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A study of the pathways of glucose oxidation of Pseudomonas aeruginosa Reid, K. Garth 1959

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A STUDY OP THE PATHWAYS OF GLUCOSE OXIDATION OF PSEUDOMONAS AERUGINOSA b y K. Garth. R e i d B.A. A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t o f 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 o f M a s t e r o f S c i e n c e i n A g r i c u l t u r a l M i c r o b i o l o g y i n t h e D i v i s i o n o f A n i m a l S c i e n c e We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e s t a n d a r d r e q u i r e d f r o m c a n d i d a t e s f o r t h e d e g r e e o f M a s t e r o f S c i e n c e The U n i v e r s i t y o f B r i t i s n C o l u m b i a A u g u s t 1959 ABSTRACT A n e f f o r t h a s b e e n made t o d e m o n s t r a t e t h a t t h e m a j o r p a t h w a y f o r g l u c o s e o x i d a t i o n i n Pseudomonas  a e r u g i n o s a (ATCC 9027) i n v o l v e s t h e s e q u e n c e o f r e a c t i o n s : g l u c o s e — > • g l u c o n a t e — 2 - k e t o g l u c o n a t e >• 2-keto-6-p h o s p h o g l u c o n a t e — 6 - p h o s p h o g l u c o n a t e . I t a p p e a r s h o w e v e r , t h a t e x t r a c t s o f t h i s o r g a n i s m a r e c a p a b l e o f p h o s p h o r y l a t i n g g l u c o s e d i r e c t l y , t h a t i s , t o y i e l d g l u c o s e - 6 - p h o s p h a t e a n d s u b s e q u e n t l y 6 - p h o s p h o g l u c o n a t e . A s t u d y o f t h i s l a t t e r p a t h w a y was f e l t t o be n e c e s s a r y i n o r d e r t o e v a l u a t e t h e l i k e l i h o o d o f i t b e i n g a m a j o r a l t e r n a t i v e t o t h e e s t a b l i s h e d n o n - p h o s p h o r y l a t e d p a t h w a y . S i n c e i t i s known t h a t g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e f r o m P. a e r u g i n o s a a n d o t h e r m i c r o o r g a n i s m s a s w e l l a s f r o m c e r t a i n a n i m a l t i s s u e s e x h i b i t s a m a r k e d s e n s i t i v i t y t o v a r i o u s n u c l e o t i d e s p a r t i c u l a r l y t o a d e n o s i n e t r i p h o s p h a t e . A s t u d y o f t h i s i n h i b i t i o n was made i n o r d e r t o a s s e s s t h e p o s s i b l e r o l e t h a t t h i s s e n s i t i v i t y may p l a y i n d e t e r m i n i n g t h e i m p o r t a n c e o f t h i s p a t h w a y a s t h e m a j o r r o u t e o f g l u c o s e o x i d a t i o n . Enzyme f r a c t i o n a t i o n s t u d i e s r e v e a l e d t h a t h e x o k i n a s e a n d g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e c o u l d be separated either by an ethanol fractionation or by an alkaline ammonium sulfate fractionation. The best separation of dehydrogenase was obtained using ethanol although hexokinase could only be isolated using the alkaline ammonium sulfate method. Ce l l free extracts of P. aeruginosa oxidize glucose to 2-keto-gluconate but carry the reaction no further. This represents a consumption of ly<M of oxygen per yuM glucose. In the presence of ATP the amount of oxygen consumed was reduced to a maximum of 0.5 per yt<M glucose, indicating the accumulation of a compound less oxidized than 2-ketogluconic acid. 6-phosphogluconate appeared to conform to the requirements of such a compound. Chromatographic analysis of reaction mixtures containing ATP revealed the accumulation of a phosphorylated compound which could not be identified. Under i n vitro conditions both pathways appear to be operable but the non-phosphorylated pathway accounts for most of the glucose in the metabalizing organism. In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the'Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of n Q i r y l n g The University of B r i t i s h Columbia, Vancouver 8, Canada. D a t e Septemhpr 3 , 1959 ACKNOWLEDGEMENT I wish, t o e x p r e s s my s i n c e r e t h a n k s t o D r . J . J . R . C a m p b e l l f o r h i s d i r e c t i o n a n d e n c o u r a g e m e n t d u r i n g t h e c o u r s e o f t h i s w ork. TABLE OP CONTENTS Page HISTORICAL REVIEW 1 MATERIALS AND METHODS 11 O r g a n i s m 11 C e l l F r e e P r e p a r a t i o n s 11 Enzyme A s s a y s 13 G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e 1$ P h o s p h o h e x o i s o m e r a s e 14 2 - K e t o - 6 - p h o s p h o g l u c o n a t e r e d u c t a s e 14 H e x o k i n a s e 15 G l u c o n o k i n a s e 16 2 - K e t o g l u c o n o k i n a s e 16 Enzyme F r a c t i o n a t i o n 17 P r o t a m i n e s u l f a t e 17 Ammonium s u l f a t e 18 A l k a l i n e ammonium s u l f a t e 19 A l c o h o l f r a c t i o n a t i o n 19 C a l c i u m p h o s p h a t e g e l . . 20 M a n o m e t r i c P r o c e d u r e s . . . 20 A n a l y t i c a l P r o c e d u r e s 21 P r o t e i n d e t e r m i n a t i o n 21 G l u c o s e d e t e r m i n a t i o n 22 2 - K e t o g l u c o n a t e d e t e r m i n a t i o n 23 P y r u v a t e d e t e r m i n a t i o n 24 C h r o m a t o g r a p h y 25 Page EXPERIMENTAL RESULTS AND DISCUSSION 31 Enzyme S t u d i e s w i t h C e l l E x t r a c t s 31 Enzyme P u r i f i c a t i o n 42 M a n o m e t r i c S t u d i e s 45 SUMMARY 58 BIBLIOGRAPHY 60 HISTORICAL REVIEW Studies by Stokes and Campbell (58) and by Wood and Schwerdt (63) have revealed that gluconic and 2-ketogluconic acids are intermediates i n the oxidation of glucose by Pseudomonas aeruginosa (ATCC 9027). It has also been shown that the system of terminal respiration i s the tricarboxylic acid cycle (9) (11) and that the organism possesses a deviation from this cycle involving glyoxylate (10). Using labelled substrates, Wang, Stern and Gilmour (61) have concluded that P. aeruginosa oxidizes gluconate almost exclusively by the Entner-Doudoroff pathway which involves 6-phosphogluconate. This information appears to be contradictory to the conclusion that glucose i s oxidized by way of 2-ketogluconate. However, Blackwood and Blakley (3) have shown that Leuconostoc mesenteroides ferments 2-ketogluconate by a sequence of reactions involving the phosphorylation of this compound and then i t s subsequent reduction to 6-phosphogluconic acid. An effort w i l l be made i n the present work to determine whether or not a similar sequence of reactions could account for the oxidation of glucose and gluconate i n P. aeruginosa 9027. 2 I t i s w e l l known t h a t v a r i o u s s p e c i e s o f Pseudomonas a n d o t h e r a e r o b e s w i l l a c c u m u l a t e g l u c o n i c a c i d and 2 - k e t o g l u c o n i c a c i d i n t h e i r g r o w t h medium u n d e r c e r t a i n c o n d i t i o n s ( 3 6 ) ( 3 9 ) ( 4 7 ) ( 5 0 ) ( 5 2 ) . A l t h o u g h a c c u m u l a t i o n o f t h e s e compounds i s n o t p r o o f t h a t t h e y a r e m e t a b o l i c i n t e r m e d i a t e s , l a t e r w ork d o n e i n t h i s l a b o r a t o r y ( 3 8 ) a n d e l s e w h e r e ( 1 3 ) ( 6 3 ) w i t h c e l l f r e e e x t r a c t s h a s e s t a b l i s h e d g l u c o n i c and 2 - k e t o g l u c o n i c a c i d s a s members o f a n o n - p h o s p h o r y l a t e d p a t h w a y o f g l u c o s e o x i d a t i o n i n P. a e r u g i n o s a . E n t n e r a n d S t a n i e r ( 2 5 ) h o w e v e r , h a v e s t a t e d t h a t 2 - k e t o g l u c o n a t e may n o t be a n i n t e r m e d i a t e i n t h e m a j o r p a t h w a y o f g l u c o s e o x i d a t i o n i n P. f l u o r e s c e n s . T h e y b a s e d t h e i r a r g ument on t h e f a c t t h a t u l t r a v i o l e t ' i r r a d i a t e d a s p a r a g i n e grown c e l l s w o u l d t a k e up l i t t l e o r no o x y g e n i n t h e p r e s e n c e o f g l u c o n i c o r 2 - k e t o g l u c o n i c a c i d s , a l t h o u g h g l u c o s e was s t i l l o x i d i z e d . S i n c e u l t r a v i o l e t r a d i a t i o n i s known t o i n h i b i t a d a p t i v e enzyme s y n t h e s i s , i t was c o n c l u d e d t h a t g l u c o s e w o u l d n o t be u t i l i z e d b y way o f t h e p a r t i a l l y i n a c t i v a t e d 2 - k e t o g l u c o n i c p a t h w a y , s i n c e g l u c o s e o x i d a t i o n i t s e l f was n o t i m p a i r e d . T h e y a l s o p o i n t e d o u t t h a t c e l l s grown on g l u c o s e , w o u l d o x i d i z e g l u c o s e o r g l u c o n a t e a t a h i g h i n i t i a l r a t e ( a p p r o x i m a t e l y t e n t i m e s t h e r a t e o f 2 - k e t o g l u c o n a t e o x i d a t i o n ) . C a m p b e l l a n d K i t o s ( 5 ) a n d a l s o C l a r i d g e a n d Werkman ( 1 4 ) h o w e v e r , c o u l d n o t r e p r o d u c e 3 t h i s two p h a s e r e s p o n s e . I n c o n t r a s t , C a m p b e l l a n d K i t o s (5) f o u n d t h a t c e l l s grown on a s p a r a g i n e , g l u c o s e , o r 2 - k e t o g l u c o n a t e w o u l d a l w a y s p r o d u c e t h e same a d a p t i v e l a g when a l l o w e d t o a c t o n 2 - k e t o g l u c o n a t e . By u s i n g s t r e p t o m y c i n i n p l a c e o f u l t r a v i o l e t l i g h t as a n i n h i b i t o r o f a d a p t i v e enzyme f o r m a t i o n , t h e y f o u n d t h a t g l u c o s e grown c e l l s c o u l d o x i d i z e 2 - k e t o g l u c o n a t e a t t h e same r a t e a s 2 - k e t o g l u c o n a t e grown c e l l s . I t was t h e r e f o r e c o n c l u d e d t h a t t h e a d a p t i v e r e s p o n s e t o 2 - k e t o g l u c o n a t e b y g l u c o s e grown c e l l s was due t o some " t i m e c o n s u m i n g r e a c t i o n " t a k i n g p l a c e p r i o r t o t h e a c t u a l o x i d i a t i o n o f 2 - k e t o g l u c o n a t e , r a t h e r t h a n t h e s y n t h e s i s o f a i enzyme. E n t n e r and S t a n i e r a g r e e d t h a t 2 - k e t o g l u c o n a t e a c c u m u l a t e d d u r i n g t h e o x i d a t i o n o f g l u c o s e b u t r a t i o n a l i z e d t h i s a s b e i n g t h e r e s u l t o f a s p l i t p a t h w a y where t h e m a i n s y s t e m i s b y way o f g l u c o n i c a c i d w i t h o n l y a smaTl p o r t i o n g o i n g b y w a y . o f 2 - k e t o g l u c o n i c a c i d . T h e s e w o r k e r s f o u n d t h a t c o n s i d e r a b l y more o x y g e n was consumed d u r i n g t h e r a p i d i n i t i a l p h a s e t h a n w o u l d be r e q u i r e d t o o x i d i z e g l u c o s e t o 2 - k e t o g l u c o n i c a c i d . T h e y c o n c l u d e d t h a t t h e p r o b a b l e r a t e l i m i t i n g i n t e r m e d i a t e w o u l d be a compound o x i d i z e d more t h a n 2 - k e t o g l u c o n a t e . C a m p b e l l an d K i t o s (5) h o w e v e r , h a v e n o t b e e n a b l e t o c o n f i r m t h e m a n o m e t r i c d a t a o f E n t n e r a n d S t a n i e r . T h e y f o u n d t h a t r e s t i n g c e l l s o f P. f l u o r e s c e n s (A312) o x i d i z e d g l u c o s e and 2 - k e t o g l u c o n a t e c o m p l e t e l y a n d a t a r a p i d r a t e . Wood a n d S c h w e r d t (64) f o u n d t h a t e x t r a c