u m i n a t e d t u b e s was c a . 300 f o o t c a n d l e s . L i g h t was e x c l u d e d fro m t h e d a r k t u b e s b y w r a p p i n g i n alum inum f o i l . As shown i n t h e t a b l e t h e r e was no d i f f e r e n c e b e tw e e n t h e q u a n t i t i e s o f g l y o x y l a t e p r o d u c e d in th e l ig h t and t h e d a r k . 33 FIGURE LEGENDS F ig u r e l a - I d . A d so rp tio n S p e c tra of th e 2 , U -d in itro p h e n y lh y d ra z o n e s of P y r u v a t e , a - K e t o g l u t a r a t e , G l y o x y l a t e and t h e P r o d u c t o f th e I s o c i t r i t a s e R e a c tio n . The m e a s u r e m e n ts w e r e made a f t e r t r e a t m e n t o f t h e k e t o - a c i d s w i t h 2 , U- d i n i t r o p h e n y l h y d r a z i n e a s d e s c r i b e d i n A n a l y t i c a l M e th o d s . F ig u re 2 . Is o c itrita se : Enzyme A s s a y C u rv e . c rib e d f o r th e i s o c i t r i t a s e a ssa y . F i g u r e 3- Time Course o f I s o c i t r i t a s e R e a c t i o n . C o n d itio n s as d e sc rib e d fo r th e i s o c i t r i t a s e assay except th a t in c u b a tio n was a s i n d i c a t e d . F i g u r e U- Is o c itrita s e : pH Optimum. C o n d i t i o n s a s f o r i s o c i t r i t a s e a s s a y e x c e p t t h a t a s e r i e s o f 5 m in u te i n c u b a t i o n s was used a t th e in d ic a te d p H 's. Enzyme, Ammonium S u l f a t e C. C o n d itio n s as S e e T a b le I . des­ F ig u re 5 a~ 5b. I s o c i t r i t a s e : C o fa c to r R eq u irem en ts. C o n d itio n s as f o r i s o c i t r i t a s e a s s a y . F i g u r e 5a-, c y s t e i n e - H C l v a r i e d ; F i g u r e 5 6 , m agnesium c h l o r i d e v a r i e d . Enzyme, Ammonium S u l f a t e C. F ig u re 6 . F ig u re 7 . Is o c itrita se : E f f e c t of S u b s tra te C o n c e n tra tio n . C o n d itio n s as f o r i s o c i t r i t a s e a s s a y e x c e p t t h a t s u b s t r a t e c o n c e n t r a t i o n s w ere as i n d i c a t e d . Enzyme, Ammonium S u l f a t e C Iso c itrita se : A c tiv a tio n E nergy. C o n d itio n s as f o r i s o ­ c i t r i t a s e a s s a y e x c e p t t h a t t e m p e r a t u r e s w e re a s i n d i c a t e d . Enzyme, Ammonium S u l f a t e C. 3h SPECTRUM OF PYRUVATE 2 , 4 — DNPH SPECTRUM OF ofKETOOLUTRATE 2 , 4 - D N P H .200 F ig u re l a F ig u re lb OPTICAL DENSITY .1oo SPECTRUM OF 2 , 4 - DNPH OF UNKNOWN SPECTRUM OF GLYOXYLATE 2 ,4 - D N P H .4 0 0 F ig u r e 1 c F ig u re Id O TIME .3 0 0 A FT E R ca EO M INS. O TIME A F T E R ca EO M INS. .200 A h im X ma x > 4 5 5 w y t 400 450 500 550 400 450 WAVELENGTH C m y u ] 500 ■ 4 55 nri/* - 550 35 4.0 ISOCITRITASE: ENZYME ASSAY CURVE TIME COURSE OF ISOCITRITASE REACTION Figure 2 ■" M0LES GLYOXYLATE / 10 MINS. s.o Id 5 > X §o a4 2.0 IO 1.0 S.O 4.0 ISOCITRITASE S.O is U N IT S 30 4B •0 78 TIME ,M M IN U T E 8 ISOCITRITASE: COFACTOR REQUIREMENTS ISOCITRITASE-. pH OPTIMUM F ig u re 5 a 9 MING. Figure U 4.M0LE8 GLYOXYLATE/ iM CYSTEINE /■■. F i g u r e £b S.0 10.0 I i.o 10 3.0 4.0 SO SO 36 F ig u re 6 ISOCITRITASE: EF FE CT OF S UBST RAT E CONCENTRATION (Km = 4 . 8 X I 0 * 4 M) 50 40 30 20 1.0 2.0 3.0 4 .0 5.0 6.0 CsT x io® ISOCITRITASE: ACTIVATION ENERGY A = 1 0 , 7 0 0 CAL. PER MOLE O X > h- o o — i UJ > z o t~ o< U oci 0.5 u. O o O o — I .0 0 3 2 .0 0 3 3 .0 0 3 4 RECIPROCAL OF ABSOLUTE TEMPERATURE .0 0 3 5 (|/T ) 37 PART IT G l y c i n e - A l a n i n e T ra n s a m in a se 1) A n a l y t i c a l Methods Amino A c i d s : G ly c in e and a l a n i n e w ere d e te r m in e d by e l u t i o n o f t h e i r n i n h y d r i n r e a c t i o n p r o d u c t s from p a p e r c h ro m ato g ram s, f o llo w e d b y s p e c t r o p h o t o m e t r i c m easurem en ts a t 575 wu, a c c o r d i n g t o t h e method of Kay e t a l . ( 1 9 5 6 ) . The chrom atogram s f o r q u a n t i t a t i v e work w ere r u n u n i d i r e c t i o n a l l y u s i n g Whatman No. 1 f i l t e r p a p e r w h ich had b e e n p r e v i o u s l y w ashed . The wash was c a r r i e d o u t u s i n g IN a c e t i c a c i d f o ll o w e d by t h r e e r i n s e s i n g l a s s d i s t i l l e d w a t e r and one i n p r o p a n o l am m o nia-w ater ( 6 t 3 : l ) . The l a t t e r was u s e d as t h e s o l v e n t sy stem f o r a l l q u a n t i t a t i v e work . F o r t h e q u a l i t a t i v e d e t e c t i o n o f amino a c i d s i n v o lv e d i n t r a n s ­ a m i n a t i o n r e a c t i o n s , o n e - d i m e n s i o n a l p a p e r chrom atogram s w ere r u n u s i n g b u t a n o l - e t h a n o l - w a t e r (l_|.:l:5 )j p r o p a n o l- a m m o n ia - w a te r ( 6 : 3 ?l ) and b u t a n o l - a c e t i c a c i d - w a t e r ( iqs 1 s5) an ^ t h e s p o t s d e t e c t e d b y s p r a y i n g w i t h 0 . 1 p e r c e n t n i n h y d r i n i n 95 p e r c e n t e th a n .o l. P y r u v i c A c id t P y r u v ic a c i d was d e te r m in e d c o l o r i m e t r i c a l l y a c c o r d ­ in g t o t h e method o f S tr a u b as o u t l i n e d by Green e t a l . s lig h t m o d ific a tio n . (19U5) w i t h To 1 . 0 ml o f t h e sam ple c o n t a i n i n g 0 . 1 t o 1 . 0 uM p y r u v a t e , i n a c a l i b r a t e d t e s t t u b e was added 1 . 0 ml p o t a s s i u m h y d r o x i d e (100 gm p e r 60 ml w a t e r ) , f o llo w e d by 0 . 5 ml o f 2 . 0 p e r c e n t s a l i c a l d e h y d e i n 95 P e r c e n t e t h a n o l . A f t e r a t e n m in u te i n c u b a t i o n a t 3 7 °G, t h e s o l u t i o n was made up t o 10 m l, c o o le d i n an i c e b a t h and 30 t h e c o l o r m e a s u re d u s i n g a N o. l±2. f i l t e r m e te r. As p o i n t e d o u t b y G re e n e t a l . i n a K le tt-S u m m e r s o n c o l o r i ­ (19U5>) t h e c o l o r y i e l d fro m a g iv e n am o u n t o f p y r u v a t e v a r i e d som ew hat fro m one d e t e r m i n a t i o n t o t h e n e x t in a fa s h io n n o t e a s ily c o n tr o lle d . I t w as t h e r e f o r e n e c e s s a r y t o i n c l u d e a p y r u v a t e s t a n d a r d i n a l l d e t e r m i n a t i o n s an d t o c a l c u l a t e t h e am ount i n t h e s a m p le t h e r e f r o m . K l e t t r e a d i n g s w e re fo u n d t o b e a r a l i n e a r r e l a t i o n s h i p t o p y r u v a t e c o n c e n t r a t i o n u p t o ^ a . 1 . 0 uM i n a 1 .0 ml s a m p le . The r e a c t i o n w as s p e c i f i c f o r p y r u v a t e . G ly o x y l i c and P y r u v i c A c i d s ; G l y o x y la t e and p y r u v a t e w e re a s s a y e d s p e c t r o p h o t o m e t r i c a l l y i n m i x t u r e s a s t h e i r 2 ,U “ d i n i t r o p h e n y l h y d r a z o n e s u s i n g t h e sam e p r o c e d u r e d e s c r i b e d p r e v i o u s l y f o r d e t e r m i n i n g g l y o x y l a t e a lo n e . From t h e s t a n d a r d c u r v e s o b t a i n e d f o r t h e DNPH d e r i v a t i v e s o f e a c h a c i d a t lj.90 a nd 5 U0 Mu* a s im u lt a n e o u s e q u a t i o n w as d e r i v e d w h ic h p e r m itte d th e c a l c u l a t i o n o f b o th t h e i r c o n c e n tr a tio n s : l . £ 6 x + 2 .2 £ y = A 1 .5>0x * 1 .]f7y = B w h e re ^ x - uM p y r u v a t e p e r s a m p le y = uM g l y o x y l a t e p e r sa m p le A = o p t i c a l d e n s i t y a t U90 mu B = o p t i c a l d e n s i t y a t 5U0 mu. G l y c i n e - A l a n i n e T r a n s a m in a s e : G l y c i n e - a l a n i n e t r a n s a m i n a s e w as d e te r m in e d i n a 1 . 0 m l a s s a y m ix tu r e c o n t a i n i n g : 10 uM so d iu m g l y o x y l a t e m o n o h y d ra te ; 10 uM L - a l a n i n e ; I4.0 uM p h o s p h a te b u f f e r , pH 8 . 0 ; 39 5 uM magnesium c h l o r i d e ; and _ca. 10 ug p y r i d o x a l - 5 - p h o s p h a t e . A fte r a t e n m in u te i n c u b a t i o n a t 3 7 °C, t h e r e a c t i o n was s to p p e d b y t h e a d d itio n of S trau b T est r e a g e n ts . The b l a n k f o r t h e c o l o r m easurem ent c o n t a i n e d a l l a d d i t i o n s e x c e p t enzym e. Such c o n tro l is n ecessary to c o r r e c t f o r a s m a l l amount o f c h e m ic a l t r a n s a m i n a t i o n w h ich o c c u r s b e tw e e n g l y o x y l a t e and a l a n i n e i n t h e s t r o n g a l k a l i u se d i n t h e t e s t . A u n i t o f t r a n s a m i n a s e a c t i v i t y = t h a t amount o f enzyme c a t a l y z i n g t h e p r o d u c t i o n o f 1 -0 uM p y r u v a t e i n t e n m in u te s i n t h e above p r o t o c o l . S p e c i f i c a c t i v i t y = t h e number o f u n i t s o f a c t i v i t y p e r m i l l i g r a m p ro te in . P r o t e i n was d e t e r m in e d as p r e v i o u s l y d e s c r i b e d . 2) P r e l i m i n a r y O b s e r v a t i o n s D u rin g t h e s t u d i e s on i s o c i t r i t a s e i t was fou n d t h a t , when u s i n g c r u d e p r e p a r a t i o n s and l o n g i n c u b a t i o n p e r i o d s (o n e t o two h o u r s ) , a - k e t o g l u t a r a t e and p y r u v a t e w ere f r e q u e n t p r o d u c t s o f s i d e r e a c t i o n s i n v o l v in g g ly o x y la te . I t seemed l i k e l y th^at a t r a n s a m i n a t i o n b e tw e e n g l y o x y l a t e and end og en ou s amino a c i d s was r e s p o n s i b l e . To v e r i f y t h i s a s s u m p tio n g l y o x y l a t e was i n c u b a t e d w i t h a number o f amino a c i d s ( L - g l u t a m a t e , L -g lu t^ im in e , L - a s p a r a t e , L - a l a n i n e , L - t y r o s i n e , L - l y s i n e , L - p r o l i n e , L - l e u c i n e , L - h i s t i d i n e , L - a r g i n i n e and L - t h r e o n i n e ) i n t h e p r e s e n c e of b o i l e d and u n b o i l e d c r u d e enzyme. A p p r o p r i a t e c o n t r o l s o m i t t i n g one o r t h e o t h e r presum ed s u b s t r a t e w ere a l s o i n c u b a t e d i n e a c h c a s e . T rans­ a m i n a t i o n was d e t e c t e d by means o f p a p e r c h ro m a to g ra p h y o f t h e r e a c t i o n m i x t u r e s f o ll o w e d by n i n h y d r i n s p r a y . An e n z y m a tic t r a n s a m i n a t i o n ho o c c u r r e d o n ly i n t h o s e r e a c t i o n m i x t u r e s c o n t a i n i n g L - a l a n i n e . E v id e n tly th e a - k e t o g l u t a r a t e d e te c te d d u rin g th e i s o c i t r i t a s e runs was p r o d u c e d p a r t i a l l y a s a r e s u l t of a d i r e c t c h e m ic a l t r a n s a m i n a t i o n b e tw e e n g l y o x y l a t e and endogenous g l u t a m a t e and p a r t i a l l y as a r e s u l t o f an e n z y m a tic t r a n s a m i n a t i o n m e d ia te d by a l a n i n e s i n c e an a l a n i n e g l u t a m a t e t r a n s a m i n a s e was a l s o p r e s e n t ; S in c e i t glyoxyla+.R a la n in e g ly c in e p y ru v ate L - g l u t a m a te seemed p o s s i b l e t h a t an a l a n i n e - g l y o x y l a t e t r a n s a m i n a t i o n m ig h t c o n s t i t u t e an i m p o r t a n t '’p u l l i n g ' ’ r e a c t i o n f o r t h e r e m o v a l of g l y o x y l a t e p ro d u c e d by i s o c i t r i t a s e , f u r t h e r s t u d i e s w ere c a r r i e d out