t s o f P. f l u o r e s c e n s w o u l d o x i d i z e g l u c o s e - 6 - p h o s p h a t e , 6 - p h o s p h o g l u c o n a t e and p e n t o s e p h o s p h a t e s a s w e l l a s g l u c o s e a n d g l u c o n i c a c i d s . T r e a t m e n t o f t h e p r e p a r a t i o n i n s u c h a way a s t o i n a c t i v a t e t h e g l u c o s e ~ 6 - p h o s p h a t e o x i d i z i n g s y s t e m h a d l i t t l e e f f e c t o n t h e q u a n t i t a t i v e c o n v e r s i o n o f g l u c o s e o r g l u c o n a t e t o 2 - k e t o g l u c o n a t e . T h e y f o u n d a l s o t h a t t h e s e e x t r a c t s w o u l d r e d u c e DPN"*" o r TPN i n t h e p r e s e n c e o f g l u c o s e - 6 - p h o s p h a t e , 6-phospho-g l u c o n a t e o r r i b o s e - 5 - p h o s p h a t e h u t n o t i n t h e p r e s e n c e o f g l u c o s e , g l u c o n a t e , 2 - k e t o g l u c o n a t e , r i b o s e o r f r u c t o s e d i p h o s p h a t e e i t h e r i n t h e p r e s e n c e o r a b s e n c e o f ATP; i n d i c a t i n g t h e a b s e n c e o f h e x o k i n a s e o r g l u c o n o k i n a . s e (63). T h e s e w o r k e r s c o n c l u d e d t h a t t h e p a t h w a y o f o x i d a t i o n o f p h o s p h o r y l a t e d compounds i s i n d e p e n d e n t o f t h e n o n p h o s p h o r y l a t e d p a t h w a y . N a r r o d a n d Wood (49) l a t e r d e m o n s t r a t e d t h e p r e s e n c e o f g l u c o n o k i n a s e and 2 - k e t o g l u c o n o k i n a s e i n P. f l u o r e s c e n s . C l a r i d g e a n d Werkman (15) c o n f i r m e d much o f t h e d a t a p r e s e n t e d b y Wood a n d S c h w e r d t (64) b u t were a b l e t o d e m o n s t r a t e a h e x o k i n a s e i n P. a e r u g i n o s a t h u s p r e s e n t i n g a l i n k b e t w e e n t h e p h o s p h o r y l a t e d p a t h w a y a n d t h e n o n p h o s p h o r y l a t e d ^ The f o l l o w i n g a b b r e v i a t i o n s a r e u s e d i n t h i s p r e s e n t a t i o n A D P — a d e n o s i n e d i p h o s p h a t e ; A T P — a d e n o s i n e t r i p h o s p h a t e ; O T P — c y s t o s i n e t r i p h o s p h a t e ; G - T P — g u a n o s i n e t r i p h o s p h a t e ; I T P — i n o s i n e t r i p h o s p h a t e ; U T P — u r i d i n e t r i p h o s p h a t e ; D P N — 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 ; D P N H — 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 ( r e d u c e d f o r m ) ; T P N — t r 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 T r i s — T r i s ( h y d r o x y m e t h y l ) a minomethane. 5 'pathway a t t h e h e x o s e l e v e l . K l e i n a n d D o u d o r o f f (37) d e m o n s t r a t e d a n a d a p t i v e g l u c o k i n a s e i n P. p u t r i f a c i e n s . D e l e y (19) h a s p r e s e n t e d e v i d e n c e f o r 2 - k e t o g l u c o k i n a s e i n many a e r o b i c g e n e r a o f m i c r o o r g a n i s m s i n c l u d i n g Pseudomonas w h i l e D o i , H a l v o r s o n a n d C h u r c h (21) f o u n d t h a t e x t r a c t s o f B a c i l l u s c e r e u s s p o r e s w o u l d p h o s p h o r y l a t e 2 - k e t o g l u c o n a t e b u t n o t g l u c o s e o r g l u c o n i c a c i d . The u n i q u e n a t u r e o f t h e n o n p h o s p h o r y l a t e d p a t h w a y o f g l u c o s e o x i d a t i o n l i e s i n t h e f a c t t h a t a p p a r e n t l y no e n e r g y i s g a i n e d b y t h e c e l l d u r i n g t h e two o x i d a t i v e s t e p s f r o m g l u c o s e t o 2 - k e t o g l u c o n a t e . C a m p b e l l , L i n n e s a n d E a g l e s (6) a n d C a m p b e l l , R a m a k r i s h n a n , L i n n e s a n d E a g l e s (8) f o u n d t h a t g l u c o s e , g l u c o n a t e a n d 2 - k e t o g l u c o n a t e w o u l d s u p p o r t e q u a l amounts o f g r o w t h o f P. a e r u g i n o s a when t h e s e compounds were u s e d as c a r b o n s o u r c e s f o r g r o w t h . T h i s w o u l d i n d i c a t e t h e r e _ i s no d i f f e r e n c e i n a v a i l a b l e e n e r g y b e t w e e n t h e d i f f e r e n t o x i d a t i o n l e v e l s . F u r t h e r e v i d e n c e f o r t h i s i s t h e f a c t t h a t t h e s e o x i d a t i o n s d e p e n d u p o n n e i t h e r p y r i m i d i n e n u c l e o t i d e s n o r f l a v o r p r o t e i n s f o r h y d r o g e n t r a n s p o r t (54-)- (63). C a m p b e l l e t a l (8) were u n a b l e t o d e m o n s t r a t e any t r a n s f e r o f i n o r g a n i c p h o s p h o r u s t o t h e a d e n y l i c a c i d s y s t e m b y e x t r a c t s o f P. a e r u g i n o s a d u r i n g t h e o x i d a t i o n o f g l u c o s e t o 2 - k e t o g l u c o n a t e . T h e r e i s no e v i d e n c e i n t h e s e p a p e r s t o i n d i c a t e t h a t t h e s t e p s f r o m g l u c o s e t o 2 - k e t o g l u c o n a t e 6 y i e l d e n e r g y t o t h e m e t a b o l i s i n g o r g a n i s m . Wood a n d S c h w e r d t ( 6 $ ) f o u n d t h a t t h i s p a t h w a y i n P. f l u o r e s c e n s was p a r t i a l l y s e n s i t i v e t o 10 ^ M c y a n i d e , i n d i c a t i n g a c y t o c h r o m e l i n k e d s y s t e m . R a m a k r i s h n a n a n d C a m p b e l l ( 5 4 ) f o u n d p y o c y a n i n t o be a g o o d h y d r o g e n a c c e p t o r f o r g l u c o n i c d e h y d r o g e n a s e i n P. a e r u g i n o s a . D o i e t a l ( 2 1 ) ho w e v e r , h a v e p u r i f i e d a DPN l i n k e d g l u c o s e d e h y d r o g e n a s e f r o m e x t r a c t s o f B. c e r e u s s p o r e s . The p a t h w a y o f g l u c o s e o x i d a t i o n seems t o be q u i t e w e l l e s t a b l i s h e d i n t h e s e p r e p a r a t i o n s . G l u c o s e i s o x i d i z e d t o 2 - k e t o g l u c o n a t e v i a g l u c o n i c a c i d f o l l o w e d b y p h o s p h o r y l a t i o n i n t h e p r e s e n c e o f ATP and M g ; + + t o y i e l d 2 - k e t o - 6 - p h o s p h o g l u c o n a t e ( 2 K 6 P G ) . T h i s compound i s t h e n r e d u c e d i n t h e p r e s e n c e o f DPN t o g i v e 6 - p h o s p h o g l u c o n a t e w h i c h i n t u r n i s o x i d i z e d f u r t h e r i n a v a r i e t y o f p o s s i b l e p a t h w a y s , t h e mos t i m p o r t a n t b e i n g a n E n t n e r - D o u d o r o f f a n d a P e n t o s e c y c l e m e c h a n i s m . T h e r e i s no i n d i c a t i o n t h a t e i t h e r g l u c o s e o r g l u c o n a t e a r e p h o s p h o r y l a t e d d i r e c t l y . T he a u t h o r s p o i n t o u t t h a t 2K6PG may be d i s s i m i l a t e d d i r e c t l y b y a n a s y e t unknown m e c h a n i s m . S e v e r a l w o r k e r s h a v e d e m o n s t r a t e d a 2K6PG r e d u c t a s e ; B l a c k w o o d a n d B l a k l e y (3) i n L e u c o n o s t o c m e s e n t e r o i d e s a n d P r a m p t o n and Wood ( 2 8 ) i n P. f l u o r e s c e n s . The p r e s e n c e o f a 2 - k e t o - 6 -p h o s p h o g l u c o n a t e r e d u c t a s e i n t h e e x t r a c t s o f t h e s e o r g a n i s m s i s v e r y i m p o r t a n t i n p r o v i d i n g a l i n k b e t w e e n 'the phosphorylated compounds, and thereby increasing the importance of 6-phosphogluconate as a key intermediate (20) (24) (46) (63). At this point the very important data of Wang et a l (61) must be considered. Using a radiorespirometric technique they found that the CO2 produced by whole c e l l s of P. aeruginosa from s p e c i f i c a l l y labelled glucose had a different isotope content than the CO2 from similarly labelled gluconic acid. That i s , the C-j-and of gluconic acid were converted into CO2 at equivalent rates whereas the from glucose was converted to CO2 at a much faster rate than C ^ . Their data indicated that the oxidation of gluconic acid takes place almost entirely by way of an Entner-Doudoroff 3-3 s p l i t mechanism of 6-phosphogluconate, whereas glucose oxidation probably consists of a combination of the Entner-Doudoroff and Pentose cycle mechanisms with the former playing the predominant role. Lewis et a l (46) presented evidence indicating that i n P. fluorescens and P. aeruginosa the Entner-Doudoroff pathway accounted for, from one-third to one-half of the glucose oxidized while the pentose cycle probably accounted for most of the balance. Blackwood and Blakley (3) demonstrated that Leuconostoc  mesenteroides metabolized gluconate to yi e l d equimolar amounts of C0 9 and ethanol plus acetate and lactate; whereas 8 2-ketogluconate gave equimolar amounts of CC^, acetate and lactate. In both cases Cj always appeared as CC^ and Cg as the methyl carbon in lactate. The original pathway described by Entner and Doudoroff (24) in Pseudomonas saccharophila consisted of a standard hexose monophosphate shunt to 6-phosphogluconate followed by a dehydration to an hypothetical intermediate 2-keto-3-deoxy-6-phosphogluconic acid (2K3D6PG). This i n turn was s p l i t to yield pyruvate and triose phosphate. When the ce l l s were poisoned with dinitrophenol or arsenite the triose phosphate was converted to pyruvate. Kovachevich and Wood (42) have c l a r i f i e d the sequence of reactions i n P. fluorescens from 6-phosphogluconate to the products of the aldolase s p l i t . A dehydrase enzyme converts 6-phosphogluconate to the enol form followed by a v i r t u a l l y irreversible conversion to 2K3D6PG. The aldolase that s p l i t s 2K3D6PG to pyruvate and glyceraldehyde-3-phosphate (G3P) has been shown to be relatively specific, for fructose diphosphate and deoxyribose-5~ph.osph.ate were not attacked. No cofactors were required. Other organisms found to have high levels of 2K3D6PG aldolase a c t i v i t y were P. aeruginosa and Escherichia c o l i . Although small amounts of a fructose diphosphate aldolase, as well as other glycolytic enzymes such as G3P dehydrogenase (13) 5appear i h P. aeruginosa (5) (13) and i n P. fluorescens ( 5 ) , no Embden-Meyerhof scheme a p p e a r s t o o p e r a t e i n t h e s e o r g a n i s m s (1) (7) (14). C l a r i d g e a n d Werkman (15) h a v e d e m o n s t r a t e d s m a l l amounts o f d i h y d r o x y a c e t o n e among t h e o x i d a t i o n p r o d u c t s o f 2 - k e t o g l u c o n a t e i n P. a e r u g i n o s a . E x a c t l y how t h i s compound i s i n v o l v e d i n t h e m e t a b o l i s m o f t h i s o r g a n i s m i s n o t c l e a r , a l t h o u g h Hauge e t a l ( 3 0 ) h a v e d e m o n s t r a t e d a l i n k b e t w e e n d i h y d r o x y a c e t o n e m e t a b o l i s m and t h e p e n t o s e c y c l e i n A c e t o b a c t e r s u b o x y d a n s . The e x t e n t t o w h i c h a p a t h w a y i s u t i l i z e d may be g o v e r n e d t o some e x t e n t b y t h e amount o f o x y g e n a v a i l a b l e t o t h e m e t a b o l i z i n g c e l l . L o ckwood a n d S t o d o l a (47) f o u n d t h a t u n d e r c o n d i t i o n s o f h i g h a e r a t i o n 2 - k e t o g l u c o n a t e w o u l d a c c u m u l a t e i n P. f l u o r e s c e n s . I f a e r a t i o n was d e c r e a s e d 2 - k e t o g l u c o n a t e was c o n v e r t e d t o ° < - k e t o g l u t a r i c a c i d . K i t o s e t a l ( 3 6 ) h o w e v e r , f o u n d t h a t u n d e r c o n d i t i o n s o f h i g h o x y g e n t e n s i o n A. s u b o x y d a n s o x i d i z e d g l u c o s e a l m o s t e x c l u s i v e l y , a n d c o m p l e t e l y , v i a t h e p e n t o s e c y c l e . P e n t o s e p h o s p h a t e a c c o u n t e d f o r 75% o f t h e g l u c o s e u t i l i z e d . U n d e r t h e s e c o n d i t i o n s t h e p e n t o s e c y c l e a c c o u n t e d f o r n e a r l y a l l t h e CO2 p r o d u c e d . U n d e r r e d u c e d o x y g e n t e n s i o n ( a i r a t m o s p h e r e ) t h e p e n t o s e c y c l e s t i l l a c c o u n t e d f o r most o f t h e p r o d u c e d a l t h o u g h a s m a l l amount a p p a r e n t l y was p r o d u c e d b y a n o t h e r m e c h a n i s m . The t o t a l amount o f g l u c o s e o x i d i z e d t o c o m p l e t i o n h o w e v e r , was d e c r e a s e d a n d t h i s 10 d i f f e r e n c e was a c c o u n t e d f o r b y t h e a c c u m u l a t i o n o f 2 - k e t o g l u c o n i c a c i d . The p r e s e n t s t a t u s o f g l u c o s e o x i d a t i o n i n P. a e r u g i n o s a a s p r e s e n t e d i n t h i s h i s t o r i c a l r e v i e w a n d b y t h e e v i d e n c e t o be p r e s e n t e d i n t h i s t h e s i s i n d i c a t e s t h a t i t i s p r o b a b l y s i m i l a r t o t h a t f o u n d i n many r e l a t e d o r g a n i s m s . The o x i d a t i o n o f g l u c o s e t o 2 - k e t o g l u c o n a t e w i t h o u t i n v o l v i n g p h o s p h o r y l a t e d i n t e r m e d i a t e s a p p e a r s t o be a common p a t h w a y among many g e n e r a ; i n c l u d i n g P seudomonas, X a n t h o m o n a s , E s c h e r i c h i a , A e r o b a c t e r , A c e t o b a c t e r , B a c i l l u s , P a r a c o l o b a c t r u m a n d S e r r a t i a (19) ( 2 1 ) . A l t h o u g h g l u c o n o k i n a s e a n d 2 - k e t o g l u c o n k i n a s e h a v e b e e n d e m o n s t r a t e d i n P, f l u o r e s c e n s , w h i c h i s g e n e r a l l y c o n s i d e r e d t o be v e r y s i m i l a r t o P. a e r u g i n o s a (5)» o n l y t h e h e x o k i n a s e h a s b e e n d e m o n s t r a t e d i n e x t r a c t s o f P, a e r u g i n o s a 9027. I n s p i t e o f t h i s i t seems p r o b a b l e t h a t g l u c o s e i s d e g r a d e d b y way o f p a t h w a y s s i m i l a r t o t h a t i n L. m e s e n t e r o i d e s (3) ( 4 ) P. f l u o r e s c e n s ( 2 8 ) o r B. c e r e u s s p o r e s ( 2 1 ) ; w h e r e b y g l u c o s e , g l u c o n a t e and 2 - k e t o g l u c o n a t e a r e m e t a b o l i z e d b y way o f 6 - p h o s p h o g l u c o n a t e a n d l i n k e d e n z y m a t i c a l l y w i t h g l u c o s e a n d 2 - k e t o g l u c o n a t e v i a t h e i r p h o s p h o r y l a t e d d e r i v a t i v e s . T h e r e a p p e a r s t o be g o o d e v i d e n c e f o r t h e o p i n i o n t h a t 6 - p h o s p h o g l u c o n a t e i s t h e n d e g r a d e d p r i m a r i l y b y an E n t n e r - D o u d o r o f f a n d s e c o n d a r i l y b y a W a r b u r g - D i c k e n s p a t h w a y . MATERIALS AND METHODS O r g a n i s m The o r g a n i s m u s e d t h r o u g h o u t t h e s e e x p e r i m e n t s was a s t r o n g l y p i g m e n t i n g s t r a i n o f Pseudomonas a e r u g i n o s a (ATCC 9027). I t was m a i n t a i n e d on a g l y c e r o l p e p t o n e a g a r medium w i t h f r e s h t r a n s f e r s b e i n g t a k e n a s r e q u i r e d f r o m l y o p h i l i z e d s t o c k c u l t u r e s . The medium u s e d f o r l a r g e y i e l d s o f c e l l s f o r e x p e r i m e n t a l p u r p o s e s was o f t h e f o l l o w i n g c o m p o s i t i o n : D i p o t a s s i u m h y d r o g e n p h o s p h a t e , 0.15%; ammonium d i h y d r o g e n p h o s p h a t e , 0.30%; f e r r o u s s u l f a t e , 0.5 P ' P ' E i ' ; y e a s t e x t r a c t , 0.1%; g l u c o s e , 0.5%; magnesium s u l f a t e h e p t a h y d r a t e , 0.1% ( a d d e d a f t e r s t e r i l i z a t i o n ) ; 0 H 7.O. A t l e a s t two t r a n s f e r s were made t h r o u g h t h i s medium b e f o r e c e l l s were grown f o r h a r v e s t i n g . I n c u b a t i o n t e m p e r a t u r e was a l w a y s 30°C. C u l t u r e were grown e i t h e r i n 100 m l . q u a n t i t i e s i n Roux f l a s k s , f o r 18 t o 20 h o u r s o r i n 15 l i t r e q u a n t i t i e s w i t h c o n s t a n t a e r a t i o n a n d v i g o r o u s s t i r r i n g f o r 6 t o 8 h o u r s . T he r e s u l t i n g g r o w t h was 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 a t 8,000 x g i n a S e r v a l l r e f r i g e r a t e d c e n t r i f u g e . C e l l F r e e P r e p a r a t i o n s C e l l s were washed i n one o f two ways d e p e n d i n g o n t h e m e t h o d b y w h i c h c e l l f r e e e x t r a c t s (CEX) were t o be p r e p a r e d . C e l l s t o be b r o k e n b y t h e 10 K c . R a y t h e o n o s c i l l a t o r were washed o n c e i n M/30, pHV.O p h o s p h a t e b u f f e r and c o n c e n t r a t e d t o a p p r o x i m a t e l y 1/6 t h e o r i g i n a l g r o w t h v o l u m e . The c e l l s were t h e n w a s h e d a g a i n w i t h 0.2% KC1 a n d r e s u s p e n d e d i n t h i s d i l u e n t a t t h e r a t e o f 200 mg./ml. 50 m l . a l i q u o t s o f t h i s s u s p e n s i o n were e x p o s e d t o t h e o s c i l l a t o r f o r 12 m i n u t e s f o l l o w e d b y n e u t r a l i z a t i o n w i t h 0.2N KOH and c e n t r i f u g a t i o n a t 12,000 x g t o remove d e b r i s and w h o l e c e l l s . C e l l s t o be c r u s h e d b y t h e Hughes P r e s s ( 3 2 ) were washed o n c e i n d i s t i l l e d w a t e r t h e n o n c e i n a s o l u t i o n o f 0.2 mg.% g l u t a t h i o n e . The r e s u l t i n g c e l l p a s t e was p a c k e d i n o p e n e n d e d g l a s s v i a l s o f a p p r o p r i a t e d i m e n s i o n s . E a c h v i a l c o n t a i n e d 6 t o 8 g r a m s - o f c e l l s (wet w e i g h t ) . T h e s e were t h e n t i g h t l y c o r k e d and f r o z e n i n a d r y i c e - e t h a n o l b a t h f o r 20 m i n u t e s . The f r o z e n c e l l s w ere s u b s e q u e n t l y s t o r e d a t - 1 8 ° C . u n t i l n e e d e d . When t h e o p e r a t i o n was c o m p l e t e d , t h e e x t r a c t was removed f r o m t h e r e s e r v o i r a n d s u s p e n d e d a t t h e r a t e o f 200 mg./ml. i n a c o l d d i l u e n t ( s l i g h t l y m o d i f i e d f r o m t h a t d e s c r i b e d b y P u l l m a n and R a c k e r (55)) o f t h e f o l l o w i n g c o m p o s i t i o n : M/20 g l y c y l g l y c i n e ; M/4- s u c r o s e ; B o v i n e serum a l b u m i n , 500 mg.%; M/20 T r i s b u f f e r . The r e s u l t i n g v i s c o u s s o l u t i o n was h o m o g e n i z e d t h o r o u g h l y w i t h a g l a s s o r t e f l o n P o t t e r - E l v e h j e m h o m o g e n i z e r . The r e s u l t i n g 13 p r e p a r a t i o n was e a s i l y p i p e t t e d and was c e n t r i f u g e d a t 12,000 x g f o r 15 m i n u t e s t o remove d e b r i s and w h o l e c e l l s . The s u p e r n a t a n t was r e m oved a n d k e p t i n i c e u n t i l r e q u i r e d . A l l Hughes p r e s s a t e s were p r e p a r e d i m m e d i a t e l y p r i o r t o u s e . Enzyme A s s a y s G e n e r a l l y , a l l a s s a y s i n v o l v i n g t h e r e d u c t i o n o r o x i d a t i o n o f p y r i d i n e n u c l e o t i d e s were c a r r i e d o u t b y m e a s u r i n g i n c r e a s e o r d e c r e a s e r e s p e c t i v e l y i n a b s o r b a n c y a t 340 i n a Beckman m o d e l DU S p e c t r o p h o t o m e t e r . A l l r e a c t i o n s t o o k p l a c e i n q u a r t z c u v e t t e s o f 1 cm. l i g h t p a t h . C h a n g e s i n o p t i c a l d e n s i t y were r e c o r d e d e v e r y 15 s e c o n d s f o r t h e f i r s t m i n u t e an d e v e r y 30 s e c o n d s t h e r e a f t e r . G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e T h i s enzyme was 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 b y a m o d i f i c a t i o n o f a p r o c e d u r e d e s c r i b e d b y K o r n b e r g and H o r e c k e r ( 4 0 ) . A t y p i c a l a s s a y p r o c e d u r e was a s f o l l o w s : R e a g e n t s C o n t r o l s T e s t M/5 T r i s b u f f e r pH 7=4 .2 .2 .2 d i s t i l l e d w a t e r .77 .68 .63 K g l u c o s e - 6 - p h o s p h a t e ( 2 5^M/ml) - .10 .10 CFX ( s o n i c a t e ) .05 - .05 TPN (.005M) .02 .02 .02 A l l reagents were added to the cuvette except the TPN and the instrument zeroed on this mixture. The TPN was added, the mixture stirred and the increase in optical density at 340 ryk recorded as described above. Substrate concentration was always maintained in sufficient excess to ensure that the rate of TPN reduction remained a function of enzyme activity, at least during the i n i t i a l stages of the reaction. Specific activities and units of activity of glucose-6-phosphate dehydrogens.se were determined by the method of Kornberg ( 4 0 ) , that i s , "A unit of enzyme is defined as that amount which causes an i n i t i a l change of optical density of 1.000 per minute at 25-30°C. Specific Activity is expressed as units per mg. protein." Phosphoglucoisomerase and Phosphomannoisomerase These enzymes were assayed in the same manner as glucose-6-phosphate except that fructose-6-phosphate and mannose-6-phosphate were substituted for glucose-6-phosphate. 