on t h i s en zy m e. 3) P a r t i a l P u r i f i c a t i o n S te p One ; P r e p a r a t i o n o f c ru d e e x t r a c t . The p l a n t m a t e r i a l from f o u r lU h o u r c u l t u r e s o f B l a s t o c l a d i e l l a w ere h a r v e s t e d as d e s c r i b e d p re v io u sly . A f t e r w e i g h i n g , t h e mats w ere hom ogenized i n g l a s s d i s ­ _4 t i l l e d w a t e r (h ml/gin) c o n t a i n i n g 10 M v e rse n e by g rin d in g w ith g la s s b e a d s (U gm b ead s/g m p l a n t s ) i n an Omnimixer c o o le d i n a s a l t i c e b a t h ( - 5 ° C ) a t 1 6 ,0 0 0 rpm f o r t h r e e m i n u t e s . A f t e r d e c a n t i n g from t h e b e a d s t h e ho m ogenate was c e n t r i f u g e d a t 2 2 ,0 0 0 x G f o r 30 m in u te s and t h e s u p e rn a ta n t used f o r f r a c t i o n a t i o n . B e c a u s e o f t h e l a r g e amounts o f endogenous amino a c i d s i n t h e c r a d e e x t r a c t , no d i r e c t e s t i m a t i o n o f t h e i n i t i a l t r a n s a m i n a s e a c t i v i t y hi c o u ld b e m ade. The e x t r a c t was t h e r e f o r e d i a l y z e d f o r 20 h o u r s a g a i n s t 0 .2M p h o s p h a te b u f f e r * pH 7 *h> at k°C* a f t e r w hich i t was a s s a y e d f o r a c tiv ity . S in c e d i a l y s i s alw ay s lo w e re d t h e p r o t e i n c o n c e n t r a t i o n th ro u g h ap p aren t d e n a tu r a tio n of in a c tiv e p ro te in * th e i n i t i a l s p e c i f i c a c t i v i t y was e x p r e s s e d a s t h e number o f m icro m o les o f p y r u v a t e p r o d u c e d by th e d ia ly z e d p r e p a r a t i o n p e r m illig ra m p r o te in in th e o r i g i n a l u n d ialy zed p r e p a r a tio n . T h is was p o s s i b l e s i n c e i t was s u b s e q u e n t l y fo u n d t h a t ev e n p r o lo n g e d d i a l y s i s of t h e enzyme i n w a t e r or b u f f e r d id n o t a f f e c t th e tra n sa m in a se a c t i v i t y . A s i m i l a r p r o c e d u r e was u s e d f o r t h e p r o ta m i n e s u l f a t e f r a c t i o n . S te p Two: P r o ta m in e S u l f a t e T re a tm e n t A. 0 .7 5 ml o f a p r o ta m i n e s u l f a t e s o l u t i o n (20 mgm/ml) was added slow ly* w i t h s t i r r i n g f o r e v e r y 100 mgm o f c ru d e e x t r a c t p r o t e i n a t 0°G. A fter a d d i t i o n a l t e n m in u te s t h e a b u n d an t p r e c i p i t a t e s tir rin g for an w hich form ed was removed b y c e n t r i f u g a t i o n a t 30*000 x G f o r 15 m i n u t e s . S te p T h r e e ; F i r s t A ceto ne F r a c t i o n a t i o n . W ith t h e t e m p e r a t u r e of t h e e x t r a c t m a i n t a i n e d a t -2°G t o 0°G enough a c e to n e (-1 0 ° C ) was added r a p i d l y d ro p w is e t o b r i n g t h e c o n c e n t r a t i o n t o 32 p e r c e n t . The s o l u t i o n was t h e n c o o le d t o - 8 °G and more a c e to n e added t o b r i n g t h e c o n c e n t r a t i o n to h i Per c e n t. A f t e r a l l o w i n g t h e t e m p e r a t u r e t o r i s e t o - 6 °C, t h e i n a c t i v e p r e c i p i t a t e w hich form ed was removed by c e n t r i f u g a t i o n a t 6*000 x G f o r t h r e e m in u te s a t - 6 °G. The c o n c e n t r a t i o n o f a c e to n e i n t h e s u p e r n a t a n t was t h e n b r o u g h t t o U? P a r c e n t w h i l e m a i n t a i n i n g t h e t e m p e r a t u r e a t - 1 0 ° C . A fte r ag ain U2 a l l o w i n g t h e t e m p e r a t u r e t o r i s e t o - 6 ° C, t h e p r e c i p i t a t e was removed b y c e n t r i f u g a t i o n as b e f o r e and d i s c a r d e d . The a c t i v e f r a c t i o n was t h e n o b t a i n e d by b r i n g i n g t h e a c e to n e c o n c e n t r a t i o n t o 57 p e r c e n t a t - 1 0 ° C . A fte r a llo w in g th e te m p e ra tu re t o r i s e t o - 6 °C, the? s o l u t i o n was- c e n t r i f u g e d and t h e s e d im e n t r e s u s ­ p en d e d i n o n e - f i f t h t h e o r i g i n a l volume of g l a s s d i s t i l l e d w a t e r . The s l i g h t l y t u r b i d , somewhat s t r a w - c o l o r e d s o l u t i o n t h u s o b t a i n e d , was t h e n d i a l y z e d 20 h o u r s a g a i n s t t h r e e ch an g es o f 100 volum es o f d i s t i l l e d w a t e r c o n t a i n i n g 10 S tep F o u r : -4 0 M v e r s e n e a t 1 G. N e g a tiv e A d s o r p t i o n w i t h Calcium P h o s p h a te G e l. Enough o f a c a lc iu m p h o s p h a te g e l s o l u t i o n ( 1 8 .6 mgm/ml) was added t o t h e d i ­ a l y z e d a c e t o n e f r a c t i o n a t 0°G t o g i v e a g e l - p r o t e i n r a t i o o f 2 mgm g e l / mgm p r o t e i n . A f t e r a l l o w i n g a d s o r p t i o n e q u i l i b r i u m t o be r e a c h e d by s t i r r i n g f o r 15 m in u te s b y hand t h e g e l was removed by c e n t r i f u g a t i o n and d i s c a rd ed . The p r o c e s s was r e p e a t e d once more u s i n g t h e same g e l - p r o t e i n r a t i o and t h e s u p e r n a t a n t o b t a i n e d by c e n t r i f u g a t i o n u s e d i n t h e n e x t s te p . S tep F i v e : F i n a l A cetone F r a c t i o n a t i o n . In o r d e r t o c o n c e n t r a t e t h e t r a n s a m i n a s e and o b t a i n some a d d i t i o n a l p u r i f i c a t i o n a second a c e to n e p r e c i p i t a t i o n was c a r r i e d o u t . W h ile m a i n t a i n i n g t h e t e m p e r a t u r e a t -5 °C t o 0 °G enough a c e t o n e ( - 10 °G) was added t o g iv e a c o n c e n t r a t i o n o f 62 p e r c e n t . The p r e ­ c i p i t a t e w hich form ed was o b t a in e d by c e n t r i f u g a t i o n and r e s u s p e n d e d i n U3 o n e - f o u r t h volum e g l a s s d i s t i l l e d w a t e r . The r e s u l t i n g enzyme s o l u t i o n was t h e n d i a l y z e d f o r 20 h o u r s a g a i n s t t h r e e c h an g es o f 500 volum es of d is tille d w a te r. The r e s u l t s o f a t y p i c a l t r a n s a m i n a s e p u r i f i c a t i o n a r e shown i n T a b le V I . U) S t o i c h i o m e t r y , R e v e r s i b i l i t y and I d e n t i f i c a t i o n o f P r o d u c ts * P y r u v a t e was v e r i f i e d as t h e k e t o a c id p r o d u c t of t h e t r a n s a m i n a ­ t i o n r e a c t i o n b e tw e e n g l y o x y l a t e and a l a n i n e by means of p a p e r chroma­ to g ra p h y of i t s 2,U -d in itro p h e n y lh y d ra z o n e in b u ta n o l- e th a n o l- w a te r a c c o r d i n g t o t h e method o f G a v a l l i n i (1950) a nd by t h e c o l o r o b t a in e d i n th e s p e c i f i c q u a n t i t a t i v e s a lic y la ld e h y d e r e a c t i o n of S tra u b (s e e A n a l y t i c a l M e th o d s ) . G ly c in e was i d e n t i f i e d by means o f p a p e r chroma­ t o g r a p h y i n p r o p a n o l ammonia, b u t a n o l - e t h a n o l - w a t e r and b u t a n o l - a c e t i c a c id -w a te r. The m o b i l i t i e s o f a l l r e a c t a n t s a r e l i s t e d i n T a b le V I I . D e t e r m i n a t i o n s o f a l l r e a c t a n t s a f t e r one and two h o u r i n c u b a t i o n s o f g l y o x y l a t e and a l a n i n e w i t h enzyme as shown i n T a b le V I I I , p e r m i t t e d d e f i n i t i o n of th e r e a c tio n as: G ly o x y l a te -h L - A la n in e ■^ G ly c in e -t P y r u v a te When an a t t e m p t was made t o d e m o n s t r a t e r e v e r s i b i l i t y b y i n c u b a t i n g 10 uM p y r u v a t e and 10 uM g l y c i n e i n t h e u s u a l r e a c t i o n m i x t u r e , no d i s a p p e a r a n c e o f p y r u v a t e c o u ld be d e t e c t e d n o r was a l a n i n e p r o d u c e d . T h i s , i n s p i t e o f t h e f a c t t h a t t h e Mfo rw a rd " r e a c t i o n p r o c e e d e d t o o n ly c a . o n e - th ir d c o n v e rsio n (s e e F ig u re 9 ) . l i k e l y t h a t p y r u v a t e o r g l y c i n e was i n h i b i t o r y . I t t h e r e f o r e seemed uu TABLE VI RESULTS OF TRANSAMINASE PURIFICATION P ro te in C o n c e n tra tio n (mgm/ml) F r a c t io n S p e c ific A c tiv ity O v er-all" P u r if i c a t i o n R ec o v ery F a c t or (p e r c e n t) 1 . Crude E x t r a c t 5 -3 0 .6 8 100 1 2 . P r o ta m i n e S u l f a t e 2 .7 1 .U0 103 2 3 . A c e to n e I 2 .2 1 1 .0 96 16 1;. G el S u p e m a t e B 0 .7 29.0 100 no 5 . A ceto n e I I 0 .9 5 5 .o 60 80 C o n d itio n s; "(T o ta l 10 uM sodium g l y o x y l a t e m o n o h y d ra te ; 10 uM L - a l a n i n e ; I4.O uM p h o s p h a te b u f f e r , pH 8 . 0 ; £ uM MgCl2; 10 u g p y r i d o x a l - 5 - p h o s p h a t e . Enzyme,, < 0 . 7 u n i t s . T o tal volume 1 m l. T e m p e ra tu re 37 C. R e a c t i o n s to p p e d a t 10 m in u te s b y a d d i t i o n of S t r a u b r e a g e n t s . u n i t s / t o t a l number o f u n i t s i n o r i g i n a l e x t r a c t ) x 100 US TABLE V II RF'S OF TRANSAMINASE REACTANTS S o lv en t R e a c ta n t B u tan o l-E th an o l G l y o x y l a t e (2,1+ DNPH) o . u o , 0 .5 7 P y r u v a t e (2,1+ DNPH) 0 . 1*8 , 0 .6 1 P r o p a n o l Ammonia B u ta n o l A c e tic G ly c in e O .36 O.lhL 0 .1 5 A la n in e 0 . J_l2 O.^O 0 .2 2 TABLE V I I I STOICHIOMETRY OF GLYOXYLATE-ALANINE TRANSAMINATION P y ru v a te uM A la n in e uM G ly o x y la t e uM Time G ly c in e uM 1 hr 2 .9 2 .7 * 2 .5 2 .8 2 hrs 3 -5 3 .6 * 3-3 3 .2 C o n d itio n s: As f o r t r a n s a m i n a s e a s s a y e x c e p t t h a t i n c u b a t i o n was f o r t i m e s i n d i c a t e d . R e a c t io n s to p p e d w i t h 0 .0 9 ml 2N HC1. Enzyme: Gel s u p e r n a t a n t B . "'D eterm ined b y c a l c u l a t i o n from o p t i c a l d e n s i t i e s a t H90 and 5U0 mu and S t r a u b t e s t ( a v e r a g e d ) . U6 R e v e r s i b i l i t y was s u b s e q u e n t l y d e m o n s t r a te d by means of t h e f o l l o w ­ in g e x p e r i m e n t s . Two p a r a l l e l s e r i e s o f f o u r r e a c t i o n t u b e s each w ere s e t up a c c o r d i n g t o t h e u s u a l p r o t o c o l and a f t e r i n i t i a t i o n o f t h e r e a c t i o n a t u b e from each s e t was s to p p e d a t f i v e m in u te i n t e r v a l s up t o 20 m i n u t e s . I n one s e t 20 uM g l y c i n e i n 0 . 1 ml was added a t t e n m in u te s t o t h e t u b e s t o b e s to p p e d a t l £ and 20 m i n u t e s . The r e s u l t s o f t h i s e x p e r i m e n t a r e shown i n F i g u r e 10 w here i t may b e s e e n t h a t g ly c in e bro u g h t about apparent r e v e r s a l . That t h i s was s o , was con­ f ir m e d i n a n o t h e r e x p e r im e n t i n w hich 2 .0 uM p y r u v a t e and 20 uM g l y c i n e w e re i n c u b a t e d f o r one h o u r i n t h e u s u a l r e a c t i o n m i x t u r e . Under t h e s e c i r c u m s t a n c e s t h e r e was a d i s a p p e a r a n c e o f 0 .2 8 uM p y r u v a t e w i t h a c o r r e s p o n d i n g a p p e a r a n c e o f a l a n i n e as d e m o n s tr a te d by q u a l i t a t i v e p a p e r c h r o m a to g r a p h y . 