2-keto-6-phosphogluconate reductase This assay depends upon the oxidation of DPNH in the presence of cell extract and 2-keto-6-phosphogluconate. A typical protocol is shown. T r i s b u f f e r pH7.4 M/5 .2 .2 .2 d i s t i l l e d w a t e r • 72 .71 .67 DPNH (.002M) .04 .04 .04 K 2 - k e t o - 6 - p h o s p h o g l u c o n a t e (25 yAM/ml.) - .05 .05 CFX (Hughes p r e s s ) .04 - .04 The r e a c t i o n m i x t u r e was z e r o e d a t a n o p t i c a l d e n s i t y o f 0.300 i n t h e a b s e n c e o f c e l l e x t r a c t . The r e a c t i o n was t h e n i n i t i a t e d b y a d d i t i o n o f enzyme. P o t a s s i u m 2-keto-6 - p h o s p h o g l u c o n a t e was p r e p a r e d f r o m t h e b a r i u m s a l t b y p r e c i p i t a t i o n o f t h e b a r i u m w i t h ILjSO^ i n a c i d s o l u t i o n . H e x o k i n a s e H e x o k i n a s e was d e t e r m i n e d b y m e a s u r i n g TPN r e d u c t i o n i n t h e p r e s e n c e o f h e x o s e , ATP, Mg + +, c e l l f r e e e x t r a c t and an e x t e r n a l s o u r c e o f g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e . B e c a u s e o f t h e i n h i b i t o r y e f f e c t o f n u c l e o t i d e s on g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , a s h a s b e e n shown i n e x t r a c t s f r o m s e v e r a l o r g a n i s m s (20) (63), t h i s e x t e r n a l s o u r c e o f enzyme i s n e c e s s a r y . C o m m e r c i a l g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , o b t a i n e d f r o m S i g m a C h e m i c a l Co". , h a d an a c t i v i t y o f 330 K o r n b e r g u n i t s / g r a m . T h i s p r e p a r a t i o n , h o w e v e r , was v e r y h i g h i n h e x o k i n a s e a c t i v i t y w h i c h l e d t o some e r r o n e o u s r e s u l t s d u r i n g e a r l y e x p e r i m e n t s . By h e a t i n g a t 50°C. f o r 5 t o 10 m i n u t e s t h e 16 h e x o k i n a s e was a l m o s t c o m p l e t e l y i n a c t i v a t e d l e a v i n g g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e a t a h i g h l e v e l . G l u c o n o k i n a s e and 2 - k e t o g l u c o n o k i n a s e H e x o k i n a s e , g l u c o n o k i n a s e a n d 2 - k e t o g l u c o n o k i n a s e may a l s o be d e t e r m i n e d i n a more a c c u r a t e f a s h i o n b y m e a s u r i n g m a n o m e t r i c a l l y t h e r e l e a s e o f CC^ f r o m b i c a r b o n a t e b u f f e r . The method u s e d was t h a t o f C i f f e r i , B l a k l e y a n d S i m p s o n ( 1 2 ) f o r t h e d e t e r m i n a t i o n o f 2 - k e t o g l u c o n o k i n a s e i n L . m e s e n t e r o i d e s ; m o d i f i e d f r o m t h e o r i g i n a l m e t h o d d e s c r i b e d b y C o l o w i c k a n d K a l c k a r ( 1 6 ) . T h i s m ethod i s b a s e d on t h e f a c t t h a t t h e t r a n s f e r o f one mole o f p h o s p h a t e f r o m A TP t o a n a p p r o p r i a t e s u b s t r a t e r e s u l t s i n t h e l i b e r a t i o n o f one a c i d e q u i v a l e n t . T h i s may t h e n be m e a s u r e d m a n o m e t r i c a l l y b y t h e r e l e a s e o f COg f r o m b i c a r b o n a t e b u f f e r m a i n t a i n e d a b o v e pH7.5» 9 0 0 M g + + Q 0 0 R-OH + R-O-P-O-P-O-^-O" >- R-O-P-O-P-0 + R-O-P-0" + H + 6~ 6" 0~ k i n a s e 6~ 6" 6" ( s u b s t r a t e ) + (ATP) (ADP) + ( J o ^ J J d ? l a t e d W a r b u r g v e s s e l s were s e t up a c c o r d i n g t o t h e f o l l o w i n g p r o t o c o l . R e a g e n t s F i n a l C o n t r o l s T e s t Amount D i s t i l l e d w a t e r .5 .4- .4-E . D . T . A . ( E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ) pH 7 . 7 1 0 y*M . 1 . 1 . 1 . 1 NaHCO^ pH7c7 60yt(M o l o l . 1 . 1 Hughes P r e s s p r e p a r a t i o n . 3 . 3 ° 3 . 3 M g C l ^ ^ O pH7.0 3 0 y<M o l . 1 . 1 . 1 g l u c o s e , g l u c o n a t e o r 2 - k e t o - ) g l u c o n a t e pH7 .0 ) s i d e 2 0 « M - - o l o l A T P pH7 .7 ) a r m 20 yitS. - . 1 - . 1 f r e s h l y c u t w h i t e p h o s p h o r o u s c e n t e r w e l l S o d i u m g l u c o n a t e was p r e p a r e d f r o m t h e l a c t o n e b y a l k a l i n e h y d r o l y s i s w i t h NaOH. S o d i u m 2 - k e t o g l u c o n a t e was p r e p a r e d b y t r e a t m e n t o f t h e c a l c i u m s a l t w i t h Dowex 50H+ f o r m i o n - e x c h a n g e r e s i n f o l l o w e d b y n e u t r a l i z a t i o n w i t h NaOH. The g a s p h a s e was 9 5 % N 2 a n d 5 % C 0 2 . Enzyme F r a c t i o n a t i o n P r o t a m i n e s u l f a t e A c e l l f r e e e x t r a c t p r e p a r e d b y s o n i c o s c i l l a t i o n o f g l u c o s e grown c e l l s was p l a c e d i n an i c e b a t h . C r y s t a l l i n e ammonium s u l f a t e was a d d e d w i t h c o n s t a n t s t i r r i n g u n t i l a f i n a l c o n c e n t r a t i o n o f M/ 1 5 was r e a c h e d . The pH was t h e n a d j u s t e d t o 6.0 w i t h N / 1 a c e t i c a c i d . N u c l e o p r o t e i n m a t e r i a l was r e m o v e d b y t h e s t e p w i s e 18 a d d i t i o n o f p r o t a m i n e s u l f a t e s o l u t i o n c o n t a i n i n g 20 mg./ml. A f t e r e a c h a d d i t i o n a s m a l l a l i q u o t ( 1 m l . ) was r e m o v e d , c e n t r i f u g e d a n d a 280/260 a b s o r p t i o n r a t i o , o f t h e s u p e r n a t a n t d e t e r m i n e d . When t h e r a t i o o f 0.7 was r e a c h e d no f u r t h e r a d d i t i o n o f p r o t a m i n e s u l f a t e was made. A f t e r c e n t r i f u g a t i o n o f t h e w h o l e p r e p a r a t i o n a t 12,000 x g f o r 20 m i n u t e s i n t h e r e f r i g e r a t e d c e n t r i f u g e i t was d i a l y s e d a g a i n s t M/200 pH 7.6 p h o s p h a t e b u f f e r . The v o l u m e o f t h e b u f f e r was m a i n t a i n e d a t 20 t i m e s t h a t o f t h e m a t e r i a l t o be d i a l y z e d . I t was a l l o w e d t o d i a l y s e o v e r n i g h t o r f o r f o u r h o u r s w i t h c o n s t a n t s t i r r i n g a n d a t l e a s t one c o m p l e t e c h a n g e o f b u f f e r . A l l d i a l y s e s were c a r r i e d o u t a t 4-°C. The m a t e r i a l was t h e n c e n t r i f u g e d a n d t h e s u p e r n a t a n t a s s a y e d f o r p r o t e i n a n d enzyme a c t i v i t y . I t was t h e n u s e d f o r f u r t h e r f r a c t i o n a t i o n s o r f r o z e n a t - 1 8 ° C . u n t i l r e q u i r e d . Ammonium S u l f a t e The c e l l e x t r a c t a f t e r 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 was s e p a r a t e d i n t o v a r i o u s f r a c t i o n s w i t h c r y s t a l l i n e ammonium s u l f a t e . The s a l t was g r a d u a l l y a d d e d t o t h e p r e p a r a t i o n w i t h c o n s t a n t s t i r r i n g i n a n i c e b a t h u n t i l 2 5 % o f s a t u r a t i o n h a d b e e n r e a c h e d . The p r e p a r a t i o n r e m a i n e d i n t h e i c e b a t h f o r a n a d d i t i o n a l 10 m i n u t e s , t h e n - c e n t r i f u g e d a t 12,000 x g f o r 20 minutes. The precipitate was redissolved in M/200 pH 7.6 phosphate buffer and dialyzed against buffer as described above. This fraction was designated at the 0/25 cut. The supernatant was separated further, i n the same manner, into 25/38, 38/50, 50/60 and 60/90 fractions. Protein and enzyme a c t i v i t i e s were determined on a l l fractions. Alkaline saturated ammonium sulfate A saturated alkaline ammonium sulfate solution (pH7.4 with NH^GH) was used to fractionate the protamined extracts on the recombined fractions from the above procedure i n much the same way as solid ammonium sulfate. The fractions obtained were 0/20, 20/26, 26/34 and 3V45. Alcohol fractionation Extracts either treated f i r s t with protamine sulfate or recombined fractions from previous procedures were fractionated with 95% cold ethanol by the method of McDonald (48). The c e l l extract was placed in a dry ice-ethanol bath to maintain as cold a temperature as possible. With constant s t i r r i n g , 1 ml. molar acetate buffer (pH5»4) was added for every 19 mis. extract. Cold 95% ethanol was then added u n t i l 10% concentration of ethanol was reached. The precipitate was then removed by centrifugation 12,000 x g f o r 20 m i n u t e s a t =2 C. The p r e c i p i t a t e was r e s u s p e n d e d , d i a l y s e d a n d a s s a y e d i n t h e u s u a l manner. S i m i l a r l y 10/20 and 20/30 f r a c t i o n s w e r e made. C a l c i u m p h o s p h a t e g e l S i n c e most o f t h e enzyme a c t i v i t y was f o u n d t o he i n t h e 20/30 ammonium s u l f a t e c u t , t h i s f r a c t i o n was u s e d f o r f u r t h e r a t t e m p t s a t f r a c t i o n a t i o n "by a d s o r p t i o n a n d e l u t i o n f r o m c a l c i u m p h o s p h a t e g e l (35)° Maximum p u r i f i c a t i o n o f g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e was o b t a i n e d w i t h t h e f o l l o w i n g p r o c e d u r e ; h o w e v e r , no s e p a r a t i o n c o u l d be e f f e c t e d b y t h i s m e t h o d . T h r e e t u b e s were s e t up c o n t a i n i n g 0%, 43.75% a n d 50% c a l c i u m p h o s p h a t e g e l , enzyme p r e p a r a t i o n and w a t e r t o make a v o l u m e o f 0.8 m l . A d s o r p t i o n was a l l o w e d t o t a k e p l a c e f o r 10 m i n u t e s a t 0°C. w i t h c o n s t a n t s t i r r i n g . The g e l was r e m o v e d b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t t e s t e d f o r p r o t e i n a n d enzyme a c t i v i t y . The p r e c i p i t a t e was w a s h e d o n c e w i t h d i s t i l l e d w a t e r t h e n e l u t e d w i t h M/50 pH7»5 p h o s p h a t e b u f f e r . P r o t e i n a n d enzyme a c t i v i t i e s w e r e t h e n d e t e r m i n e d on t h e s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n . M a n o m e t r i c P r o c e d u r e s W a r b u r g v e s s e l s c o n t a i n i n g t h e f o l l o w i n g t y p i c a l r e a c t i o n m i x t u r e were s e t u p . 21 W a r b u r g Cup C o n t e n t s F i n a l Amount E n d o g e n o u s T e s t M / l T r i s b u f f e r pH7.4 200 JKM. ©2 ©2 .2 .2 d i s t i l l e d w a t e r 1.76 1.31 1.36 .91 ATP (25/tM/ml.) M g + + (100 mg./ml.) NaP (1.5M) l O y ^ M 25 JAM 60 yl/M 10 juM .4 - ^ .05 .04* .04 .04 .4 .05 .04 g l u c o s e o r 2 - k e t o g l u c o n a t e ( s i d e arm) = _ .4 .4 Hughes p r e s s p r e p a r a t i o n 1.0 1.0 1,0 1.0 KOH ( 2 0 % ) ( c e n t e r w e l l ) .15 .15 .15 .15 C o n t r o l f l a s k s were r u n i n e a r l i e r e x p e r i m e n t s w i t h NaP as a v a r i a b l e . A n a l y t i c a l P r o c e d u r e s P r o t e i n d e t e r m i n a t i o n P r o t e i n was d e t e r m i n e d b y t h e method o f S u t h e r l a n d , C o r i , H a y n e s and O l s e n (59) m o d i f i e d f r o m K a b a t a n d M a y e r (34). 0.5 m l . o f a p r o t e i n s o l u t i o n a p p r o p r i a t e l y d i l u t e d was a d d e d t o 3 m i s . o f a f r e s h l y p r e p a r e d a l k a l i n e c o p p e r s o l u t i o n o f t h e f o l l o w i n g c o m p o s i t i o n . 2% CuS04.5H20 4% Na K t a r t r a t e 4% N a 2 CO^ The r e a c t i o n m i x t u r e was i n c u b a t e d 15 m i n u t e s i n a 45°C w a t e r b a t h . To t h i s was a d d e d 0.3 m l . o f a 1:3 d i l u t i o n 2; o f F o l i n - C i o c a l t e a u p h e n o l r e a g e n t i n w a t e r . The s o l u t i o n was m i x e d b y i n v e r s i o n a n d i n c u b a t e d a t room t e m p e r a t u r e f o r 15 m i n u t e s . O p t i c a l d e n s i t y was m e a s u r e d a t 650 i n a F i s h e r e l e c t r o p h o t o m e t e r . The r e s u l t s were a p p l i e d a g a i n s t a s t a n d a r d c u r v e a n d t h e c o r r e s p o n d i n g v a l u e s i n mg. p r o t e i n o b t a i n e d . The s e n s i t i v i t y o f t h i s t e s t i s 0-15 Y P e r 0<>5 m l . s a m p l e . G l u c o s e d e t e r m i n a t i o n G l u c o s e was d e t e r m i n e d b y t h e g l u c o s t a t m e thod w i t h r e a g e n t s o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p . D e p r o t e i n i z a t i o n was b y t h e method o f N e l s o n ( 5 1 ) . R e a g e n t s : ( a ) % ZnSO^ . ? H 2 0 ; 0 . 