5 ) Enzyme P r o p e r t i e s S ta b ility : The enzyme may be s t o r e d f o r a t l e a s t a week a t U°C w ith o u t lo s s of a c t i v i t y . F r o z e n ( - 1 8 ° ) sam ples of t h e enzyme s t o r e d f o r tw o months showed no l o s s o f a c t i v i t y . The enzyme i s a l s o s t a b l e t o p r o lo n g e d d i a l y s i s a g a i n s t v a r i o u s b u f f e r s o l u t i o n s i n d i s t i l l e d w a te r. E f f e c t o f pH: H ig h e s t a c t i v i t y i s o b t a i n e d a t a b o u t pH 8 -5> . However, s i n c e as shown i n F i g u r e 11 c o n s i d e r a b l e n o n e n z y m a tic t r a n s ­ a m i n a t i o n o c c u r s i n t h i s pH r a n g e , a l l r o u t i n e a s s a y s o f t h e a c t i v i t y o f t h e enzyme w ere c a r r i e d o u t a t pH 8 . 0 . U7 R e a c tio n R a te : As i l l u s t r a t e d i n F i g u r e 8 t h e amount o f p y r u v a t e p ro d u c e d i s d i r e c t l y p r o p o r t i o n a l t o t h e q u a n t i t y o f enzyme up t o 0 . 7 u n its. A l l a s s a y s w ere c a r r i e d o u t i n t h i s r a n g e s i n c e t h e method f o r t h e d e t e r m i n a t i o n o f p y r u v a t e w hich was u s e d , was s u i t a b l e o n ly i n t h e r a n g e up t o 1 uM o f p y r u v a t e . A t y p i c a l tim e c o u r s e c u r v e f o r t h e e n z y m a tic t r a n s a m i n a t i o n r e a c t i o n i s shown i n F i g u r e 9* The r e a c t i o n was found t o p r o c e e d t o o n ly c a . o n e - th ir d c o n v e rs io n . A c t i v a t o r s and I n h i b i t o r s : N e i t h e r p r o lo n g e d d i a l y s i s a g a i n s t d i s t i l l e d w a t e r , p h o s p h a te b u f f e r o r a 50 p e r c e n t s a t u r a t e d s o l u t i o n o f ammonium s u l f a t e c a u s e d t h e enzyme t o e x h i b i t any s t i m u l a t i o n by t h e a d d i t i o n o f p y r i d o x a l - 5 - p h o s p h a t e '. The l a t t e r p r o c e d u r e d i d c a u se t h e i r r e v e r s i b l e l o s s o f c a . 30 p e r c e n t of t h e o r i g i n a l a c t i v i t y . P r e c i p i t a t i o n o f t h e enzyme b y d i a l y s i s a g a i n s t s a t u r a t e d ammonium s u l f a t e f o r t e n h o u r s f o llo w e d b y d i a l y s i s f o r t h r e e d ay s a g a i n s t 0 .02M a c e t a t e b u f f e r , pH U .0 ( Y o u a t t , 1958) cau sed c o m p lete and i r r e v e r s i b l e l o s s o f a l l a c t i v i t y w hich c o u ld n o t be r e s t o r e d ev en by p r o lo n g e d i n c u b a t i o n i n t h e p r e s e n c e o f p y r i d o x a l p h o s p h a te and v a r i o u s m e ta l io n s ( F e ^ “ , Mg"1', Cu ") ( M e t z l e r , 195U) • I n common w i t h o t h e r p y r i d o x a l p h o s p h a te c o n t a i n i n g enzym es, g ly c in e - a l a n i n e t r a n s a m i n a s e was i n h i b i t e d by b o t h h y d ro x y la m in e and p o t a s s i u m c y a n i d e (G olow ick and K a p la n , 1953) ( T a b l e I X ) . However, _ 2 i n o r d i n a t e l y h ig h c o n c e n t r a t i o n s o f t h e l a t t e r w e re r e q u i r e d (10 M ). -3 H y d ro x y lam in e i n h i b i t e d c o m p l e t e l y a t 10 M and c a . [±0 p e r c e n t a t 10 The i n h i b i t i o n o f t h e l a t t e r c o n c e n t r a t i o n was c o m p l e t e l y overcome by M. U6 TABLE IX INHIBITION OF TRANSAMINASE uM P y ru v a te A d d itio n s P e r Gent I n h ib i t io n C o n tro l 0 .65 0 10 M KCN 0 10 M KCN 0 .6 3 0 .05 92 10 M H ydro xy lam ine 0 .00 100 10 M H yd rox ylam in e 0 .36 10 M NH20H + 1 0 p h o s p h a te 0 .6 5 U2 0 C o n d itio n s: M p y rid o x a l As f o r t r a n s a m i n a s e a s s a y e x c e p t f o r a d d i t i o n s as i n d i c a t e d . Enzyme: Gel S u p e r n a t a n t B . H9 _ 3 10 M p y r id o x a l p h o s p h a te . Iso n ia z id (D a v is o n , 19^6; Y o u a t t , 1958) a n o t h e r r e p o r t e d i n h i b i t o r o f v i t a m i n B 6 enzymes d i d n o t i n h i b i t even a t 10“ 2M. R e q u ir e m e n ts f o r a m e ta l io n h av e so f a r no t b e e n d e m o n s t r a te d f o r any t r a n s a m i n a s e . e x c e p tio n . The g l y c i n e - a l a n i n e t r a n s a m i n a s e was found t o be no D i a l y s i s a g a i n s t d i s t i l l e d w a t e r and v a r i o u s b u f f e r s c o n t a i n i n g v e r s e n e f a i l e d t o r e v e a l t h e need f o r a m e t a l l i c c o f a c t o r . S u b stra te S p e c if ic ity ; In o rd e r to t e s t th e s p e c i f i c i t y of th e p a r t i a l l y p u r i f i e d p r e p a r a t i o n s , i n c u b a t i o n s w ere c a r r i e d o u t w i t h a number of s u b s t r a t e p a i r s . The a p p e a r a n c e o f p r o d u c t s was d e t e c t e d b y means o f q u a l i t a t i v e p a p e r ch ro m a to g ra p h y w i t h e x c e p t i o n s as n o t e d i n t h e T a b le X . L - a l a n i n e c o u ld n o t be r e p l a c e d b y any o t h e r amino a c i d i n s y ste m s co n ta in in g g ly o x y la te . D - a l a n i n e was c o m p l e t e l y i n a c t i v e . Though t h e o r i g i n a l c ru d e e x t r a c t c o n t a i n e d c o n s i d e r a b l e g l u t a m a t e - a l a n i n e t r a n s a m i n a s e a c t i v i t y i t was c o m p le t e l y l a c k i n g i n t h e p u r i f i e d p r e p a r a ­ tio n . An e n z y m a tic t r a n s a m i n a t i o n b etw een p y r u v a t e and a s p a r a g i n e , h o w e v e r, was d e t e c t e d . F u r t h e r work w i l l h a v e t o be done t o d e t e r m in e w h e th e r t h i s i s due t o t h e same o r a d i f f e r e n t enzyme th o u g h s t r u c t u r a l c o n s i d e r a t i o n s would seem t o s u g g e s t t h a t a s e p a r a t e enzyme i s i n v o l v e d . E f f e c t of L ig h t: A ssay s o f t r a n s a m i n a s e a c t i v i t y w ere c a r r i e d o u t i n t h e l i g h t and i n t h e d a r k u n d e r t h e same c o n d i t i o n s u s e d p r e v i o u s l y f o r l i g h t - d a r k assay s of i s o c i t r i t a s e . a r e p r e s e n t e d i n T a b le X I . The r e s u l t s o f t h i s e x p e rim e n t No s t i m u l a t i o n by l i g h t was found t o o c c u r . 50 TABLE X ENZYME SPECIFICITY OF GLYCINE-ALANINE TRANSAMINASE K e to A c id Amino A cid P y ru v a te G ly c in e L -is o le u c in e L -s e rin e L -v a lin e L -le u c in e L -ly s in e L -p h e n y l a l a n i n e L -p ro lin e L - g lu ta m in e L -a s p a ra g in e L -c y s te in e L -a rg in in e L - try p to p h a n +— — L -a la n in e D -a la n in e L -p h e n y l a l a n i n e L -c y s te in e .L - v a lin e L - 1 r y p t o p h an L -s e rin e L - l e u c in e L - l y s in e L -th re o n in e L -a s p a rta te L - g lu ta m a te L -is o le u c in e L -a s p a ra g in e + — O x a la c e ta te L - a l a n i n e '' - a -K e to b u ty ra te L - a l a n i n e '' L -g ly c in e — a -K e to g lu ta ra te L - a l a n i n e '' - G ly o x y la te • C o n d itio n s ; R e s u lt As f o r t r a n s a m i n a s e a s s a y e x c e p t t h a t ; i n c u b a t i o n w as f o r 2 h o u r s ; i n c u b a t i o n s o f p y r u v a t e c o n ta i n e d 2 uM p y r u v a t e and 20 uM o f am in o a c i d . A l l o t h e r s c o n t a i n e d 10 uM e a c h o f k e t o a c i d and am ino a c i d . Enzym e; A c e to n e I I . ^ A c t i v i t y d e te r m in e d b y S tr a u b t e s t f o r p y r u v a t e . t a t i v e p a p e r c h r o m a to g r a p h y . A ll o th e rs b y q u a li­ TABLE X I IN VITRO EFFECT OF LIGHT ON TRANS AMINASE" uM P y r u v a t e / l O m in u te s C o n d itio n s; L ig h t Dark 0 .7 6 0 .7 5 As f o r t h e t r a n s a m i n a s e a s s a y , e x c e p t t h a t l i g h t t u b e s w ere s u b j e c t e d t o l i g h t o f i n t e n s i t y _ca. 300 f o o t c a n d l e s . Dark t u b e s w ere s h i e l d e d w i t h aluminum f o i l . Enzyme: Gel s u p e r n a t a n t B . "A verage o f d u p l i c a t e d e t e r m i n a t i o n s . 52 FIGURE LEGENDS F ig u re 8 . T r a n s a m in a s e : Enzyme A ssay C u rv e. t r a n s a m i n a s e a s s a y ( s e e T a b le V I ) . S upernat ant B . F i g u r e R. Time Coursre o f G l y c i n e - A l a n i n e T r a n s a m i n a t i o n . C o n d it i o n s as f o r t r a n s a m i n a s e a s s a y e x c e p t t h a t i n c u b a t i o n tim e s w ere as i n d i c a t e d . R e a c t i o n s to p p e d w i t h 0 .0 9 ml 2N HC1. Enzyme: Gel S u p e r n a t a n t B. C o n d i t i o n s as f o r Enzyme: Gel F i g u r e 1 0 . R e v e r s i b i l i t y o f G l y c in e - A l a n i n e T r a n s a m i n a t i o n . Two p a r a l l e l s e r i e s o f f o u r r e a c t i o n t u b e s each w ere s e t up u s i n g t h e c o n d i t i o n s d e s c r i b e d f o r t h e t r a n s a m i n a s e . The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f s u b s t r a t e s and a t u b e fro m each s e t s to p p e d a t 9 m in u te i n t e r v a l s up t o 20 m i n u t e s . As shown i n one s e t 20 uM g l y c i n e i n 0 . 1 ml was added t o t h e 19 and 20 m in u te t u b e s . Enzyme: Gel S u p e rn a te n t B . F i g u r e 11- T r a n s a m in a s e : E f f e c t o f Hydrogen Ton C o n c e n t r a t i o n . C o n d i t io n s as f o r t r a n s a m i n a s e a s s a y e x c e p t U0 uM t r i s b u f f e r was u s e d from pH 7*2 t o pH 8 -U and I4O UM b o r a t e b u f f e r from pH 8 .J4 t o pH 1 0 . Chem ical c o n t r o l s w e re i n c u b a t e d f o r e a c h pH and p y r u v a t e d e te r m in e d u s i n g t h e pH 7 . 2 r e a c t i o n m i x t u r e as c o n t r o l . Enzyme: Gel Sup e r n a t a n t B . 53 c or >: o z x o Q> O< uj co z UO 51 to < 6 , 9 and 12 h o u r c u l t u r e s r e s p e c t i v e l y . At t h e a p p o i n t e d t i m e s t h e c u l t u r e s w ere examined m i c r o s c o p i c a l l y ( F i g u r e s 12 t o 17) and t h e n h a r v e s t e d by f i l t r a t i o n , w e ig h e d , and a l i q u o t s u s e d f o r t h e d e t e r m i n a t i o n o f d r y w e ig h t and s o l u b l e p r o t e i n . P ro te in d e t e r m i n a t i o n s w ere c a r r i e d o u t on 2 2 ,0 0 0 x G s u p e r n a t a n t s o b t a i n e d by c e n t r i f u g a t i o n o f Omnhmixer h o m o g e n a te s . The grow th c u r v e s ( F i g u r e s 18 and 19) w ere o b t a i n e d by a d j u s t i n g t h e w e i g h t s t o a c o n s t a n t ino cu lu m b ased on t h e t h r e e h o u r c u l t u r e . Growth was fo u n d t o b e e x p o n e n t i a l t h r o u g h o u t t h e grow th p e r i o d as i n d i c a t e d by a l o g a r i t h m i c p l o t o f t h e d r y w e ig h t d a t a ( F i g u r e 1 8 ) . M ic r o s c o p ic e x a m i n a t i o n r e v e a l e d t h a t t h e p l a n t s w ere a t a l l s t a g e s o f q u i t e u n i f o r m m orphology ( F i g u r e s 12 t o 17) and s i z e e x c e p t t h a t a t t w e l v e h o u r s , when some p l a n t s w ere b e g i n n i n g t o g e r m in a t e , v a r i a t i o n s d id a p p e a r (F ig u re 1 7 )- From t h e s e o b s e r v a t i o n s i t was c l e a r t h a t su ch 58 c u l t u r e s w ere t h e r e s u l t o f t h e v e r y n e a r l y s y n c h ro n o u s grow th o f a s in g le g e n e ra tio n of o rd in a ry c o lo r le s s p l a n t s . The p r o t e j n c o n c e n ­ t r a t i o n s i n t h e p l a n t s a t v a r i o u s ag es a r e p r e s e n t e d i n T a b le X I I I . 3) R e l a t i o n s h i p B etw een Endogenous R e s p i r a t o r y R a te and S ta g e o f Developm ent o f O r d i n a r y C o l o r l e s s P l a n t s Endogenous R e s p i r a t i o n o f Z o o sp o res and E f f e c t o f Added S u b s t r a t e and I n h i b i t o r s : C a n tin o and H y a tt (1953o) h av e a l r e a d y e s t a b l i s h e d t h e p r e s e n c e o f m ost o f t h e enzymes a s s o c i a t e d w i t h t h e K r e b s ' c y c l e i n m a tu re o r d i n a r y c o l o r l e s s p l a n t s . B ec au se o f t h i s and t h e c l o s e a s s o c i a t i o n o f i s o c i t r i t a s e and C02 f i x a t i o n w i t h t h e t r i c a r b o x y l i c a c i d c y c l e , i t was o f p a r e n t h e t i c a l i n t e r e s t , b e f o r e p r o c e e d i n g w i t h f u r t h e r s t u d i e s on i s o c i t r i t a s e , t r a n s a m i n a s e , and d e v e lo p m e n t t o o b t a i n some i n d i c a t i o n o f t h e t y p e o f m e ta b o lism e x h i b i t e d by z o o s p o r e s . R e s p i r a t i o n s t u d i e s w ere c a r r i e d o u t u s i n g a c o n v e n t i o n a l W arburg c o n s t a n t volum e r e s p i r o m e t e r c o n t a i n i n g a t o t a l of 2 .7 ml i n t h e f l a s k s . Oxygen u p t a k e and c a r b o n d i o x i d e e v o l u t i o n m e a s u re m e n ts , w ere done s i m u l t a n e o u s l y a t 30 °C u s i n g i d e n t i c a l f l a s k s w i th and w i t h o u t 0 . 2 ml t e n p e r c e n t KOH r e s p e c t i v e l y and c o n t a i n i n g 100 uM p h o s p h a te b u f f e r pH 6 . 5 * C o r r e c t i o n f o r c a r b o n d i o x i d e r e t e n t i o n by t h e b u f f e r was made a c c o r d i n g t o t h e m ethod of Jo h n so n as d e s c r i b e d by U m b re it _et _al. and t h e r e s p i r a t o r y q u o t i e n t s c a l c u l a t e d u s i n g t h e f o r m u la o f U m b reit _et a l . ( 1 9 5 7 ) * A number o f d e t e r m i n a t i o n s w ere done on z o o s p o re r e s p i r a t i o n . The endogenous Q02 o f z o o s p o r e s as an a v e r a g e of many e x p e r im e n ts was fou nd t o b e 102 u l 0 2 p e r h o u r p e r mgm d r y w e ig h t a t pH t e m p e r a t u r e 30°C. 6 - 5 t o 7 -0 and The r e s p i r a t o r y q u o t i e n t was c a l c u l a t e d t o be 0 . 8 7 . 59 The a d d i t i o n o f t h e f o l l o w i n g i n t e r m e d i a t e s d id n o t a f f e c t t h e r e s p i r a ­ to ry ra te j g lu co se , s u c c in a te , a c e ta te , m a la te , a -k e to g l u t a r a te , p y r u v a t e , g l u t a m a t e and i s o c i t r a t e . . A rs e n ite at a c o n c e n tra tio n of I4. x 10 — 4 M i n h i b i t e d oxygen u p t a k e U.5 p e r c e n t ( T a b le X I I ) . As would b e e x p e c te d m a lo n a te d id n o t t h e n e u t r a l r a n g e o f pH b u t a t pH5 t r a t i o n o f 2 x 10 per c e n t. _3 - 5 ( Q o 0 a f f e c t t h e oxygen u p t a k e i n = 22) m a lo n a te a t a co n c e n - M i n h i b i t e d c o m p le t e l y a t U x 10 _4 i t i n h i b i t e d 50 S u c c in a te a t te n tim es th e c o n c e n tra tio n re tu rn e d oxygen u p t a k e t o endogenous l e v e l s . Thus p r e s u m p t i v e e v i d e n c e h a s b e e n o b t a in e d f o r a. f u n c t i o n i n g t r i c a r b o x y l i c a c id c y c l e i n z o o s p o r e s . However, s i n c e t h e a d d i t i o n o f v a r i o u s s u b s t r a t e s f a i l e d t o s t i m u l a t e endogenous oxygen u p t a k e f u r t h e r i n v e s t i g a t i o n s , n o t w i t h i n t h e s c o p e o f t h i s w ork , w i l l be n e c e s s a r y f o r c o n firm a tio n . Endogenous R e s p i r a t i o n o f D e v e lo p in g P l a n t s : C an tin o ( 1 9 5 5 ) S in c e Brown and had found a v e r y much lo w e r Q0 ^ f o r m a tu re O.G. p l a n t s ( c a . 9 u l 0 2/mgm d r y w t / h r ) t h a n r e p o r t e d h e r e f o r z o o s p o r e s . Q0 ^ d e t e r m i n a t i o n s w ere done a t v a r i o u s s t a g e s of a s i n g l e g e n e r a t i o n c u l t u r e . A s e r i e s o f 500 ml E rle n m e y e r f l a s k s c o n t a i n i n g 300 ml PYG and e q u ip p e d w i t h a e r a t i o n d e v i c e s w ere each i n o c u l a t e d w i t h a h eav y s u s p e n s i o n o f sw arm ers and i n c u b a t e d a t 27°C. At f o u r h o u r i n t e r v a l s up t o t h e b e g i n n i n g o f g e r m i n a t i o n one of t h e f l a s k s was removed and t h e p l a n t m a t e r i a l h a r v e s t e d b y c e n t r i f u g a t i o n i n 250 ml c e n t r i f u g e b o ttle s . The m a t e r i a l was t h e n washed t w i c e i n d i s t i l l e d w a t e r by 60 TABLE X I I THE EFFECT OF INHIBITORS ON ZOOSPORE RESPIRATION A d d itio n s C o n c e n tra tio n None 56 u l / h o u r - U 10- 4 M X 1— 1 CO A rse n ite 0 2 U ptake M a lo n a te k x 10~4M M a lo n a te h x 10~4M S u ccin ate k M a lo n a te 1| x 10 S u c c in a te 8 x 1 0 " 3M i - — u u 1 i None u l/h o u r P e r Cent In h ib itio n H6 u l / h o u r - 2U u l / h o u r U8 37 u l / h o u r 20 U6 u 1 0 2/ h o u r None 'Zt X 10* "m _ 4 M F l a s k c o n t a i n e d 100 uM g l y c y l g l y c i n e b u f f e r pH 5*5* 0 *U nil swarm er s u s p e n s io n ., 0 . 2 ml 10% KOH i n c e n t e r w e l l and a d d i t i o n s as i n d i c a t e d . T e m p e ra tu r e 30 C. 61 c e n t r i f u g a t i o n and a f t e r rem o v in g an a l i q u o t f o r a d r y w e ig h t d e t e r m i n ­ a t i o n r e s u s p e n d e d I n 0 .01M p h o s p h a te b u f f e r pH 6 -5 f o r e s t i m a t e s o f t h e r a t e o f oxygen u p t a k e . As shown i n F i g u r e 20 t h e Q0 ^ d i d d e c r e a s e a s t h e p l a n t s d e v e lo p e d . However, e v e n as t h e y n e a r m a t u r i t y i t i s s t i l l a p p r e c i a b l e and some­ w hat h i g h e r t h a n v a l u e s p r e v i o u s l y r e p o r t e d u s i n g m u l t i p l e g e n e r a t i o n c u l t u r e s w h ich c o n t a i n e d a good d e a l o f b i o l o g i c a l l y i n a c t i v e m a t e r i a l and dead p l a n t s . U) R e l a t i o n s h i p o f I s o c i t r i t a s e and T ra n s a m in a s e A c t i v i t i e s t o D evelopm ent and M o rp ho genesis D i s t r i b u t i o n o f t h e Enzymes a t V a r i ous S ta g e s i n t h e L i f e C y c le : E s t i m a t e s w e re made of i s o c i t r i t a s e and t r a n s a m i n a s e a c t i v i t i e s in e x t r a c t s p r e p a r e d from r e s i s t a n t s p o r a n g i a , o r d i n a r y c o l o r l e s s p l a n t s and z o o s p o r e s from o r d i n a r y c o l o r l e s s p l a n t s . E x c ep t i n t h e c a s e of z o o s p o r e i s o c i t r i t a s e , a l l e s t i m a t e s w ere made on h ig h speed s u p e r n a t a n t s p r e p a r e d as d e s c r i b e d e l s e w h e r e . B ec au se o f t h e h i g h l e v e l s o f endo­ genous amino a c i d s ( p a r t i c u l a r l y a l a n i n e ( L o v e t t , 1959)., i t was n o t p o s s i b l e t o a s s a y th e zoospores d i r e c t l y f o r i s o c i t r i t a s e . The i s o c i t r i ­ t a s e a c t i v i t y of z o o s p o re e x t r a c t s was d e t e r m in e d b y a s s a y i n g t h e f r a c t i o n o b t a i n e d by a d d in g 0 .6 5 volum es of s a t u r a t e d ammonium s u l f a t e t o an e x t r a c t p r e p a r e d u s i n g t h e c a r b o ru n d u m - g la s s g r i n d i n g m i x t u r e . R e p r e s e n t a t i v e d a t a a r e p r e s e n t e d i n T a b le XIV. t i e s f o r b o t h enzymes a r e g i v e n f o r a l l s t a g e s . S p e c ific a c t i v i ­ A c tiv ity in a dry w e ig h t b a s i s a r e a l s o g iv e n f o r a l l s t a g e s e x c e p t R .S . inhere, b e c a u s e 62 TABLE X I I I SOLUBLE PROTEIN Age (H ours) S o lu b le P r o te in (mgrn/gm) 0 (Z oospores) 371 3 393 6 350 9 380 12 312 TABLE XIV DISTRIBUTION OF ISOCITRITASE AT VARIOUS STAGE OF THE LIFE CYCLE Iso c itrita s e U n its p e r U n its p e r Mgm P r o t e i n Gm Dry Wt S t a g e of D ev elo pm en t T ran s am inas e U n its p e r U n its p e r Mgm P r o t e i n Gm Dry Wt Z o o sp o re s 82 0 .2 3 271 0 .7 9 M ature 0 . C. 5 5 0 ,2b 193 O .58 R.S . ( I4 d a y s ) C o n d itio n s: 0 .5 0 0 .9 9 As f o r a s s a y s o f r e s p e c t i v e enzymes d e s c r i b e d p r e v i o u s l y . 63 o f t h e d i f f i c u l t y i n o b t a i n i n g c o m p le te b r e a k a g e , a c c u r a t e d r y w e i g h t a c t i v i t y r e l a t i o n s h i p s c o u ld n o t be o b t a i n e d . B o th enzymes w ere d e t e c t a b l e a t a l l s t a g e s of t h e l i f e c y c l e e x a m in e d . tie s Though i t h a s n o t b e e n p o s s i b l e t o o b t a i n s u f f i c i e n t q u a n t i ­ o f R . S . z o o s p o r e s f o r p h y s i o l o g i c a l s t u d i e s t h e a s s u m p tio n h a s b e e n t h a t t h e y do n o t d i f f e r from t h o s e o f O.C. p l a n t s . R e l a t i o n s h i p o f T ra n s a m in a se and I s o c i t r i t a s e A c t i v i t i e s t o t h e D evelopm ent o f O .C. P l a n t s : The dev elo p m en t o f t h e enzymes d u r i n g t h e gro w th o f s i n g l e g e n e r a t i o n c u l t u r e s was t r a c e d by means o f ' a s e r i e s o f e x p e r i m e n t s i n w h ich c u l t u r e s o f v a r i o u s a g e s , i n c u b a te d i n t h e l i g h t and t h e d a r k , w ere a s s a y e d f o r e n z y m a tic a c t i v i t i e s . The m ethods f o r i n o c u l a t i n g and h a r v e s t i n g c u l t u r e s w ere as d e s c r i b e d i n t h e growth c u rv e e x p e r i m e n t . Crude e x t r a c t s w ere p r e p a r e d u s in g t h e Omnimixer as i n t h e p u r i f i c a t i o n o f i s o c i t r i t a s e e x c e p t that- t e n r a t h e r t h a n 5 ml o f 0.1M p h o s p h a t e b u f f e r pH 7 was u s e d p e r gram wet w e i g h t . I s o c i t r i t a s e was a s s a y e d d i r e c t l y as d e s c r i b e d e a r l i e r and t r a n s ­ a m in a se was a s s a y e d a f t e r d i a l y s i s v s . 0 .02M p h o s p h a te b u f f e r pH 8 . 