3 N B a ( 0 H ) 2 . The c o n c e t r a t i o n o f z i n c s u l f a t e i s s u c h t h a t a d e f i n i t e p i n k c o l o r i s p r o d u c e d i n t h e p r e s e n c e o f p h e n o l p h t h a l e i n when 5 m i s . o f t h e s o l u t i o n i s t i t r a t e d w i t h 4.7-4.8 m i s . o f t h e B a ( 0 H ) 2 s o l u t i o n . ( b ) G l u c o s t a t r e a g e n t ; 5 mg. p e r o x i d a s e , 125 mg. g l u c o s e o x i d a s e i n 80 m i s . o f „005M p h o s p h a t e b u f f e r , p H 7 . 0 . To t h i s i s a d d e d 1 m l . o f a 1% s o l u t i o n o f 0-d i a n i s i d i n e i n m e t h a n o l . F i l t e r c l e a r . ( c ) m H C l A 0 . 5 m l . a l i q u o t was t a k e n f r o m e a c h r e a c t i o n m i x t u r e t o be a s s a y e d . To e a c h was a d d e d 3 .5 m i s . d i s t i l l e d w a t e r , 23 0 . 5 m l . ZnSO^ s o l u t i o n and f i n a l l y 0 . 5 m l . o f t h e B a ( 0 H ) 2 s o l u t i o n . The r e s u l t i n g p r e c i p i t a t e was r e m o v e d b y c e n t r i f u g a t i o n . To 1 m l . o f t h e s u p e r n a t a n t , 4 m i s . o f t h e g l u c o s t a t r e a g e n t were a d d e d . The r e a c t i o n was a l l o w e d t o p r o c e e d f o r 10 m i n u t e s a t room t e m p e r a t u r e t h e n s t o p p e d w i t h one d r o p o f 4N H C l . O p t i c a l d e n s i t y was m e a s u r e d a t 400 yt. i n a Beckman m o d e l DU s p e c t r o p h o t o m e t e r . The v a l u e s o b t a i n e d were c o m p a r e d t o a s t a n d a r d c u r v e c o n s t r u c t e d f r o m d i l u t i o n s o f a known g l u c o s e s t a n d a r d . The r a n g e o f t h e s t a n d a r d c u r v e was f r o m . 0 5 t o .25y*M g l u c o s e p e r 0 . 5 m l . s a m p l e . 2 - K e t o g l u c o n a t e d e t e r m i n a t i o n 2 - k e t o g l u c o n a t e was d e t e r m i n e d a s t h e q u i n o x a l i n e d e r i v a t i v e b y r e a c t i o n w i t h O - p h e n y l e n e d i a m i n e h y d r o c h l o r i d e a c c o r d i n g t o a method m o d i f i e d f r o m L a n n i n g a n d C o h e n ( 4 4 ) . To 0 . 1 m l . o f t h e r e a c t i o n m i x t u r e w e r e a d d e d 0.8 m l . d i s t i l l e d w a t e r and 0 . 1 m l . c o l d 6 0 % w/w p e r c h l o r i c a c i d . The r e s u l t i n g p r e c i p i t a t e was r e m o v e d b y c e n t r i f u g a t i o n i n a M i s c o m i c r o c e n t r i f u g e p r e v i o u s l y c h i l l e d t o 4 ° C . The s u p e r n a t a n t was n e u t r a l i z e d w i t h 5N KOH. The r e s u l t i n g p r e c i p i a t e was c e n t r i f u g e d a n d w a shed o n c e w i t h 0 . 5 m l . c o l d d i s t i l l e d w a t e r . The s u p e r n a t a n t s were c o m b i n e d an d made up t o 2.0 m i s . To t h i s was a d d e d 1 m l . o f a s o l u t i o n o f O - p h e n y l e n e d i a m i n e c o n t a i n i n g 15 mg./ml. i n 0 . 2 5 N H C l . The t u b e s were t h e n p l a c e d i n b o i l i n g w a t e r f o r 30 m i n u t e s , c o o l e d t o room t e m p e r a t u r e and o p t i c a l d e n s i t i e s d e t e r m i n e d a t 330rvy* a n d 360»^» . S t a n d a r d s were r u n s i m u l t a n e o u s l y w i t h e a c h d e t e r m i n a t i o n and a s t a n d a r d c u r v e c o n s t r u c t e d . S e n s i t i v i t y o f t h e a s s a y i s f r o m 10 t o 1 0 0 ^ p e r 0.1 m l . s a m p l e . P y r u v a t e d e t e r m i n a t i o n P y r u v a t e was d e t e r m i n e d b y t h e method o f S e g a l , B l a i r a n d Wyngaarden (56). R e a g e n t s : ( a ) 7% w/w p e r c h l o r i c a c i d ( b ) 5N KOH ( c ) O.IM, pH7.4 p h o s p h a t e b u f f e r ( d ) .002 DPNH ( e ) L a c t i c d e h y d r o g e n a s e i n O.IM N a C l ( 1:1000 d i l u t i o n ) The l a s t two r e a g e n t s were o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p . 0 . 5 m l » o f e a c h r e a c t i o n m i x t u r e was d e p r o t e i n i z e d w i t h an e q u a l v olume o f c o l d 7% p e r c h l o r i c a c i d , c e n t r i f u g e d i n t h e m i c r o c e n t r i f u g e a t 4 ° C . a n d t h e p r e c i p i t a t e d i s c a r d e d . The s u p e r n a t a n t was c a u t i o u s l y a d j u s t e d f r o m pH 3-4 w i t h 5N KOH, c e n t r i f u g e d a n d t h e p r e c i p i t a t e a g a i n d i s c a r d e d . 0 . 5 m l . o f t h e s u p e r n a t a n t was a d d e d t o 0.04 m l . , .002M DPNH a n d 0.22 m l . w a t e r i n a q u a r t z c u v e t t e . T h i s was t h e n z e r o e d a t 34 0 r y u . w i t h a n o p t i c a l d e n s i t y o f 0.400 i n t h e Beckman DU. Enzyme was a d d e d a n d t h e r e d u c t i o n i n o p t i c a l d e n s i t y r e c o r d e d a t ' r e g u l a r t i m e i n t e r v a l s u n t i l t h e r e a c t i o n h a d s t o p p e d (7-9 m i n u t e s ) . The c o n c e n t r a t i o n o f p y r u v a t e was c a l c u l a t e d f r o m k n o w l e d g e o f t h e m o l a r e x t i n c t i o n c o e f f i c i e n t o f DPNH a n d t h e m o l e c u l a r w e i g h t o f p y r u v i c a c i d a c c o r d i n g t o t h e f o l l o w i n g r e l a t i o n s h i p . n e t c hange i n O.D.@ 34-0"*y*.X m o l e c u l a r w e i g h t p y r u v i c a c i d , (88 gm mol"- 1) m o l a r e x t i n c t i o n c o e f f i c i e n t o f DPNH ( 6 . 2 2 x l C r c m m o l " ) mg. p y r u v i c a c i d / 0 . 5 m l . s a m p l e . C h r o m a t o g r a p h y ( a ) C h r o m a t o g r a p h i c a n a l y s i s o f W a r b u r g c u p c o n t e n t s f o r p h o s p h o r y l a t e d compounds r e q u i r e d t h e f o l l o w i n g p r e p a r a t i v e t r e a t m e n t . To 10 m i s . o f e a c h r e a c t i o n m i x t u r e , 0 . 5 m l . c o l d 6 0 % w/w p e r c h l o r i c a c i d was a d d e d ; t h e m i x t u r e was c e n t r i f u g e d a n d t h e s u p e r n a t a n t a d j u s t e d t o pH 4 w i t h 5N KOH. The t u b e s were c h i l l e d t o 0 ° C . a n d c e n t r i f u g e d . The s u p e r n a t a n t was r e m o v e d a n d c o n c e n t r a t e d i n a C r a i g f l a s h e v a p o r a t o r t o a p p r o x i m a t e l y 2 m i s . A f t e r c e n t r i f u g a t i o n , t h e s u p e r n a t a n t was t r e a t e d w i t h 0.2 m l . ( e x c e s s ) m o l a r b a r i u m a c e t a t e and 3 m i s . c o l d 95% e t h a n o l . The t u b e s w e r e a l l o w e d t o r e m a i n i n i c e f o r a t l e a s t 15 m i n u t e s b e f o r e c e n t r i f u g a t i o n . The p r e c i p i t a t e was washed o n c e w i t h c o l d 95% e t h a n o l t h e n d r i e d i n v a c u o . The w h i t e b a r i u m i n s o l u b l e m a t e r i a l was t h e n d i s s o l v e d i n a minimum o f w a t e r ( 1 m l . ) a n d made s l i g h t l y a c i d w i t h HCl. Metal cations were removed with Dowex 50H + (2). The supernatant was removed, adjusted to pH 8.0 with N.H4OH and evaporated i n vacuo to as small a volume as possible (approximately 0.1 ml). In l a t e r experiments acid washed K o r i t was used to reduce the nucleotide content i n the rea c t i o n mixtures where ATP had been used. This step was introduced at the point where the barium insoluble materials were redissolved. About 2.7 mg. of Norit p e r ^ M ATP or 92 mg. per 10 mis. of r e a c t i o n mixture reduced the nucleotide content by 90% as determined by 260 absorption. (b) Where phosphate esters were involved, #1 Whatman paper was f i r s t washed with NHC1 and 0.2% Versene e i t h e r by allowing the mixture to run o f f the paper by normal descending chromotography or by simply passing the paper through the s o l u t i o n . A f t e r the spots were applied and allowed to dry, the sheets were suspended i n the chromatogram tanks i n the presence of the solvent f o r 30 minutes before p l a c i n g the solvent i n the troughs. (c) Solvent systems f o r the separation of phosphate esters (17). i . Methanol 60: NH^OH 10: water 30 i i . Propanol 60: HCOOH 30: water 10 i i i . Methanol 80: HCOOH 15: water 5 ( d ) S p r a y s i . D e m o n s t r a t i o n o f p h o s p h a t e s ( 2 9 ) 6 0 % w/w p e r c h l o r i c a c i d 1 p a r t NHC1 2 p a r t s 4% w/v ammonium m o l y b d a t e 5 p a r t s d i s t i l l e d w a t e r 11 p a r t s C h r o m a t o g r a m s were d r i e d a t room t e m p e r a t u r e , s p r a y e d w i t h t h e s o l u t i o n d e s c r i b e d , t h e n h e a t e d f o r 10 m i n u t e s b y s u s p e n d i n g i n a i h o t a i r o v e n . The p a p e r s were t h e n s t e a m e d f o r 1 m i n u t e w i t h o u t e x c e s s i v e w e t t i n g o r u n t i l t h e p a p e r was l e s s b r i t t l e . The c h r o m a t o g r a m s were t h e n e x p o s e d t o u l t r a v i o l e t l i g h t u n t i l t h e c h a r a c t e r i s t i c b l u e s p o t s o f p h o s p h a t e compounds a p p e a r e d . i i . D e m o n s t r a t i o n o f k e t o s e s ( 3 8 ) . O r c i n o l 0.5 gm. T r i c h l o r o a c e t i c a c i d 15 gm. W a t e r s a t u r a t e d n - b u t a n o l 100 m i s . The c h r o m a t o g r a m s were h e a t e d t o 1 0 5 ° C f o r 15-20 m i n u t e s . The s p o t s were c h a r a c t e r i s t i c f o r v a r i o u s compounds, k e t o h e p t o s e s - b l u i s h - g r e e n k e t o h e x o s e s - y e l l o w i i i . D e m o n s t r a t i o n o f k e t o h e x o n i c a c i d s (17)• O - p h e n y l e n e d i a m i n e 400 mg. c o n c e n t r a t e d h y d r o c h l o r i c a c i d 0.65 m l . d i s t i l l e d w a t e r 3 m i s 95% e t h a n o l 16 m i s C h r o m a t o g r a m s were h e a t e d a t 100° C f o r l e s s t h a n 3 m i n u t e s g i v i n g c h a r a c t e r i s t i c s p o t s . 2 - k e t o - 6 - p h o s p h o g l u . c o n a t e - g r e e n , no f l u o r e s c e n c e . 2-keto-3-deoxy-6 - p h o s p h o g l u c o n a t e - y e l l o w - g r e e n , g r e e n f l u o r e s c e n c e . 2 - k e t o g l u c o n a t e - g r e e n , g r e e n f l u o r e s c e n c e . 31 EXPERIMENTAL RESULTS AND DISCUSSION I Enzyme S t u d i e s w i t h C e l l E x t r a c t s I n o r d e r t o u n d e r s t a n d t h e i n t e r m e d i a t e s t e p s i n g l u c o s e o x i d a t i o n more f u l l y a t t e n t i o n was f i r s t f o c u s e d o n t h e enzymes w h i c h a t t a c k g l u c o s e a n d t h o s e w h i c h a t t a c k g l u c o s e - 6 - p h o s p h a t e . D i c k e n s a n d G l o c k r e p o r t e d t h a t t h e g l u c o s e - 6 - p h o s p h a t e (G6P) d e h y d r o g e n a s e o f r a b b i t l i v e r was s e n s i t i v e t o ATP. Wood a n d S c h w e r d t (63) c o u l d n o t d e m o n s t r a t e t r 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 (TPN) r e d u c t i o n b y e x t r a c t s o f P. ' f l u o r e s c e n s i n t h e p r e s e n c e o f g l u c o s e and a d e n o s i n e t r i p h o s p h a t e ( A T P ) , e v e n when an e x t e r n a l s o u r c e o f G6P d e h y d r o g e n a s e was a d d e d a n d c o n c l u d e d t h a t t h i s o r g a n i s m d i d n o t p o s s e s s h e x o k i n a s e . T h e y a l s o p o i n t e d o u t t h a t b e c a u s e o f t h e s e n s i t i v i t y o f t h e d e h y d r o g e n a s e t o ATP, g l u c o s e i s p r o b a b l y n o t o x i d i z e d b y a p a t h w a y i n v o l v i n g t h i s enzyme, b u t r a t h e r , b y way o f g l u c o n a t e a n d 6 - p h o s p h o g l u c o n a t e (6PG) o r 2 - k e t o g l u c o n a t e . I n t h e p r e s e n t work i t was d e c i d e d t h a t t h e i n h i b i t o r y m e c h a n i s m o f ATP s h o u l d be i n v e s t i g a t e d i n o r d e r t o e v a l u a t e t h e i m p o r t a n c e o f g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e i n t h e o x i d a t i o n o f g l u c o s e . As i n d i c a t e d i n T a b l e I s e v e r a l n u c l e o t i d e s i n h i b i t G6P d e h y d r o g e n a s e , a l t h o u g h A TP a p p e a r s t o b e t h e most e f f e c t i v e . W i t h t h e e x c e p t i o n o f g u a n o s i n e t r i p h o s p h a t e (GTP) a l l t h e n u c l e o t i d e s t e s t e d d i s p l a y e d m a r k e d i n h i b i t i o n o f t h e enzyme. I t s h o u l d be n o t e d t h a t ATP was a much more e f f e c t i v e i n h i b i t o r t h a n ADP. T a b l e I The e f f e c t o f v a r i o u s n u c l e o t i d e s on g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e a c t i v i t y . A O . D . @ 34-0 f o r 1 m i n . R e a c t i o n m i x t u r e - .108 p l u s A T P .25uM " 1 .25/*M .134-.070 .016 p l u s ADP .5C>M " " 1 . 5 iff - .124 .088 .056 p l u s I T P 1.5*M " CTP " UTP " u GTP 11 - . 0 3 0 .043 .078 .160 I t was f o u n d t h a t t h e a d d i t i o n o f magnesium i o n s r e s u l t e d i n r e a c t i v a t i o n o f t h e s y s t e m . T h i s r e v e r s a l o f i n h i b i t i o n h o wever was d e p e n d e n t u p o n t h e p o i n t a t w h i c h t h e M g + 4 w a s a d d e d ( T a b l e I I ) . I f A T P a n d Mg+"* were a d d e d t o t h e r e a c t i o n m i x t u r e a t t h e same t i m e t h e magnesium was l e s s e f f e c t i v e t h a n when i t was a d d e d one m i n u t e a f t e r t h e r e a c t i o n h a d b e e n i n i t i a t e d . E v e n a l a r g e e x c e s s o f magnesium w o u l d n o t r e v e r s e t h e i n h i b i t o r y a c t i o n o f A T P when a d d e d a t t h e same t i m e a s t h e n u c l e o t i d e . S i m i l a r concentrations of magnesium were much more effective i n reversing the action of ADP than of ATP. Table II ++ Effect of Mg on the inhibition of glucose-6-phosphate dehydrogenase by nucleotides. A O.D. ® 340 for 1 min. Reaction mixture - .108 Plus ATP 1 .25 MM - .016 plus ATP 1.25»M and 5uM Mg - .042 plus ATP 1.25^M and 5>M Mg44 - .130 (added after 1 minute) plus ATP .5«M and 50uM Mg++ - .006 plus ADP - .028 plus ADP 2.5^M and Mg++ - .120 The a b i l i t y of magnesium to reverse the inhibitory action of different nucleotides may be a function of the relative a f f i n i t i e s of these nucleotides for magnesium. In order to test the effects of Mg on the reactions under study, i t was added to the reaction mixture and was found to inhibit enzyme ac t i v i t y (Table III). Table III Effect of Mg++ and Versene on glucose-6-phosphate dehydrogenase, A O.D. @ 340 for 1 min. Reaction mixture - .108 plus Mg++ 5*M - .034 plus Versene 5uM - .078 plus Mg44 5/»M and Versene 1CWM - .196 plus Mg"*4 5^ ,M and Versene 5rM - =170 plus Big** and Versene 1/tM - .158 The a d d i t i o n o f V e r s e n e t o 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 magnesium n o t o n l y r e v e r s e d t h i s i n h i b i t i o n h u t a c t u a l l y s t i m u l a t e d t h e r e a c t i o n . V e r s e n e a l o n e h a d o n l y a s l i g h t i n h i b i t o r y e f f e c t . I t h a s b e e n s u g g e s t e d ( 2 6 ) t h a t i n t h e p y r i m i d i n e l i n k e d " a l c o h o l " c l a s s o f d e h y d r o g e n a s e s , i n c l u d i n g G6P d e h y d r o g e n a s e , d i v a l e n t c a t i o n s f u n c t i o n b y c o m p l e x i n g t h e coenzyme t o apoenzyme t h r o u g h t h e p y r o p h o s p h a t e l i n k a g e o f t h e coenzyme. A n e x p e r i m e n t was d e s i g n e d t o t e s t t h e i m p o r t a n c e o f t h e s e "bound" i o n s . The c e l l e x t r a c t was d i a l y s e d a g a i n s t 1000 v o l u m e s o f 0.6% V e r s e n e i n 0.02M p h o s p h a t e b u f f e r a t pH7.4, f o l l o w e d b y a s e c o n d d i a l y s i s a g a i n s t 1000 v o l u m e s o f 0.6% V e r s e n e i n 0.9% KC1 s o l u t i o n (55). E a c h d i a l y s i s was r u n f o r 20 h o u r s a t 4 ° C . The d i a l y s a t e was c e n t r i f u g e d a n d t h e s u p e r n a t a n t a s s a y e d f o r G6P d e h y d r o g e n a s e . I t was d i s c o v e r e d t h a t enzyme a c t i v i t y was e v i d e n t o n l y i n t h e p r e s e n c e o f Mg + + ( P i g . 1 ) . I t i s e v i d e n t f r o m t h e d a t a o b t a i n e d w i t h t h e d i a l y z e d e x t r a c t t h a t magnesium i s r e q u i r e d f o r enzyme a c t i v i t y 7 b u t f r o m work w i t h t h e c r u d e p r e p a r a t i o n s i t was e v i d e n t t h a t e x c e s s i v e amounts o f magnesium a r e i n h i b i t o r y . I f V e r s e n e was a d d e d t o t h e r e a c t i o n m i x t u r e c o n t a i n i n g t h e d i a l y z e d p r e p a r a t i o n a n d magnesium, no enzyme a c t i v i t y was a p p a r e n t . A T P c o u l d b e s u b s t i t u t e d f o r V e r s e n e . K o r n b e r g ( 4 0 ) f o u n d t h a t P i g . I. A l l c u v e t t e s c o n t a i n : T r i s " b u f f e r ; w a t e r ; g l u c o s e ~ 6 - p h o s p h a t e ; d i a l y s e d c e l l e x t r a c t p r e p a r a t i o n ; TPN .150 .125 .100 o ro .075 § .050 -025 0 0 Fig 5yxM Mg added 5yuMMg+ATP or Versene 30 6 0 90 Seconds 120 150 Magnesium activation of G6P dehydrogenase in dialysed cell extracts. magnesium was r e q u i r e d f o r maximum G6P d e h y d r o g e n a s e a c t i v i t y i n f r a c t i o n a t e d e x t r a c t s o f y e a s t . I t i s w e l l known (33) t h a t magnesium w i l l r a p i d l y f o r m a c h e l a t e w i t h ATP, p o l y p h o s p h a t e s , p h o s p h o r i c e s t e r s , i n o r g a n i c p h o s p h o r o u s , h y d r o x y a c i d s , a m i n e s a n d amino a c i d s . I n f a c t , 0.1M p h o s p h a t e h a s "been f o u n d t o i n h i b i t y e a s t G6P d e h y d r o g e n a s e (4-0). I t a p p e a r s t h a t a n y c h e l a t i n g a g e n t w h i c h w i l l c o m p l e x t h e e s s e n t i a l magnesium a s s o c i a t e d w i t h t h e enzyme w i l l compete w i t h TPN f o r t h i s m e t a l . When A T P and TPN a r e a d d e d t o g e t h e r t o a r e a c t i o n m i x t u r e t h e r e i s a c o m p e t i t i o n b e t w e e n t h e s e c o m p l e x i n g a g e n t s f o r t h e magnesium o f t h e enzyme. S i n c e A T P i s g e n e r a l l y a d d e d p r i o r t o TPN, a n d i n a f i v e t o t w e n t y - f i v e f o l d e x c e s s , i t i s l i k e l y t o t i e up t h e m a j o r i t y o f t h e magnesium. E x a c t l y how t h e a d d i t i o n o f e x c e s s magnesium r e v e r s e s A T P i n h i b i t i o n i s n o t c l e a r b u t i t may i n v o l v e a m e c h a n i s m b y w h i c h A T P i s c h e l a t e d a n d e f f e c t i v e l y r e m o v e d t h u s a l l o w i n g more TPN t o be a v a i l a b l e . A d d i t i o n o f magnesium i o n s a l o n e t o a r e a c t i o n m i x t u r e r e s u l t s i n t h e c h e l a t i o n o f TPN a n d g l u c o s e - 6 - p h o s p h a t e t h u s e f f e c t i v e l y r e m o v i n g b o t h coenzyme a n d s u b s t r a t e f r o m p o s s i b l e a t t a c h m e n t t o t h e apoenzyme. W h i l e s t u d y i n g t h e s p e c i f i c i t y o f G6P d e h y d r o g e n a s e t o w a r d s s u b s t r a t e s i t was n o t i c e d t h a t f r u c t o s e - 6 - p h o s p h a t e ( F 6 P ) a n d m a n n o s e - 6 - p h o s p h a t e (M6P) w o u l d r e d u c e TPN i n t h e p r e s e n c e o f c e l l e x t r a c t s . I t was e v i d e n t t h a t a p h o s p h o g l u c o i s o m e r a s e a n d a p h o s p h o m a n n o i s o m e r a s e were p r e s e n t i n t h e s e e x t r a c t s . The p r e s e n c e o f t h e s e enzymes i s i m p o r t a n t i n i n t e r -c o n v e r t i n g t h e h e x o s e s b u t t h e i r p r e s e n c e a l s o e x p l a i n s how t h e h e x o s e s c a n be c o n n e c t e d t o p e n t o s e s . E a g o n (23) h a s shown i n e x t r a c t s o f P. a e r u g i n o s a t h a t mannose i s p r o b a b l y d i s s i m i l a t e d e i t h e r b y d i r e c t p h o s p h o r y l a t i o n t o M6P o r b y i s o m e r i z a t i o n t o f r u c t o s e f o l l o w e d b y p h o s p h o r y l a t i o n t o F 6 P . I n e i t h e r c a s e b o t h t h e m a n n o k i n a s e a n d m a n n o i s o m e r a s e enzymes w e r e a d a p t i v e . P h o s p h o m a n n i s o m e r a s e o n t h e o t h e r h a n d was a c o n s t i t u t i v e enzyme. S i n c e b o t h p h o s p h o h e x o i s o m e r a s e s a r e f r e e l y r e v e r s i b l e b o t h G6P a n d F 6 P c a n a c t a s s u b s t r a t e s f o r p h o s p h o m a n n o i s o m e r a s e . I n m u s c l e (57) t h e e q u i l i b r i u m b e t w e e n t h e s e compounds i s a s shown. G6P ^ ^ F 6 P ^ = M6P 60 30 20 P r e v i o u s work i n t h i s l a b o r a t o r y ( 6 0 ) showed t h a t P. a e r u g i n o s a (120 NA) h a d a h e x o k i n a s e . However, t h e G6P d e h y d r o g e n a s e o f t h i s o r g a n i s m d i d n o t d i s p l a y t h e same s e n s i t i v i t y t o n u c l e o t i d e s as e i t h e r t h e 9027 s t r a i n o r P. f l u o r e s c e n s A312 (63). T h e r e was no d i f f i c u l t y i n d e m o n s t r a t i n g TPN r e d u c t i o n b y t h e s e e x t r a c t s i n t h e p r e s e n c e o f g l u c o s e , ATP a n d Mg . No e x o g e n o u s s u p p l y o f G6P d e h y d r o g e n a s e was r e q u i r e d . T h i s work r e v e a l e d t h a t ATP a n d t o a l e s s e r d e g r e e CTP b u t n o t I T P o r UTP w o u l d s e r v e a s coenzyme i n t h e p h o s p h o r y l a t i o n o f g l u c o s e . The a d d i t i o n o f ADP a l s o r e s u l t e d i n TPN r e d u c t i o n a f t e r a s h o r t l a g p e r i o d . T h i s was p r o b a b l y t h e r e s u l t o f a m y o k i n a s e r e a c t i o n . A t t h i s t i m e P^  a e r u g i n o s a 9027 was t e s t e d i n t h e same way and was f o u n d t o c o n t a i n no h e x o k i n a s e . I t was n o t r e a l i z e d u n t i l l a t e r h o w e v e r , t h a t t h e p r e s e n c e o f n u c l e o t i d e t r i p h o s p h a t e s w o u l d i n h i b i t G6P d e h y d r o g e n a s e . I n o r d e r t o overcome t h i s d i f f i c u l t y an e x o g e n o u s s o u r c e o f G6P d e h y d r o g e n a s e , o b t a i n e d c o m m e r c i a l l y , was a d d e d t o t h e r e a c t i o n m i x t u r e . T h i s p r e p a r a t i o n was r e l a t i v e l y i n s e n s i t i v e t o n u c l e o t i d e s ( P i g . 