0 f o r 26 h o u r s and t h e s p e c i f i c a c t i v i t y o f t h e c ru d e e x t r a c t e x p r e s s e d as p r e v i o u s ly . A c t i v i t i e s p e r gram d r y w e ig h t and p e r p l a n t w ere c a l c u l a t e d from c o n v e r s i o n f a c t o r s o b t a i n e d from t h e g ro w th c u rv e e x p erim en t. I t must b e n o te d t h a t t h e r e was c o n s i d e r a b l e v a r i a t i o n i n t h e r e s u l t s o b t a i n e d i n ru n s done a t d i f f e r e n t t i m e s . T h is i s a t t r i b u t a b l e t o a l a c k o f a d e q u a t e means o f s t r i c t t e m p e r a t u r e c o n t r o l . C o n s e q u e n tly , 6u w h i l e tim e was u s e d as t h e c r i t e r i o n o f t h e d ev elo p m en t o f c u l t u r e s i t was n o t alw ays an a c c u r a t e r e f l e c t i o n o f p h y s i o l o g i c a l , m a t u r i t y . S in c e no c o n s i s t e n t d i f f e r e n c e s i n e i t h e r i s o c i t r i t a s e o r t r a n s ­ a m in ase a c t i v i t i e s w e re found b e tw e e n c u l t u r e s grown i n t h e l i g h t and t h e d a r k t h e d a t a f o r b o t h c o n d i t i o n s o f g ro w th were p o o le d i n o b t a i n i n g t h e p l o t t e d a v e r a g e s th o u g h d a t a from l i g h t and d a r k a r e i n d i c a t e d as such in th e g r a p h s . F i g u r e s 2 1 , 2 2, 23 and 21+ sum m arize t h e r e s u l t s . The number of i s o c i t r i t a s e u n i t s p e r gram d r y w e i g h t and p e r m i l l i g r a m p r o t e i n d e c r e a s e s d u r i n g t h e f i r s t t h r e e h o u r s o f g ro w th , re m a in s low u n t i l n i n e h o u r s and t h e n r i s e s a g a i n t o i n i t i a l l e v e l s ( F i g u r e 2 2 ) . When t h e d a t a w ere p l o t t e d on a p e r p l a n t b a s i s ( F i g u r e 21) i t was found t h a t t h e number o f u n i t s p e r p l a n t rem ain ed r e l a t i v e l y c o n s t a n t up t o c a . t h r e e t o six: h o u r s and t h e n i n c r e a s e d a lm o s t e x p o n e n t i a l l y d u r i n g t h e l a t t e r h a l f o f t h e g e n e r a t i o n tim e d e m o n s t r a t i n g t h a t n e t s y n t h e s i s o f i s o ­ c i t r i t a s e d o e s n o t o c c u r u n t i l r e l a t i v e l y l a t e i n d e v e lo p m e n t. G l y c i n e - a l a n i n e t r a n s a m i n a s e on t h e o t h e r hand th o u g h d r o p p in g somewhat i n t h e number o f a c t i v i t y u n i t s p e r gram d ry w e ig h t o f p l a n t m a t e r i a l and m i l l i g r a m p r o t e i n as shown i n F i g u r e 23, i s ho w ever, a p p a r e n t l y s y n t h e s i z e d from t h e v e r y b e g in n i n g o f p l a n t d e v e lo p m e n t. As shown i n F i g u r e 21+ t h i s s y n t h e s i s i s e x p o n e n t i a l . The r e l a t i o n s h i p b e tw e e n enzyme s y n t h e s i s and o v e r - a l l s y n t h e s i s o f p l a n t m a t e r i a l i s shown i n F i g u r e 25 R e l a t i o n s h i p o f T ra n s a m in a se and I s o c i t r i t a s e A c t i v i t i e s t o t h e D evelopm ent o f R .S . P l a n t s : The p r o c e d u r e and c o n d i t i o n s u s e d h e r e 65 f o r t h e g ro w th o f s e m is y n c h r o n o u s c u l t u r e s of r e s i s t a n t s p o r a n g i a a r e t h o s e d e v e lo p e d by L o v e t t ( 1 9 5 9 ) • A tw elv e l i t e r f l a s k c o n ta in in g t e n l i t e r s o f PYG c o n t a i n i n g 0 .7 5 gm sodium b i c a r b o n a t e p e r l i t e r was i n o c u l a t e d w i t h 10 ml o f a z o o s p o r e s u s p e n s i o n ( swarmer d e n s i t y = 20) and i n c u b a t e d w i t h v i g o r o u s a e r a t i o n a t 25°C . Commencing a t 2b h o u r s and a t 12 h o u r i n t e r v a l s t h e r e a f t e r sam p les l a r g e enough t o y i e l d c a . two grams o f p l a n t m a t e r i a l w ere w ith d ra w n by means of a s i p h o n . A fter h a rv e s tin g c e l l f r e e e x tr a c ts w ere p r e p a r e d as d e s c r i b e d p r e v i o u s l y , e x c e p t t h a t a s i x r a t h e r t h a n t h r e e m in u te g r i n d i n g p e r i o d was u s e d b e c a u s e o f t h e much t h i c k e r w a l l s o f R . S . a s compared t o 0.C* O th e r w is e i s o c i t r i t a s e and t r a n s a m i n a s e a c t i v i t i e s w ere a s s a y e d as p r e v i o u s l y . The r e s u l t s o f two s u c h ru n s a r e shown i n F i g u r e s 26 and 27 . As may b e s e e n i n F i g u r e 26 t h e s p e c i f i c a c t i v i t y o f i s o c i t r i t a s e re m a in s f a i r l y c o n s t a n t t h r o u g h t h e f i r s t 2b h o u r s . However, b e g i n n i n g a t 36 h o u r s t h e r e i s a s h a r p i n c r e a s e i n a c t i v i t y w i t h a peak b e i n g r e a c h e d a t U8 h o u r s f o ll o w e d b y a slow d e c l i n e . The s p e c i f i c a c t i v i t y a t U8 h o u r s i s h i g h e r t h a n d e t e c t e d a t an y o t h e r s t a g e o f t h e l i f e cy cle ( c f . T a b le X I V ) . T ra n s a m in a s e a c t i v i t y was found t o b eh av e s i m i l a r l y as shown i n F i g u r e 2.?. S in c e r e s i s t a n t s p o r a n g i a m a tu re ( i . e . , a r e c a p a b l e of g e r m i n a t i o n ) u n d e r t h e s e c o n d i t i o n s a t c a . 80 h o u rs t h e l e v e l o f a c t i v i t y o f b o t h enzymes r e a c h t h e i r maxima a t a b o u t t h r e e - f i f t h s t i o n tim e . of t h e g e n e r a ­ 66 FIGURE LEGENDS C o n d i t i o n s f o r c u l t i v a t i o n and h a r v e s t o f c u l t u r e s a r e g iv e n i n d e t a i l i n t h e t e x t . M ethods f o r e n z y m a tic a s s a y s and p r e p a r a t i o n o f e x t r a c t s a r e d e s c rib e d i n d e t a i l else w h e re . F ig u re 12. Z o o s p o re s from O .C. p l a n t s ( 5 0 0 x ) . fla g e lla . Note p o s t e r i o r F i g u r e 13 . A g e r m lin g t h r e e h o u r s a f t e r i n i t i a t i o n o f grow th i n a n e a r s y n c h ro n o u s c u l t u r e ( 500 x ) . F i g u r e lU . A s i x h o u r o ld O .C . p l a n t from a s i n g l e - g e n e r a t i o n n e a r - s y n c h r o n o u s c u l t u r e ( 500 x ) . F i g u r e 15* A n i n e h o u r o ld O.C. p l a n t from a n e a r s y n c h ro n o u s c u l t u r e ( 50 Gx) . F ig u re 16 . A gro up o f s e v e r a l n i n e h o u r O .C. p l a n t s i l l u s t r a t i n g t h e u n i f o r m i t y e x h i b i t e d by p l a n t s i n n e a r s y n c h ro n o u s c u l t u r e s (2l;0x) F i g u r e 17* Twelve h o u r O .C . p l a n t s from a n e a r sy n c h ro n o u s s i n g l e g e n e r a t i o n c u l t u r e of (2l_|.0x) . Note t h a t one p l a n t h a s a l m o s t c o m p leted z o o s p o re r e l e a s e . F ig u re 1 6 . Growth Curve o f a S i n g l e G e n e r a t i o n C u l t u r e o f B la st o c la d ie ll a . F ig u re 19. Log Growth Curve o f I n d i v i d u a l P l a n t o f B l a s t o c l a d i e l l a . The p l o t t e d d a t a a r e t h e l o g a r i t h m s of t h e v a l u e s o b t a i n e d b y d i v i d i n g t h e d r y w e ig h t s o b t a i n e d a t t h e tim e s i n d i c a t e d f b y t h e c o r r e c t e d number of v i a b l e swarm ers i n t h e in o c u lu m . F ig u re 20. R e l a t i o n s h i p b e tw e e n C u l t u r e Age and Oxygen U p ta k e . Q02 = u l Oxygen consumed p e r m i l l i g r a m d r y w e ig h t p e r h o u r . F ig u re 21. Iso c itrita s e : R e l a t i o n s h i p t o Development o f O.C. P l a n t s . The amounts o f enzyme p e r p l a n t w ere c a l c u l a t e d from s p e c i f i c a c t i v i t i e s u s i n g c o n v e r s i o n f a c t o r s o b t a i n e d from t h e grow th c u r v e e x p e r i m e n t . F ig u re 22. Iso c itrita s e : R e l a t i o n s h i p t o O.C. A c t i v i t i e s p e r gram d ry w e ig h t w ere s p e c i f i c a c t i v i t i e s on t h e b a s i s of d r y w e ig h t v a l u e s d e te r m in e d i n t h e C u l t u r e D ev elo p m en t. c a l c u l a t e d fro m t h e t h e p r o t e i n p e r gram grow th c u rv e e x p e r i m e n t . 67 F i g u r e 23 T r a n s a m in a s e : R e l a t i o n s h i p t o O.C. C u l t u r e D ev elo p m en t. A c t i v i t i e s p e r gram d r y w e ig h t w ere c a l c u l a t e d by t h e same method u s e d f o r i s o c i t r i t a s e . F i g u r e 2l_(. T r a n s a m in a s e : R e l a t i o n s h i p t o Developm ent o f O.C. P l a n t s . T r a n s a m in a s e a c t i v i t i e s p e r p l a n t w ere c a l c u l a t e d on t h e same b a s i s as t h a t u se d f o r i s o c i t r i t a s e . F i g u r e 2£ Enzyme S y n t h e s i s as a F u n c t i o n of P l a n t Growth. The p l o t t e d v a l u e s w ere o b t a i n e d from t h e l o g d a t a used i n F i g u r e s 19, 21 and 21+• F i g u r e 26 Iso c itrita s e : R e la tio n sh ip F i g u r e 27 T r a n s a m in a s e : R e l a t i o n s h i p t o R .S . C u l t u r e D ev elop m en t. t o R .S . D ev elo pm en t. F i g u r e 12 Figure i k : F i g u r e 13 c F ig u re 17 j 70 _ i Q. _ l 3 O O UJ Figure I? ? 5 o LU 1 3 o C -JO tr cd cd Qr t— UJ CO CD >X CL ° 1H 9I3M AMO 010 0 3 J.H 0I3M x a o w o n o C C LU zUJ F i^ e 18 CD CD TIME (tOIX Z (H O U R S ) UJ Q 3h-C O ^ < O < O ( 1HDI3M AUQ M H t ^ o CD UJ re uj o B X o cr CD (" • " > 1H 9I3M AHO 0 0 T 71 O I3 M AMO H V U O / S l I N f l 3S V J.IU J.IO O S I ( , 0 ( X ) I N V ld / S lI N n OJ «S2 to O Pi S > a s er co (H OURS) CD t i­ AGE Figure 22 Q. Q. 2co or u j l— Q UJ ( , 0 1 X ) S l I N f l 01 s o n NV U O a 3 d S l I N f l 3 S V N IN V S N V a i A 1 I A I 1 0 V O IJIO B d S ) IN V O d H 3 d S S V lla ilO O S I 3 0 S l I N f l o I— z CO z I CD l.l — U J < -J UJ tr > UJ TIME Figure 21 0"\ CM u> - = 3 C O CL UJ cr CO cr (- ( . 0 1 X ) I N V T d W 3d S l I N f l Ol0 0 3 IN HOURS CL A 1 IA I1 0 V O ldlO S dS 72 o Q CM 1-Pv CVJ >- 0.75 I— o 0.50 to t- RUN ONE RUN TWO o 0.25 to 24 48 72 96 AGE IN HOURS F i g u r e 27 z TRANSAMINASE: RELATIONSHIP TO R. S. CULTURE DEVELOPMENT hkoucr a. 2 o 2.0 at uj a to »Hi to < z 2< to z < tr FIRST TRIAL SECOND TRIAL 24 72 45 AGE OF R. S. 96 -7k PISCUSSION AND CONCLUSIONS The f u n d a m e n t a l p u r p o s e o f t h e work o f which t h i s t h e s i s i s a p a r t , i s t o u n d e r s t a n d grow th and d i f f e r e n t i a t i o n i n te rm s of t h e u n d e r l y i n g b i o c h e m i c a l m echanism s i n v o l v e d . In p u r s u i t of t h i s g o al th e p h y s io lo g y o f B l a s t o c l a d i e l l a e m e r s o n ii h a s b e e n more t h o r o u g h l y s t u d i e d t h a n t h a t o f an y o t h e r a q u a t i c f u n g u s . E n z y m o lo g ic a l s t u d i e s on t h i s group o f o r g a n is m s h a v e b e e n r a r e and h av e n e v e r b e f o r e in v o lv e d t h e s t u d y o f enzymes a t t h e d e g r e e o f p u r i t y a c h ie v e d f o r t h e g l y c i n e - a l a n i n e t r a n s ­ a m in a se and i s o c i t r i t a s e o f B . e m e r s o n i i . T h erefo re b e fo re p ro c e e d in g w i t h a c o n s i d e r a t i o n o f t h e i r p o s s i b l e r o l e s i n l i g h t s t i m u l a t e d grow th and m o r p h o g e n e s is , a b r i e f d i s c u s s i o n o f t h e p r o p e r t i e s o f t h e s e enzymes seems a p p r o p r i a t e . The i s o c i t r i t a s e of B l a s t o c l a d i e l l a h a s b e e n found t o r e s e m b le v e r y c l o s e l y t h e a n a lo g o u s enzymes from o t h e r s o u r c e s f o r w h ich s i m i l a r l e v e l s o f p u r i f i c a t i o n have b e e n a c h i e v e d . T hu s, th o u g h i t d i f f e r s s l i g h t l y i n i t s pH optimum (pH 7 *U v s pH d .2 f o r Pseudom onas) and ammon­ ium s u l f a t e s o l u b i l i t y c h a r a c t e r i s t i c s , t h e M ic h a e l is c o n s t a n t (U-8 x 10- 4 M) i s a lm o s t i d e n t i c a l t o t h a t o:f t h e enzyme from Pseudomonas a e r u g i n o s a (S m ith and G u n s a lu s , 1 9 5 7 ) - The M ic h a e l is c o n s t a n t o f t h e y e a s t enzyme ( O ls o n , 1959) h a s b e e n d e te r m in e d u n d e r somewhat d i f f e r e n t 3 c o n d i t i o n s t o be 1 .2 x 10 M. L i k e t h e r e a c t i o n c a t a l y z e d b y t h e enzyme _ from o t h e r s o u r c e s , t h e B l a s t o c l a d i e l l a i s o c i t r i t a s e r e a c t i o n i s r e v e r s ­ i b l e and s p e c i f i c f o r t h e d - i s o m e r . The i s o c i t r i t a s e s from B l a s t o c l a d i e l l a and Pseudom onas (S m ith and G u n s a lu s , 1957) show v e r y c l o s e s i m i l a r i t y 75 i n t h e i r s a t u r a t i o n c u r v e s f o r b o t h m agnesium and c y s t e i n e , e x h i b i t i n g maximum a c t i v i t i e s a t 1.0 -3 M c o n c e n t r a t i o n s f o r b o th c o f a c t o r s . W o rth y o f m e n tio n i s t h e f a c t , n o t d i s c u s s e d b y S m ith and G u n s a lu s , t h a t t h e r e s p o n s e c u r v e f o r c y s t e i n e e x h i b i t s a maximum f o ll o w e d b y d e c r e a s i n g a c t i v i t y w ith in c r e a s in g c y s te in e c o n c e n tr a tio n . T h is phenom enon i s p r o b a b l y a t t r i b u t a b l e t o a c h e m ic a l r e a c t i o n b e tw e e n c y s t e i n e and g ly o x y la te . B r u n e i - G h a p e l l e (1 9 5 2 ) r e p o r t e d t h e o c c u r r e n c e o f s u c h a r e a c t i o n a t pH 9*0 and a b o v e . We h a v e fo u n d t h a t t h e r e i s c o n s id e ra b le d i s a p p e a r a n c e o f g l y o x y l a t e e v e n a t p h y s i o l o g i c a l h y d ro g e n i o n c o n c e n ­ t r a t i o n s i n th e p re se n c e o f a s u f f i c i e n t l y la r g e e x ce ss o f c y s te in e . O l s o n - (1 9 5 9 ) h a s r e p o r t e d a s i m i l a r o b s e r v a t i o n i n t h e c o u r s e o f s t u d i e s on y e a s t i s o c i t r i t a s e . D u rin g o u r s t u d i e s i t w as fo u n d t h a t e x t r a c t s o f B . e m e r s o n ii w e re c a p a b le o f m e d ia tin g th e r a p id c o n v e rs io n o f g ly o x y la te t o am ino g ro u p t r a n s f e r fro m a l a n i n e . g ly c in e b y S in c e t h i s m ig h t h a v e c o n s t i t u t e d a '’p u l l i n g ” r e a c t i o n i n t h e o p e r a t i o n o f t h e S . K . I , c y c l e and s i n c e , t o o u r k n o w le d g e , t h e r e h a v e b e e n n o p r e v i o u s r e p o r t s o f a t t e m p t s t o p u r i f y a g ly c in e - _ a l a n in e t r a n s a m i n a s e , f u r t h e r s t u d i e s w e re c a r r i e d o u t . By a s e r i e s p u rific a tio n o f a c e to n e f r a c t i o n a t i o n s and g e l t r e a t m e n t s a n e i g h t y - f o l d o f t h e enzym e w as a c h i e v e d . The p u r i f i e d enzym e w as fo u n d t o b e q u i t e s t a b l e and re s e m b le d o t h e r t r a n s a m i n a s e s i n e x h i b i t i n g I maximum a c t i v i t y i n t h e a l k a l i n e pH r a n g e . A tte m p ts t o d e m o n s t r a t e s t i m u l a t i o n o f a c t i v i t y b y p y r i d o x a l p h o s p h a te w e re u n s u c c e s s f u l th o u g h i t seem s l i k e l y , i n v ie w o f i t s tra n s a m in a tio n s , th a t i t i s a lm o s t u n i v e r s a l r o l e i n e n z y m a tic in v o lv e d i n t h e g l y o x y l a t e - a l a n i n e 76 " tra n s a m in a tio n as w e l l . The i n h i b i t i o n o f t h e r e a c t i o n b y h y d r o x y l a m i n e and t h e r e v e r s a l o f t h e i n h i b i t i o n b y p y r i d o x a l p h o s p h a t e w o u ld seem t o in d ic a te th a t th is is so. We h a v e c o n c lu d e d t h a t t h e enzyme i s p r o b a b l y v e r y t i g h t l y bound i n t h e h a lo e n z y m e th o u g h M e i s t e r (1 9 5 5 ) h a s p o i n t e d o u t t h e t h e o r e t i c a l p o s s i b i l i t y o f e n zy m a tic t r a n s a m i n a t i o n s in v o lv in g g ly o x y la te not re q u irin g a c o fa c to r. I n a t t e m p t s t o e s t a b l i s h s u b s t r a t e s p e c i f i c i t y i t was fo u n d t h a t n on e o f t h e am ino a c i d s t e s t e d w e re c a p a b l e o f r e p l a c i n g L - a l a n i n e i n sy ste m s c o n ta i n in g g l y o x y l a t e . D - a l a n i n e was c o m p l e t e l y i n a c t i v e . H o w e v e r, an e n z y m a t i c t r a n s a m i n a t i o n b e tw e e n p y r u v a t e and a s p a r a g i n e was f o u n d t o o c c u r I n o u r p u r i f i e d p r e p a r a t i o n . W h e th e r t h i s was due t o t h e same enzyme o r a d i f f e r e n t o n e , fro m t h a t c a t a l y z i n g t h e g l y o x y l a t e - a l a n i n e r e a c t i o n i s n o t a t p r e s e n t know n. F u rth e r stu d y w i l l be re q u ire d to c l a r i f y th is p o in t. I t was e x p e c t e d t h a t t h e e q u i l i b r i u m o f t h e g l y o x y l a t e - a l a n i n e t r a n s a m i n a t i o n w o u ld b e f a r i n t h e d i r e c t i o n o f g l y c i n e p r o d u c t i o n ( M e t z l e r e t a l M 195U) • t h a t c o n v e rsio n to H o w ev er, i n ti m e c o u r s e r u n s i t was fo u n d g l y c i n e and p y r u v a t e n e v e r e x c e e d e d c a . o n e - t h i r d c o n v e r s i o n w h i l e t h e r e v e r s e r e a c t i o n y i e l d i n g a l a n i n e was o n l y b a r e l y d e te c ta b le under c e r ta in c o n d itio n s. never a tta in e d . enzym e. T h e r e f o r e a. t r u e e q u i l i b r i u m was C l e a r l y , p y r u v a t e h a d an i n h i b i t i n g e f f e c t u p o n t h e N e v e r t h e l e s s t h e r e i s no r e a s o n t o b e l i e v e t h a t t h e e q u i l i b r i u m c o n s t a n t o f t h e e n z y m a t i c r e a c t i o n s h o u l d be d i f f e r e n t t h a n c h e m i c a l tra n sa m in a tio n s in v o lv in g g ly o x y la te . 77 O ur p r i m a r y i n t e r e s t i n t h e s t u d y o f i s o c i t r i t a s e w as b a s e d on i n d i r e c t e v id e n c e t h a t i t w as in v o lv e d i n t h e p r o p o s e d S . K . I . c y c l e m e ch a n ism f o r GQ2 f i x a t i o n a s t h e ’’p u l l i n g " r e a c t i o n t e n d i n g t o rem ove i s o c i t r a t e fro m t h e s i t e g lu ta ra te . o f th e re d u c tiv e c a rb o x y la tio n o f o -k e to - I t w as t h o u g h t t h a t g l y o x y l a t e and some o f t h e s u c c i n a t e p r o d u c e d w e re t h e n i n t e g r a t e d i n t o t h e b i o s y n t h e t i c m a c h in e ry w h ic h y ie ld s in c re a s e d g ro w th u n d e r c o n d i t i o n s o f i l l u m i n a t i o n b u t , i n t h e p r e s e n c e o f h ig h b i c a r b o n a t e c o n c e n t r a t i o n s , a r e s i s t a n t s p o ra n g iu m . M o re o v e r i t now seem s p o s s i b l e , on t h e b a s i s o f t h e h i g h s p e c i f i c a c t i v ­ ity o f t h e t r a n s a m i n a s e a s co m p ared t o i s o c i t r i t a s e and t h e p r o b a b l e e q u ilib riu m o f t h e g l y o x y l a t e - g l y c i n e c o n v e r s i o n to w a rd s g l y c i n e p r o ­ d u c t i o n , t h a t t r a n s a m i n a t i o n c o n s t i t u t e s a t l e a s t one o f t h e i m p o r t a n t r e a c t i o n s r e s p o n s i b l e f o r t h e f u r t h e r m e ta b o lis m o f g l y o x y l a t e . T h e r e f o r e , a num ber o f e x p e r im e n ts w e re c a r r i e d o u t i n a n a t t e m p t t o e l u c i d a t e t h e r o l e s o f t h e s e enzym es i n B l a s t o c l a d i e l l a . B e c a u se t h e s p e c i f i c lo c u s o f t h e l i g h t e f f e c t h a d n o t b e e n e s t a b l i s h e d , an y r e a c t i o n p re s u m e d t o b e in v o lv e d i n l i g h t s t i m u l a t e d g ro w th w as s u s p e c t . T h e r e f o r e , we f i r s t t e s t e d t h e p u r i f i e d enzym es f o r a s t i m u l a t i o n o f t t h e i r r e a c t i o n s b y w h i t e l i g h t an d e s t a b l i s h e d u n e q u i v o c a l l y t h a t n e i t h e r i s th e s p e c if ic lo c u s o f th e l i g h t e f f e c t . A m ethod w as t h e n d e v e lo p e d f o r t h e c u l t i v a t i o n o f O .C . c u l t u r e s w h ic h , on t h e b a s i s o f t h e i r m o r p h o l o g ic a l u n i f o r m i t y and t h e l o g a r i t h m i c n a t u r e o f t h e i r g ro w th c u r v e s , w e re c o n s id e r e d t o b e v e r y n e a r l y s y n ­ c h ro n o u s f o r m o s t o f t h e g e n e r a t i o n t i m e . i s fo u n d i n t h e r e c e n t r e s u l t s S u p p o rt f o r t h i s c o n te n tio n o f T u r ia n and C a n tin o ( i 9 6 0 ) w h e r e , 78 u s i n g s i m i l a r c u l t u r e s , n u c l e a r d i v i s i o n was found t o be c a . 80 p e r c e n t s y n c h ro n o u s f o r a t l e a s t t h e f i r s t s i x h o u rs o f g ro w th . I n ad- t i t i o n a new t e c h n i q u e was d e v e lo p e d t h a t p e r m i t t e d f o r th e f i r s t t i m e , b o t h f o r B . e m e r s o n i i and a q u a t i c f u n g i g e n e r a l l y , p h y s i o l o g i c a l s t u d i e s o f z o o s p o r e s u s i n g b o th m o t i l e , i n t a c t c e l l s and c e l l f r e e p r e p a r a t i o n s . M eanwhile L o v e tt (1959) a l s o e s t a b l i s h e d t h e c o n d i t i o n s f o r t h e grow th o f s y n c h ro n o u s c u l t u r e s of r e s i s t a n t s p o r a n g i a ! p l a n t s . Thus, w here p r e v i o u s p h y s i o l o g i c a l s t u d i e s aimed to w a rd s s o l v i n g d e v e lo p m e n ta l p r o b le m s w ere l i m i t e d t o t h e u s e o f h e te r o g e n e o u s p r e p a r a t i o n s o f m atu re o r d i n a r y c o l o r l e s s o r r e s i s t a n t s p o r a n g i a l p l a n t s , i t h as now become p o s s i b l e t o s t u d y b i o c h e m i c a l changes as t h e y o c c u r from t h e g e r m i n a t i o n o f t h e t i n y z o o s p o r e t o t h e d ev elo p m en t o f a m atu re p l a n t a lo n g e i t h e r o f t h e a l t e r n a t i v e d e v e lo p m e n ta l p a t h w a y s . Armed w i t h t h e s e new t e c h n i q u e s , we t h e n a tte m p te d t o f o l l o w t h e b e h a v i o r o f i s o c i t r i t a s e and g l y c i n e - a l a n i n e t r a n s a m i n a s e d u r i n g t h e d ev elo p m en t of s y n c h r o n i z e d , s i n g l e - g e n e r a t i o n c u l t u r e s o f o r d i n a r y c o l o r l e s s p l a n t s i n b o t h t h e l i g h t and t h e d a rk . By so d o in g i t was t h o u g h t t h a t t h e ch an g es t a k i n g p l a c e d u r i n g grow th and d i f f e r e n t i a t i o n , and t h e e f f e c t o f l i g h t t h e r e o n , m ig h t be r e f l e c t e d i n c o r r e s p o n d i n g chang es i n t h e two enzymes w hich would g iv e c l u e s t o t h e i r f u n c t i o n i n phenomena a s s o c i a t e d w i t h g ro w th . The r e s u l t s o f a number o f t h e s e e x p e r im e n ts f a i l e d t o r e v e a l any e f f e c t o f l i g h t on t h e a c t i v i t y l e v e l s o f e i t h e r g l y c i n e - a l a n i n e t r a n s ­ a m in a se o r i s o c i t r i t a s e . The s p e c i f i c a c t i v i t y o f i s o c i t r i t a s e was h i g h i n t h e z o o s p o r e s , d ro p p ed s h a r p l y as d