2). T h e s e d a t a i n d i c a t e t h e p r e s e n c e o f a g l u c o k i n a s e i n e x t r a c t s o f P. a e r u g i n o s a . Mannose d i d n o t c a u s e TPN r e d u c t i o n u n d e r t h e same c o n d i t i o n s . F r u c t o s e h o w e v e r , seems t o be i n a n i n t e r m e d i a t e p o s i t i o n ; a f t e r a one m i n u t e l a g , TPN was r e d u c e d a t a r a t e a p p r o x i m a t e l y e q u a l t o t h e g l u c o k i n a s e c u r v e . T h i s l a g m i g h t be t h e r e s u l t o f a number o f i n t e r m e d i a t e r e a c t i o n s t a k i n g p l a c e b e t w e e n f r u c t o s e a n d F 6 P r a t h e r t h a n a s i n g l e s t e p as i n t h e c a s e o f g l u c o s e . A m e c h a n i s m s u c h as t h a t i n l i v e r (30) m i g h t be i n v o l v e d , w h e r e i n , f r u c t o s e i s p h o s p h o r y l a t e d i n t h e 1 p o s i t i o n Fig. 2. A l l cuvettes contain: Tris buffer; water; glucose-6-phosphate dehydrogenase (8mg./ml.) heated to 50°C for 5 min.; TPN. o o plus glucose=6-phosphate a—'• D plus ATP, Mg++, fructose or glucose A - A • plus ATP, Mg++ , glucose and CPX /— ;—x plus ATP, Mg + +, fructose and CPX • • plus ATP, Mg"*+ , mannose and CPX 150 25 100 6 s § 075 ro a O .050 .025 0 0 30 60 90 Seconds-20 Fig. 2. Demonstration of hexokinase in cell extracts by measuring TPN reduction. 150 f o l l o w e d "by an a l d o l a s e s p l i t t o g l y c e r a l d e h y d e a i i d d i h y d r o x y a c e t o n e p h o s p h a t e . A f t e r p h o s p h o r y l a t i o n o f g l y c e r a l d e h y d e , t h e two, t h r e e c a r b o n u n i t s w o u l d b e r e c o m b i n e d b y a r e v e r s a l o f a l d o l a s e t o f o r m FDP; t h e n b y p h o s p h a t a s e and i s o m e r a s e r e a c t i o n s t o G6P. The o n l y r e a l e v i d e n c e f o r t h e p o s s i b i l i t y o f s u c h a p a t h w a y i s t h e known f a c t t h a t a weak PDP a l d o l a s e e x i s t s i n e x t r a c t s o f PIT a e r u g i n o s a . E a g o n (22) f o u n d t h a t o s m o t i c a l l y b u r s t p r o t o p l a s t s o f g l u c o s e grown P. a e r u g i n o s a w o u l d o x i d i z e g l u c o s e a n d f r u c t o s e b u t n o t mannose. I t seems e v i d e n t , t h e r e f o r e , t h a t t h e enzymes f o r t h e o x i d a t i o n o f f r u c t o s e a r e c o n s t i t u t i v e i n t h i s o r g a n i s m . G i u c o k i n a s e was a l s o d e m o n s t r a t e d b y m e a s u r i n g t h e CC^ r e l e a s e d f r o m b i c a r b o n a t e b u f f e r d u r i n g a p h o s p h o r y l a t i o n r e a c t i o n ( F i g . 3)« No e v i d e n c e , h o w e v e r , c o u l d be f o u n d f o r e i t h e r a g l u c o n o k i n a s e o r a 2 - k e t o g l u c o n o k i n a s e b y t h i s method. N o r c o u l d 2 - k e t o g l u c o n o k i n a s e be d e m o n s t r a t e d b y m e a s u r i n g DPNH o x i d a t i o n i n t h e p r e s e n c e o f 2 k e t o g l u c o n a t e , c e l l e x t r a c t , A T P a n d Mg"*4". I t s h o u l d be p o i n t e d o u t , h o w e v e r , t h a t i n a b i l i t y t o d e m o n s t r a t e t h e s e enzymes i s n o t s u f f i c i e n t p r o o f t o s a y t h a t t h e y do n o t o c c u r . C i f f e r i e t a l (12) c o u l d d e m o n s t r a t e 2 k e t o g l u c o n o k i n a s e i n L. m e s e n t e r o i d e s o n l y when t h i s o r g a n i s m was grown o n E a c h . W a r b u r g f l a s k c o n t a i n s : W a t e r ; E.D.T.A.; NaCO . CFX; M g C l 2 . X- x p l u s ATP and g l u c o s e A —A P l u s A T P — — P l u s g l u c o s e o — o e n d o g e n o u s 300 Minutes 3 Demonstration of glucokinase by release of CO2 ^ r o m bicarbonate buffe r. 2 - k e t o g l u c o n a t e , a l t h o u g h , t h e y c o u l d d e m o n s t r a t e g l u c o n o k i n a s e i n c e l l s grown on g l u c o s e , g l u c o n a t e , f r u c t o s e , mannose o r 2 - k e t o g l u c o n a t e . However, i n P. f l u o r e s c e n s (4 - 9 ) and i n A. s u b o x y d a n s ( 2 7 ) , 2 - k e t o g l u c o n o k i n a s e c o u l d be d e m o n s t r a t e d i n e x t r a c t s o f g l u c o s e grown c e l l s . U n t i l 2 - k e t o g l u c o n o k i n a s e c a n be d e m o n s t r a t e d i n P. a e r u g i n o s a , o t h e r , more i n d i r e c t m e thods must be r e l i e d u p o n t o p r o v i d e e v i d e n c e f o r i t s e x i s t e n c e . The p r e s e n c e o f a 2 - k e t o - 6 - p h o s p h o g l u c o n i c (2K6PG) r e d u c t a s e h e l p s t o p r o v i d e s u c h e v i d e n c e . T h i s enzyme was f o u n d i n e x t r a c t s p r e p a r e d b y t h e Hughes P r e s s , b u t b e c a u s e o f i t s g r e a t l a b i l i t y , a s s a y s h a d t o be made w i t h i n 24- h o u r s o f p r e p a r a t i o n . D e t e r m i n a t i o n s were made b y m e a s u r i n g r e d u c t i o n i n o p t i c a l d e n s i t y a t 34 -Ow^ i n t h e p r e s e n c e o f DPNH, c e l l e x t r a c t a n d 2K6PG ( T a b l e T V ) . The p r e s e n c e o f a v e r y s t r o n g DPNH o x i d a s e s y s t e m a t t i m e s made t h e a s s a y d i f f i c u l t t o i n t e r p r e t . I t was f o u n d t h a t 2K6PG r e d u c t a s e i s a h e a t l a b i l e enzyme a s s o c i a t e d w i t h t h e p a r t i c u l a t e f r a c t i o n o f t h e p r o t e i n . The e q u i l i b r i u m o f t h e r e d u c t a s e seems t o be f a r t o w a r d s r e d u c t i o n o f t h e 2 - k e t o - 6 - p h o s p h o g l u c o n a t e ( 2 1 ) a s d e m o n s t r a t e d b y t h e i n a b i l i t y t o show DPN r e d u c t i o n i n t h e p r e s e n c e o f 6 - p h o s p h o g l u c o n a t e a n d enzyme. 40 T a b l e IV E f f e c t o f h e a t a n d c e n t r i f u g a t i o n on 2 - k e t o - 6 - p h o s p h o g l u c o n a t e r e d u c t a s e 1 H e a t c e n t r i f u g a t i o n a t 12,000 x g f o r 1 h o u r 1 h o u r a t room temp. 1 m i n u t e a t 50°C 3 m i n u t e s 60°C S u p e r -n a t a n t * r e s i d u e R e a c t i o n M i x t u r e R e a c t i o n M i x t u r e + l o 2 5 M 2K6PG .086* . 1 2 5 .044 .087 .071 .075 . 0 0 9 . 0 1 5 . 0 2 3 . 0 1 3 .080 . 0 9 2 r e s u s p e n d e d i n v o l u m e e q u a l t o s u p e r n a t a n t A O.D. a t 340*vyuL f o r 1 m i n u t e The v a l u e s t a b u l a t e d f o r s p e c i f i c a c t i v i t i e s i n T a b l e V a r e o n l y f o r t h e c o m p a r i s o n o f r e l a t i v e a c t i v i t i e s o f t h e v a r i o u s enzymes i n c e l l e x t r a c t s and were n o t c a l c u l a t e d ( e x c e p t f o r G6P d e h y d r o g e n a s e ) a c c o r d i n g t o t h e s t a n d a r d e x p r e s s i o n o f s p e c i f i c a c t i v i t y i n e a c h c a s e ; f o r e x a m p l e , h e x o k i n a s e i s u s u a l l y e x p r e s s e d a s m i c r o m o l e s o f p h o s p h a t e t r a n s f e r r e d f r o m ATP t o s u b s t r a t e p e r u n i t t i m e p e r m i l l i g r a m p r o t e i n . U n d e r t h e c o n d i t i o n s o f t h i s e s s a y h o w e v e r , t h i s was n o t p o s s i b l e , t h e r e f o r e , a c t i v i t y was e x p r e s s e d i n K o r n b e r g u n i t s p e r mg. p r o t e i n , t h a t i s , c h a n g e i n o p t i c a l d e n s i t y a t 340rv^ p e r u n i t t i m e p e r mg. p r o t e i n . 41 T a b l e V R e l a t i v e a c t i v i t i e s o f v a r i o u s enzymes ( K o r n b e r g u n i t s ) A O.D. u n i t s mg. S p e c i f i c Enzyme p e r enzyme p r o t e i n A c t i v i t y ( u n i t s m i n u t e p e r p e r p e r mg. P r o t e i n ) c u v e t t e c u v e t t e G6P d e h y d r o g e n a s e .34-0 .34-0 1.1 .294 p h o s p h o g l u c o i s o m e r a s e .120 .120 1.1 .110 p h o s p h o m a n n o i s o m e r a s e .070 .070 1.1 .064 h e x o k i n a s e .044 .044 1.1 .040 2K6PG r e d u c t a s e .039 .039 a p p r o x . 0;5" .078 42 II Enzyme Purification Attempts to separate or purify enzymes of this system were largely limited to studies with glucose-6-phosphate dehydrogenase. The object was to obtain as active a preparation as possible; free of any contaminating hexokinase. Separation of these two enzymes was possible using two methods; namely, alkaline ammonium sulfate solution (Table VI), and ethyl alcohol (Table VII). The latt e r method was the most successful; giving fractions free of hexokinase a c t i v i t y with some increase i n specific activity. No effective separation was obtained with solid ammonium sulfate or calcium phosphate gel, although the latter method yielded a preparation with extremely high a c t i v i t i e s . Enzyme fractionation with alkaline ammonium sulfate solution. Table VI Fraction G6P dehydrogenase Assay Hexokinase * Crude 0/20 .278 .040 .100 0 20/26 .25 .039 26/34 0 .069 34/45 0 .202 Specific a c t i v i t i e s expressed as Kornberg units/mg. protein. It appeared that although G6P dehydrogenase could be separated from hexokinase i n the 0/20 fraction, the a c t i v i t y was greatly diminished. Hexokinase on the other hand could be effectively separated i n the 26/4-5 fraction with the 34-/45 fraction producing a five fold purification over the crude preparation. Using cold 95% ethanol, G6P dehydrogenase could be separated i n the 0/20 fraction (Table VII) with a slight increase i n specific a c t i v i t y . Hexokinase, however, could not be freed from G6P dehydrogenase by this method. Table VII Enzyme fractionation with 95% Ethanol Fraction Assay G6P dehydrogenase Hexokinase Crude .49 .045 0/10 .088 0 10/20 .62 0 20/30 .78 .130 30+ 1.96 .145 The technique of adsorption and elution from calcium phosphate gel did not result i n separation, but very high a c t i v i t i e s were obtained for both enzymes. In both the dehydrogenase and the hexokinase, a maximum 58 'fold increase i n activ i t y was obtained over the crude preparation (Table VIII). Table VIII Enzyme fractionation with Calcium Phosphate gel. Fraction G6P dehydrogenase Hexokinase Crude .286 .016 20/30 ammonium sulfate 3.46 .035 eluate from gel 16.2 .91 I l l M a n o m e t r i c S t u d i e s U s i n g d r i e d c e l l p r e p a r a t i o n s o f P. a e r u g i n o s a . S t o k e s a n d C a m p b e l l (58) were a b l e t o show t h a t g l u c o s e was o x i d i z e d e x c l u s i v e l y t o 2 - k e t o g l u c o n a t e w i t h t h e u p t a k e o f e x a c t l y l^u M o f o x y g e n p e r ^ M o f g l u c o s e . G l u c o n a t e was o x i d i z e d t o 2 - k e t o g l u c o n a t e w i t h t h e u p t a k e o f 0.5/A.M o f o x y g e n p e r ^ M o f g l u c o n a t e . 