ev elo p m en t b e g a n , rem ain ed 79 lo w u n t i l c a . s i x h o u r s , a m in a s e a c t i v i t y , and t h e n r o s e a g a i n on th e o th e r h a n d , to i n i t i a l le v e ls - c h an g e d r e l a t i v e l y l i t t l e T ran s­ d u rin g p l a n t g ro w th ; i t d ro p p e d o n ly _ca. o n e - t h i r d fro m s t a r t i n g l e v e l s i n t h e z o o s p o r e s t o t h o s e i n m a tu r e p l a n t s . A m ore m e a n in g f u l p i c t u r e f o r t h e p u r p o s e o f i n t e r p r e t a t i o n , e v o l v e s b y c o n s i d e r i n g t h e s y n t h e s i s o f t h e tw o enzym es i n t h e am o u n ts o f enzym e p e r p l a n t . as r e f le c te d Thus we fo u n d t h a t g l y c i n e - a l a n i n e t r a n s a m i n a s e w as s y n t h e s i z e d e x p o n e n t i a l l y ( F i g u r e 2 i|) th r o u g h o u t t h e g e n e r a t i o n tim e a s a l i n e a r f u n c t i o n o f p l a n t g ro w th ( F ig u r e 2 5 ) , w h i l e , i s o c i t r i t a s e s y n t h e s i s e x h i b i t e d a d e f i n i t e l a g ( F i g u r e s 21 an d 2 5 ) e x t e n d i n g t o b e tw e e n t h e t h i r d zo o sp o res. W h ile i t and s i x t h h o u r s a f t e r g e r m in a tio n o f t h e i s v e r y d i f f i c u l t t o a r r i v e a t any c o n c lu s io n s re g a rd in g g ly c in e - a la n in e tra n s a m in a s e , th e la g in i s o c i t r i t a s e s y n th e s is m ig h t o f f e r p o s s i b l e c l u e s t o i t s r o l e i n d e v e lo p m e n t. The s i g n i f i c a n c e o f t h i s l a c k o f i s o c i t r i t a s e s y n t h e s i s may b e a p p re c ia te d on t h e b a s i s o f C o m p a ra tiv e B io c h e m i s t r y . I t i s a x io m a ti c t h a t t h e c e l l m u st h a v e some w ay o f c o n t r o l l i n g i t s m e ta b o lis m and one m eans w h e re b y t h i s i s a c c o m p lis h e d i s b y t h e c o n t r o l o f enzym e s y n t h e s i s . $ A g r e a t d e a l o f w ork h a s b e e n d o n e on t h e phenom enon o f a d a p t i v e enzym e f o r m a t i o n i n m ic r o o r g a n is m s a s a p o s s i b l e m echanism f o r t h e c o n t r o l o f enzym e s y n t h e s i s - One o f t h e s i g n i f i c a n t f i n d i n g s o f t h e s e r e s e a r c h e s has been th a t th e re i s a r e m a r k a b le c o r r e l a t i o n b e tw e e n t h e n e e d f o r p a r t i c u l a r enzym e f u n c t i o n s and t h e s y n t h e s i s o f t h e enzym es i n v o l v e d . T h u s , f o r e x a m p le , i t h a s b e e n fo u n d t h a t f a c u l t a t i v e l y a n a e r o b i c b a c t e r i a ( E n g l e s b e r g and L e v y , 1 9 5 5 ) an d y e a s t ( S l o n i m s k i , 1 9 5 3 ) w hen 60 c u l t i v a t e d u n d e r a n a e r o b i c c o n d i t i o n s s y n t h e s i z e t h e K re b s' c y c le enzymes o n l y a t a v e r y low r a t e , b u t i n t h e p r e s e n c e o f oxygen a r a p i d s y n t h e s i s o f t h e w hole a r r a y o f r e s p i r a t o r y enzymes i s b r o u g h t a b o u t . Thus we m ight c o n c lu d e t h a t t h e l a c k o f s y n t h e s i s o f i s o c i t r i t a s e d u r i n g t h e e a r l y h o u r s o f d ev elo p m en t o f an O.C. p la n t-, r e f l e c t s a s i t u a t i o n i n w hich t h e r e a c t i o n i t c a t a l y z e s i s n o t r e q u i r e d , and t h a t iso c itrita se tim e . is not o p e ra tin g u n t i l r e l a t i v e l y l a t e in th e g e n e ra tio n I t would t h e r e f o r e f o l l o w t h a t C02 f i x a t i o n co u ld n o t be t a k i n g p la c e v i a th e o p e ra tio n of a S .K .I . cy cle in v o lv in g i s o c i t r i t a s e u n t i l l a t e i n d e v e lo p m e n t . A lth o u g h no s t u d i e s have y e t b e e n c a r r i e d out t o i n d i c a t e c a r b o n d i o x i d e f i x a t i o n i s so l i m i t e d , r e c e n t e v id e n c e c l e a r l y e s t a b l i s h e s t h a t t h e e f f e c t o f l i g h t on d ev elo p m en t i s n o t . Thus, T u r i a n and C a n tin o ( I9 6 0 ) h av e r e c e n t l y found by c y t o l o g i c a l s t u d i e s and c h e m ic a l a n a l y s e s t h a t DNA s y n t h e s i s and n u c l e a r d i v i s i o n t a k e p l a c e more r a p i d l y i n t h e l i g h t t h a n i n t h e d a rk th r o u g h o u t O.C. ontogeny} On t h e b a s i s o f t h e p r e c e d i n g d i s c u s s i o n and t h e e v id e n c e t h a t b o t h n u c l e i c a c i d s y n t h e s i s and t h e i s o c i t r i t a s e - d e p e n d e n t S . K . I . c y c l e a r e s t i m u l a t e d by l i g h t , we h av e ev o lv ed t h e f o l l o w i n g as a w o rk in g h y p o th e sis: l i g h t must be h a v in g i t s e f f e c t n o t d i r e c t l y on C02 f i x ­ a t i o n b u t on t h e m e t a b o l i c p a t h o r p a t h s l e a d i n g t o n u c l e i c a c i d s y n t h e s i s a n d , u l t i m a t e l y , grow th a t a p o i n t beyond t h e S . K . I . c y c l e . D u rin g t h e e a r l y p o r t i o n o f t h e g e n e r a t i o n tim e i t would seem t h a t i n c r e a s e d n u c l e i c a c i d s y n t h e s i s and o t h e r r e a c t i o n s a s s o c i a t e d w i t h i n c r e a s e d grow th a r e t a k i n g p l a c e a t t h e expense of a r e a d i l y a v a i l a b l e s o u r c e o f m e t a b o l i t e s w h ic h , a t l e a s t i n p a r t , may b e a l t e r n a t i v e l y 81 s u p p lie d b y i s o c i t r i t a s e h o u rs t h i s o r i t s p ro d u c ts . B e g in n in g a t t h r e e t o s i x r e a d i l y a v a i l a b l e s o u r c e o f p r e c u r s o r s b eco m es d e p l e t e d o r i n s u f f i c i e n t , n e c e s s i t a t i n g e i t h e r th e i n i t i a t i o n o f , o r a s h i f t t o . an a l t e r n a t i v e m e ch a n ism o f s u p p l y . S in c e t h i s p e r i o d c o r r e s p o n d s t o t h e d e c re a s e in endogenous r e s p i r a t o r y a c t i v i t y (F ig u re 2 0 ), i t i s p o s s ib le t h a t t h e s u p p ly d e f i c i e n c y i s d u e t o a d e c r e a s e i n t h e p r o d u c t i o n o f s u b s ta n c e s a s s o c ia te d w ith a f u n c tio n in g K re b s1 c y c le . In any c a s e , in re s p o n s e t o t h e re q u ire m e n t f o r an a l t e r n a t i v e s u p p ly s o u rc e i s o c i t r i t a s e s y n th e s is i s b eg u n , th u s b r in g in g i n t o o p e ra tio n th e S .K .I . c y c le , w ith a c o n s e q u e n t ir^ p re a s e i n C02 f i x a t i o n , w h ic h i s now p u l l e d b y l i g h t . T he g l y o x y l a t e t h a t i s p ro d u c e d i s a la n in e to y ie ld a t l e a s t in p a r t tra n s a m in a te d w ith g l y c i n e w h ic h m ig h t, i n t u r n , b e u s e d i n p r o d u c in g th y m in e (E lw y n and S p r i n s o n , 195H) an d p u r i n e s ( G o ld th w a it e t a l . , 1 9 5 6 ) f o r n u c le ic a c id s y n th e s is . u tiliz a tio n O t h e r , a s y e t , unknow n p a th w a y s f o r t h e o f g l y o x y l a t e , a s w e l l a s g l y c i n e and s u c c i n a t e may a l s o b e in o p e ra tio n . I n a f u r t h e r a tte m p t t o e lu c i d a te th e r o l e s o f i s o c i t r i t a s e and t r a n s a m i n a s e i n d e v e lo p m e n t, s y n c h ro n o u s c u l t u r e s o f r e s i s t a n t s p o r a n g i a l * p l a n t s w e re a l s o s t u d i e d . The r e s u l t s t h a t w e re o b t a in e d showed a n e x t r a o r d i n a r y c o r r e l a t i o n b e tw e e n t h e s p e c i f i c a c t i v i t i e s o f t h e tw o enzym es and t h e m o r p h o l o g ic a l and b io c h e m ic a l c h a n g e s w h ic h o c c u r d u r i n g t h e g e n e s i s o f a r e s i s t a n t s p o r a n g iu m . The s p e c i f i c a c t i v i t i e s o f b o t h g l y c i n e - a l a n i n e t r a n s a m i n a s e and i s o c i t r i t a s e w e re fo u n d t o i n c r e a s e s h a r p l y , b e g i n n in g a t a b o u t t h e tw e n ty —f o u r t h h o u r , r e a c h i n g maximum l e v e l s a t f o r t y - e i g h t h o u r s w h ic h 82 w ere t h r e e t o f o u r t i m e s h i g h e r t h a n a t any o t h e r s t a g e o f t h e e n t i r e l i f e c y c le . S i m u l t a n e o u s l y t h e r e i s a d r a m a t i c a p p e a r a n c e of gamma- c a r o t e n e and m e l a n i n , and i n c r e a s e d s y n t h e s i s o f l i p i d and c h i t i n , as w e l l as o t h e r s t r u c t u r a l and p h y s i o l o g i c a l chang es ( L o v e t t , 1 9 5 9 ). A l l o f t h e s e e v e n t s o c c u r a t a p p r o x i m a te ly t h r e e - f i f t h s of th e g e n e r a tio n t i m e , t h e p e r i o d beyond w hich R .S . m o rp h o g e n esis becomes an i r r e v e r s i b l e p ro cess? These r e s u l t s , th o u g h a d m i t t e d l y of a c o r r e l a t i v e n a t u r e , g iv e s i g n i f i c a n t s u p p o r t t o o u r c o n t e n t i o n t h a t i s o c i t r i t a s e i s a key com­ p o n e n t o f t h e b i c a r b o n a t e t r i g g e r m echanism. M oreover, we now have r e a s o n t o b e l i e v e t h a t g l y c i n e - a l a n i n e t r a n s a m i n a s e i s a l s o in v o lv e d in t h i s ch ain of e v e n ts . I n a t t e m p t i n g t o i n t e g r a t e t h e s e f i n d i n g s i n t o t h e o r i g i n a l h y p o th e ­ s i s o f m o r p h o g e n e s is , t h e f o l l o w i n g p i c t u r e em erges: b icarb o n ate a c ts t o s t o p and e v e n t u a l l y r e v e r s e s t h e fo rw a rd p r o g r e s s o f t h e K r e b s ’ c y c le by i n h i b i t i n g t h e two s u c c e s s i v e o x i d a t i v e d e c a r b o x y l a t i o n s m e d ia te d by a - k e t o g l u t a r a t e o x i d a s e and i s o c i t r i c d e h y d r o g e n a s e . As t h i s h ap p en s m ost o f t h e enzymes o f t h e t r i c a r b o x y l i c a c i d c y c l e d i s a p p e a r o r a r e r e n d e r e d i n o p e r a t i v e and t h e r e i s a d i s t u r b a n c e of t h e c r i t i c a l b a l a n c e o f r e a c t i o n s w hich c o n s t i t u t e t h e m e t a b o l i c a p p a r a t u s o f t h e c e l l . As a r e s u l t t h e r e i s a dje novo s y n t h e s i s o f some enzymes and i n c r e a s e d s y n t h e s i s o f o t h e r s t o e s t a b l i s h a new e q u i l i b r i u m s a t i s f a c t o r y t o t h e n eed s o f t h e c e l l . At c a . t h i r t y - s i x h o u r s t h i s r e m o d e l li n g p r o c e s s n e a r s c o m p le t io n and t h e o rg an ism becomes i r r e v e r s i b l y com m itted t o R .S . f o r m a t i o n as i s o c i t r i t a s e , g l y c i n e - a l a n i n e t r a n s a m i n a s e and o t h e r enzymes i n v o lv e d i n m o rp h o g e n e sis r e a c h maximum l e v e l s . Iso c itric 83 d e h y d r o g e n a s e , "the o n ly K r e b s 1 c y c l e enzym e w h ic h r e m a in s a c t i v e , b e g i n s t o m e d ia te w i t h g r e a t e r and g r e a t e r e f f i c i e n c y t h e r e d u c t i v e c a r b o x y la tio n o f a - k e t o g l u t a r a t e , w h ile i s o c i t r i t a s e p u l l s th e r e a c t i o n b y re m o v in g t h e i s o c i t r a t e t h a t i s p r o d u c e d . g l y o x y l a t e t h a t i s fo rm e d i s c y c le i s At l e a s t p a r t of th e c o n v e r te d t o g l y c i n e a n d , i f t h e S . K . I . i n v o l v e d , some o f t h e s u c c i n a t e i s r e c y c l e d b y w ay o f a T P N - s p e c if ic r e a c t i o n t o a - k e t o g l u t a r a t e t o a c c e p t m ore C02 and t h e r e m a in d e r i s f u r t h e r m e t a b o l i z e d . Somehow t h e s e r e a c t i o n s h e l p s u p p ly b u i ld i n g b lo c k s f o r th e s y n th e s is o f th e c h a r a c t e r i s t i c s t r u c t u r a l c o m p o n e n ts o f t h e r e s i s t a n t s p o r a n g iu m . B e tw e e n f o r t y - e i g h t an d s i x t y h o u r s t h e s y n t h e s i s o f g a m m a -c a ro te n e , m e la n in , l i p i d , c h i t i n and o t h e r a s s o c i a t e d s u b s t a n c e s n e a r s c o m p le tio n , t h e n e e d f o r i n t e r m e d i a t e s s u p p lie d by th e re d u c tiv e c a rb o x y la tio n o f a - k e to g lu ta r a te d is a p p e a r a n d , a s a r e s u l t , t h e l e v e l s o f i s o c i t r i t a s e and t r a n s a m i n a s e d e c r e a s e . I t w o u ld b e p r e m a tu r e t o d i s c u s s i n d e t a i l , w i t h t h e C o m p a ra tiv e B io c h e m ic a l l i t e r a t u r e fa te s a s a fra m e o f r e f e r e n c e , t h e p o s s i b l e m e t a b o l i c o f g l y o x y l a t e , s u c c i n a t e and g l y c i n e i n r e l a t i o n t o d i f f e r e n t i a t i o n and g r o w th . ♦ H o w ev e r, t h e f i n d i n g b y C a n tin o and H o r e n s t e i n (1 9 5 9 ) t h a t g l y o x y l a t e and s u c c i n a t e t o g e t h e r » d i r e c t p r o d u c t s o f t h e i s o c i t r i t a s e c l e a v a g e , w i l l r e p l a c e t h e l i g h t e f f e c t , and o u r d i s c o v e r y o f a d i r e c t m e t a b o l i c r o u t e fro m g l y o x y l a t e t o g l y c i n e a r e s t r o n g l y s u g g e s t i v e o f l i k e l y r o u t e s o f m e ta b o lis m . Thus K ik u c h i e t a l . (1 9 5 8 ) h a v e r e p o r t e d a n enzym e c a t a l y z e d r e a c t i o n y i e l d i n g detL ta—a m i n o l e v u l i n i c a c i d fro m s u c c i n a t e and g l y c i n e w h ic h , th o u g h t h e c o n d i t i o n s w e re n o t c l e a r l y d e f i n e d , w as s t i m u l a t e d b y l i g h t . M o re o v e r, s t u d i e s w i t h r a d i o a c t i v e " t r a c e r s h a v e i n c r i m i n a t e d d e l t a - a m i n o l e u l i n i c a c i d as an i n t e r m e d i a t e i n t h e c o n v e r s i o n o f g l y c i n e t o thy m in e and p u r i n e s (S hem in, 1956) as w e l l as c a r o t e n e s y n t h e s i s ( Mac K inney e t a l . , 1 9 5 5 ); and t h i s b r i n g s u s t o i h e r e c e n t o b s e r v a t i o n ( T u r i a n and C a n t in o , I9 6 0 ) t h a t th y m in e w i l l s u b s t i t u t e f o r l i g h t i n DNA s y n t h e s i s 1. From t h e s e f i n d i n g s we c a n n o t h e l p b u t r e g a r d as an a t t r a c t i v e one t h e p o s s i b i l i t y t h a t d e l t a - a m i n o l e u l i n i c a c i d i s t h e l i n k c o n n e c t i n g g l y o x y l a t e , g l y c i n e and s u c c i n i c a c i d t o l i g h t s t i m u l a t e d grow th an d , i n R .S . m o r p h o g e n e s is , a t l e a s t ca ro te n e s y n t h e s i s . The r e l a t i o n s h i p b e tw e e n l i g h t and grow th even i n c h l o r o p h y l l c o n t a i n i n g o rg an ism s i s p o o r l y u n d e r s to o d d i s p i t e t h e f a s c i n a t i n g and f u n d a m e n ta l b i o c h e m i c a l s t u d i e s b e i n g c a r r i e d o u t by w o rk e rs a l l over t h e w o r ld . The b i o c h e m i s t r y o f m o rp h o g e n esis i s such a young s c i e n c e t h a t o n l y a few w o r k e rs w o rk in g w i t h fe w e r o rg an ism s a r e engaged i n i t s s tu d y . I t i s no w onder t h e n , t h a t our knowledge i s y e t so f r a g m e n t a r y and our i n t e r p r e t a t i o n s so s p e c u l a t i v e . N e v e r t h e l e s s , such i n t e r p r e ­ t a t i o n s h a v e t h e v a l u e t h a t t h e y w i l l le a d t o f u r t h e r work w hich someday w i l l s o l v e t h e p u z z l e o f m o rp h o g e n e s is , such as t h a t in v o lv e d in t h e b io lo g y of B la s to c la d ie lla e m e rso n ii. 85 SUMMARY i' 1 ) The enzym e i s o c i t r i t a s e , w h ic h h a d b e e n p o s t u l a t e d t o o c c u r i n B l a s t o c l a d i e l l a « h a s b e a n p u r i f i e d fro m e x t r a c t s (ja . f i f t y - f o l d o f t h i s o rg a n is m and some o f i t s p r o p e r t i e s s t u d i e d . 2 ) A s e c o n d e n zy m e , g l y c i n e - a l a n i n e t r a n s a m i n a s e , w h ic h h a s n o t b e e n p r e v i o u s l y d e s c r i b e d , w as p u r i f i e d c a . e i g h t y - f o l d and i t s p r o p e r t i e s s i m i l a r l y e x a m in e d . 3 ) New m e th o d s w e re d e v e lo p e d w h ic h p e r m i t t e d , f o r t h e f i r s t tim e among t h e a q u a t i c f u n g i , e n z y m o lo g ic a l s t u d i e s a t a l l s t a g e s o f t h e g ro w th o f B l a s t o c l a d i e l l a , fro m t h e z o o s p o r e s t o t h e m a tu re o r d i n a r y c o lo rle s s p la n t. Ij.) The s y n t h e s e s o f i s o c i t r i t a s e as re fle c te d i n a c t i v i t y m e a s u re m e n ts , w e re f o llo w e d d u r i n g t h e d e v e lo p ­ m en t o f O .G . and R .S . p l a n t s . th e r e s u lts and g l y c i n e - a l a n i n e t r a n s a m i n a s e , An a t t e m p t w as t h e n made t o i n t e g r a t e o b ta in e d i n t o an i n t e r p r e t a t i o n o f th e f u n c tio n o f th e s e enzym es i n m o rp h o g e n e s is and l u m i s y n t h e s i s . 86 LITERATURE CITED A l t e r rib e r g , R . A . and H o u se w r ig h t, R . D. T ra n sa m in a ses In sm ooth B r u c e l l a a b o r t u s , s t r a i n 1 9 - J . B i o l . Chem., 201*: 1 5 9 -1 6 7 , 1 9 5 3 A r r h e n iu s , S . Q u a n t it a t iv e Laws i n B i o l o g i c a l C h e m istr y . G- B e l l and S o n s , L t d . , L ondon, 1 9 1 5 . Awapara, J - and S e a l e , B . D i s t r i b u t i o n o f tr a n s a m in a s e s J - B i o l . Chem ., 191**1*97-502, 1 9 5 2 . B a r n e r , H . D . and G a n tin o , E . C. s p e c ie s o f B la s t o c la d ie lla . in r a t o r g a n s . N u tr itio n a lr e la tio n s h ip s in a Am. J o u r . B o t . , 3 9 s 71 *6 - 7 5 1 . new B a x t e r , C. F . and R o b e r ts , E . The g a m m a -a m in o b u ty r ic -a -k e to g lu ta r ic a c id tr a n s a m in a s e o f b e e f b r a i n . J . B i o l . Chem., 2 3 3 :1 1 3 5 -1 1 3 9 1958. Broym , D . H . and G a n tin o , E . C. B l a s t o c l a d i e l l a e m e r s o n ii. The o x id a t i o n o f m a la te b y Amer. J o u r . B o t . 1*2:337-31*1* 1955* B r u n e i- C h a p e lle , G e n v ie v e , Sur l e m eta b o lism e de I ’ a c id g l y o x y l i q u e . Comptes r e n d u s , A cad . S c i . ( P a r i s ) , 2 3 l4.2 l l 4.6 6 , -1 9 5 2 . Cammarata, P . S . and Cohen, P . P . The sc o p e o f t h e tr a n s a m in a tio n r e a c t i o n i n a n im a l t i s s u e . J . B i o l . Chem., I 8 7 s 1*39 - 14-5 2 , 1 9 5 0 . Cammarata, P . S . and Cohen, P . P . F r a c t io n a t io n and p r o p e r t ie s o f g l u t a m i c - o x a l a c e t i c tr a n s a m in a s e . J . B i o l . Chem., 1 9 3 :5 3 -6 2 .. 1 9 5 1 C am p b ell, L . L . T r a n sa m in a tio n o f amino a c id s w it h g l y o x y l i c a c id in b a c te r ia l e x tr a c ts . J . B a ct . 7 1 * 8 1 - 8 3 * 1956. ♦ C am p b ell, J . J . R -, S m ith , R . A . .and E a g le s , B - A . A d e v i a t i o n from t h e c o n v e n t io n a l t r i c a r b o x y l i c a c id c y c le i n Pseudom onas a e r u g in o s a . B io c h im . B io p h y s . A c ta , 11:5914.* 1 9 5 3 C a n e lla k i s , Z . N . and Cohen, P . P . P u r i f i c a t i o n s t u d i e s o f t y r o s i n e a - k e t o g l u t a r i c tr a n s a m in a s e . J . B i o l . Chem., 2 2 2 : 5 3 - 6 2 , 1 9 5 6 . C a n tin o , E . C. M eta b o lism and m o rp h o g en esis i n a new B l a s t o c l a d i e l l a . A n to n ie v . L eeuw enhoek, 1 7 * 3 2 5 -3 6 2 , 1 9 5 1 C a n tin o , E - C. The b io c h e m ic a l n a tu r e o f m o r p h o g en etic p a t t e r n s in B l a s t o c l a d i e l l a . Amer. N a t . , 86:399-14-01** 1 9 5 2 . 87 G a n tin o , C . The r o l e o f m e ta b o lis m and ct-k e t o g l u t a r a t e o x id a s e i n g ro w th and d i f f e r e n t i a t i o n o f t h e a q u a t i c P h y c o m y c e te , B l a s t o G l a d i e l l a e m e r s o n i i , T r ^ n s . 'N - Y . A cad . S c i . ’, 1 £ : 1 5 9 -1 6 3 , 1 9 5 3 . G a n tin o , E . G. The r o l e o f m e ta b o lis m i n m o r p h o lo g ic a l d i f f e r e n t i a t i o n o f th e w a te r fu n g u s B la s to c la d ie 11a e m e r s o n ii. P r o c . I n t e r n a t . B o t. G o n g r. J u l y , 1951* P a r i s , F r a n c e . l~* G a n tin o , E . G. The r e l a t i o n b e tw e e n c e l l u l a r m e ta b o lis m and m o rp h o g e n e s is i n B l a s t o c l a d i e l l a . M y c o lo g ia , 1 8 :2 2 5 -2 1 * 0 , 1 9 5 6 . G a n tin o , E . C*. L i g h t a t i m a l a t e d g ro w th an d G02 f i x a t i o n i n a c h l o r o ­ p h y l l - l e s s f u n g u s , B l a s t o c l a d i e l l a . P r o c . 2nd I n t . C o n g r. P h o t o b i o l . j p p . U 53-U 56, 195>7 G a n tin o , E . G . D i s c u s s i o n o f b i o c h e m ic a l s y s te m s in v o lv e d i n d i f f e r e n t a t i o n o f f u n g i. In s Symposium on b i o c h e m i s t r y o f m o rp h o g e n e s is ; P r o c . 1 t h I n t e r n a t . G o n g r. f o r B io c h e m ., ( i n p r e s s ) , 1 9 5 8 . G a n tin o , E . G. R e l a t i o n s o f m e ta b o lis m t o c e l l d e v e lo p m e n t. I n : E n c y c lo p e d i a o f P l a n t P h y s io lo g y (G e rm a n y ), JL5: ( i n p r e s s ) , I 9 6 0 . G a n tin o , E . G. and H o r e n s t e i n , E . A . C y to p la s m ic e x c h a n g e w i t h o u t g a jn e tic c o p u l a t i o n i n t h e w a t e r m old B l a s t o c l a d i e l l a e m e r s o n i i . A m er. N a t . 8 8 :1 1 3 -1 5 1 * 19 5 1 G a n tin o , E . G. and H o r e n s t e i n , E . A . The r o l e o f k e t o _ g l u t a r a t e and p o l y p h e n o l o x id a s e i n t h e s y n t h e s i s o f m e la n in d u r i n g m o rp h o g e n e s is i n B l a s t o c l a d i e l l a e m e r s o n i i . Phys-. P l a n t . , _ 8 :1 8 9 -2 2 1 , 1 9 5 5 G a n tin o , E . G. and H o r e n s t e i n , E . A . The s t i m u l a t o r y e f f e c t o f l i g h t u p o n g ro w th and C02 f i x a t i o n i n B l a s t o c l a d i e l l a . I . th e S .K .I . c y c l e . M y c o lo g ia , 1 8 * 7 7 7 -7 9 9 * 1 9 5 6 a . G a n tin o , E - G. and H o r e n s t e i n , E . A. Gamma and t h e c y to p la s m ic c o n t r o l * o f d i f f e r e n t i a t i o n i n B l a s t o c l a d i e l l a . 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