2 - k e t o g l u c o n a t e was n o t o x i d i z e d . T h e y c o u l d n o t d e m o n s t r a t e t h e a c c u m u l a t i o n o f a p h o s p h o r y l a t e d compound. I n l a t e r work, u s i n g e x t r a c t s p r e p a r e d b y Hughes P r e s s , Hogankamp (31) f o u n d t h a t i n t h e p r e s e n c e o f ATP t h e amount o f o x y g e n t a k e n up d u r i n g t h e o x i d a t i o n o f g l u c o s e was r e d u c e d b y o n e - h a l f ; t h a t i s , t o 1 atom o f o x y g e n . I n t h e p r e s e n c e o f A T P a n d a p y r a s e , t h e f u l l t h e o r e t i c a l o x y g e n u p t a k e o f 2 atoms o f o x y g e n was r e s t o r e d . The i m m e d i a t e r e a c t i o n was t h a t ATP was i n some way i n t e r f e r i n g w i t h t h e c o n v e r s i o n o f g l u c o n i c a c i d t o 2 - k e t o g l u c o n i c a c i d r e s u l t i n g i n t h e a c c u m u l a t i o n o f g l u c o n i c a c i d . I t was f o u n d , h o w e v e r , t h a t i n t h e p r e s e n c e o f ATP t h e e x t r a c t s w o u l d o x i d i z e g l u c o n i c a c i d w i t h t h e u p t a k e o f o n l y one atom o f o x y g e n . ADP i n t h e p r e s e n c e o f a h i g h e n e r g y p h o s p h a t e g e n e r a t i n g s y s t e m p r o d u c e d t h e same e f f e c t a s ATP. O t h e r n u c l e o t i d e t r i p h o s p h a t e s were n o t t e s t e d . I n a n a t t e m p t t o c l a r i f y t h i s phenomenon, s e v e r a l m a n o m e t r i c e x p e r i m e n t s , u s i n g Hughes p r e s s e x t r a c t s o f g l u c o s e grown c e l l s , were p e r f o r m e d . I n t h e e x p e r i m e n t 46 p r e s e n t e d i n T a b l e IX g l u c o s e and 2 - k e t o g l u c o n a t e were u s e d a s s u b s t r a t e s i n t h e p r e s e n c e a n d a b s e n c e o f A T P 4.4 and Mg . Cup c o n t e n t s w e r e t h e n a n a l y z e d . T a b l e IX E f f e c t o f A T P on t h e o x i d a t i o n o f g l u c o s e a n d 2 - k e t o g l u c o n a t e compounds r e m a i n i n g Oo u p t a k e R e a c t i o n M i x t u r e + ICytM g l u c o s e + 10yi»M g l u c o s e jlOywM A T P + lOykM 2 - k e t o g l u c o n a t e + IOMM 2 - k e t o g l u c o n a t e ; r lOykl M A T P 173 98 0 12 7-7 4.4 0 g l u c o s e 2 - k e t o - p y r u v a t e g l u c o n -a t e -A •M - 7 * ,M 0 0.6 6.0 7-8 9.6 16.3 .007 0 .004 0 R e a c t i o n m i x t u r e c o n t a i n s : T r i s b u f f e r ; w a t e r ; M g + + ; CPX. G l u c o s e was o x i d i z e d p r i m a r i l y t o 2 k e t o g l u c o n a t e . S i n c e a l l t h e g l u c o s e h a s d i s a p p e a r e d t h e b a l a n c e was assumed t o be g l u c o n i c a c i d , t h a t i s , s i n c e 6.0ynM 2 - k e t o g l u c o n a t e = 134yi£ 0 2 ; a n d i t i s a s s u m e d 4.0yuM g l u c o n a t e = 4 ^M / 0 2 , t h e n t h e t o t a l 179yft/ 0 2 i s v e r y c l o s e t o t h e o b s e r v e d amount (173/^)« As e x p e c t e d , no o x y g e n was consumed i n t h e p r e s e n c e o f 2 - k e t o g l u c o n a t e a n d a n e g l i g i b l e c h a n g e i n r e s i d u a l 2 - k e t o g l u c o n a t e c o n c e n t r a t i o n was d e t e c t e d . A c o n s i d e r a b l y d i f f e r e n t s i t u a t i o n e x i s t e d when ATP was 4-7 a d d e d t o t h e r e a c t i o n m i x t u r e s . O x y g e n u p t a k e w i t h g l u c o s e a s s u b s t r a t e was r e d u c e d t o t h e p r e d i c t e d 1 atom p e r m o l e o f s u b s t r a t e a d d e d and 94-% o f t h e g l u c o s e was u t i l i z e d . U n f o r t u n a t e l y t h e v a l u e s o b t a i n e d f o r r e s i d u a l 2 - k e t o g l u c o n a t e i n t h e p r e s e n c e o f ATP a r e c o m p l e t e l y s p u r i o u s . I t was n o t r e a l i z e d u n t i l much l a t e r t h a t u n l e s s t h e pH o f t h e r e a c t i o n m i x t u r e s was r i g i d l y m a i n t a i n e d a t n e u t r a l i t y d u r i n g t h e a s s a y p r o c e d u r e r i b o s e w o u l d i n t e r f e r e . I n t h i s c a s e i t was t h e r i b o s e m o i e t y o f ATP. I n some c a s e s , h o w e v e r , t h i s d i f f i c u l t y was n o t e n c o u n t e r e d . T h i s a s s a y n e v e r t h e l e s s c a n n o t be c o n s i d e r e d d e p e n d a b l e when ATP h a s b e e n u s e d i n t h e r e a c t i o n m i x t u r e . I n t h e a b s e n c e o f ATP h o w e v e r , t h e method was f o u n d t o be r e l i a b l e . I n t h e p r e s e n c e o f ATP, g l u c o s e was a l m o s t c o m p l e t e l y u t i l i z e d , y e t , t h i s u t i l i z a t i o n r e q u i r e d t h e e q u i v a l e n t o f o n l y 1 atom o f o x y g e n . T h i s w o u l d i m p l y t h a t some i n t e r m e d i a t e compound h a s a c c u m u l a t e d . T h i s compound i s p r o b a b l y n o t g l u c o n i c a c i d b e c a u s e o f t h e f a c t t h a t g l u c o n a t e i s r e a d i l y o x i d i z e d i n t h e p r e s e n c e o f ATP; t h e r e i s a l s o e v i d e n c e t o show t h a t t h e a c c u m u l a t e d compound i s p h o s p h o r y l a t e d . I t must, t h e r e f o r e , be some o t h e r compound r e q u i r i n g a n e t o x i d a t i o n e q u i v a l e n t o f 1 atom o f o x y g e n . S u c h a compound i s 6-p h o s p h o g l u c o n a t e . A l t h o u g h o t h e r s a r e p o s s i b l e , t h i s i n t e r m e d i a t e i s t h e ' m o s t : p r o b a b l e f o r i m m e d i a t e i n v e s t i g a t i o n m a i n l y b e c a u s e o f i t s e s t a b l i s h e d i m p o r t a n c e i n i n t e r m e d i a t e m e t a b o l i s m . T h e r e a r e t h r e e mechanisms b y w h i c h t h i s 48 TPN e compound c o u l d a r i s e f r o m g l u c o s e . ATP M3" 1. g l u c o s e I g l u e o s e - 6 - p h o s p h a t ^ _ 2^. +H 20 6 - p h o s p h o g l u c o n o l a c t o n e ^ z ± r 6 - p h o s p h o g l u c o n a t e . -H 20 +H 20 ATP, Mg"^+ 2. g l u c o s e — ; > — £ - g l u c o n o l a c t o n e g l u c o n a t e =>--2H -ir2o 6 - p h o s p h o g l u c o n a t e . +H 20 3. g l u c o s e >- S - g l u c o n o l a c t o n e •• g l u c o n a t e -2H -H pO -2H ATP, Mg DPN 2 - k e t o g l u c o n a t e >- 2 - k e t o - 6 - p h o s p h o g l u c o n a t e +2H 6 - p h o s p h o g l u c o n a t e . T h e r e a r e p r o s and c o n s f o r t h e f e a s i b i l i t y o f e a c h o f t h e s e p a t h w a y s a n d t h e r e i s o f c o u r s e t h e p o s s i b i l i t y t h a t a l l may o c c u r , d e p e n d i n g u p o n s p e c i f i c c o n d i t i o n s . The f i r s t r e a c t i o n i s s u p p o r t e d b y c o n s i d e r a b l e d a t a . B o t h g l u c o k i n a s e a n d g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e enzymes h a v e b e e n d e m o n s t r a t e d i n e x t r a c t s o f t h i s o r g a n i s m . T h e r e i s ho w e v e r t h e f a c t t h a t t h e d e h y d r o g e n a s e w o u l d be p a r t i c u l a r l y s e n s i t i v e t o t h e h i g h A TP c o n c e n t r a t i o n s i n v o l v e d i n t h e s e r e a c t i o n s a nd t h e r e f o r e no o x y g e n u p t a k e w o u l d be e x p e c t e d . T h i s i n i t s e l f c o u l d be c o n s t r u e d a s an e x p l a n a t i o n f o r t h e o c c a s i o n a l o b s e r v a t i o n t h a t some e x t r a c t p r e p a r a t i o n s , i n t h e p r e s e n c e o f ATP and g l u c o s e , w o u l d t a k e up v i r t u a l l y no o x y g e n , y e t a n a l y s i s o f c u p c o n t e n t s a l w a y s r e v e a l e d a c o m p l e t e u t i l i z a t i o n o f g l u c o s e . A n e x p e r i m e n t was p e r f o r m e d 49 t o t e s t t h e e f f e c t s o f v a r i o u s c o n c e n t r a t i o n s o f A T P o n o x y g e n c o n s u m p t i o n b y c e l l e x t r a c t s i n t h e p r e s e n c e o f g l u c o s e ( T a b l e X ) . T a b l e X E f f e c t o f ATP c o n c e n t r a t i o n on o x y g e n c o n s u m p t i o n b y c e l l e x t r a c t s i n t h e p r e s e n c e o f g l u c o s e . 1 W Oo u p t a k e a n a l y s i s o f cup c o n t e n t s g l u c o s e yul 2KG p h o s p h a t e e s t e r s 1. 2. 3. 4. 2.5 5 10 120 80 73 38 5-4 3.9 3.3 1.7 4.7 2.8 0 0 5.3 6.8 0 0 + + The v a l u e s g i v e n f o r o x y g e n u p t a k e a r e n o t f i n a l i n t h e f i r s t two c a s e s s i n c e t h e r e a c t i o n s h a d n o t gone t o c o m p l e t i o n . A g a i n i n t h e p r e s e n c e o f ATP, u n c e r t a i n v a l u e s were o b t a i n e d f o r 2 - k e t o g l u c o n a t e . The i m p o r t a n t f e a t u r e o f t h e s e d a t a , h owever, i s t h e e f f e c t o f A T P o n t h e f a t e o f g l u c o s e . I n t h e a b s e n c e o f ATP g l u c o s e i s b e i n g q u a n t i t a t i v e l y c o n v e r t e d t o 2 - k e t o g l u c o n a t e , b u t , w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f A T P t h e r e i s a g r a d u a l d i m i n u t i o n o f o x y g e n u p t a k e a c c o m p a n i e d b y a g r a d u a l d e c r e a s e i n r e s i d u a l g l u c o s e . C h r o m a t o g r a p h i c a n a l y s i s o f c u p c o n t e n t s f o r p h o s p h o r y l a t e d compounds r e v e a l e d t h e a p p e a r a n c e o f f a i n t "blue s p o t s . T h e s e s p o t s o c c u r r e d when ATP h a d b e e n u s e d i n t h e r e a c t i o n f l a s k s . I n l a t e r e x p e r i m e n t s t h e p r e s e n c e o f M/50 f l u o r i d e was f o u n d t o e n h a n c e 5< the accumulation of these phosphates. When run concurrently with known markers, these spots appeared to correspond best with glucose-6-phosphate (Table XI). Table XI . Chromatographic analysis of cup contents from experiment described in Table X. Standards Rf values and color of spots glucose-6-phosphate .61 blue fructose-6-phosphate .62 purple-blue 6-pho sphogluc onat e .56 blue A T P .55 blue A D P .31 blue inorganic phosphate .44 yellow-green Unknowns l o (no A T P ) .45 (yellow-green) 2. (2O5^LM A T P ) .45 (yellow-green) 3. (5 *M A T P ) o59(faint blue); .45(yellow-green); .36 (blue) 4. (IOUM ATP) ,59(blue); .45(yellow-green); ,35(blue); ' .31(blue) N.B. Solvent system used was: methanol 60; ammonia 10; water 30. Spray was acid-molybdate. Since only one solvent system was used i n this experiment, identification of there spots as glucose-6-phosphate was only tentative. These data would indicate that i n the presence of increasing concentrations of ATP, the pathway of glucose oxidation shifts from the non-phosphorylated pathway to phosphorylation at the hexose level; with or w i t h o u t s u b s e q u e n t o x i d a t i o n . T h i s s u g g e s t i o n i s s u p p o r t e d i n p a r t b y t h e r m o d y n a m i c e v i d e n c e (4-3) w h i c h w o u l d i n d i c a t e t h a t i n t h e p r e s e n c e o f a p h o s p h o r y l a t i n g s y s t e m a p a t h w a y t o g l u c o s e - 6 - p h o s p h a t e w o u l d have p r e f e r e n c e b e c a u s e t h e f r e e e n e r g y o f f o r m a t i o n o f t h i s compound f r o m g l u c o s e and ATP i s c o n s i d e r a b l y l o w e r ( - 5 . 1 K g c a l / m o l ) t h a n t h e A F ° o f g l u c o n i c a c i d f r o m g l u c o s e (-2 .7 K g c a l / m o l ) . I t i s a l s o o f i n t e r e s t t o n o t e t h a t t h e f r e e e n e r g y ohange o f g l u c o n o l a c t o n e f r o m g l u c o s e i s +2 . 30 or. +3.66 K g c a l / m o l w h i c h c o n s t i t u t e s a c o n s i d e r a b l e p o t e n t i a l b a r r i e r c o m p a r e d t o t h e a l t e r n a t e p h o s p h o r y l a t e d s t e p . The s e c o n d r e a c t i o n s e q u e n c e , t h a t i s , o x i d a t i o n o f g l u c o s e t o g l u c o n a t e f o l l o w e d b y p h o s p h o r y l a t i o n h a s t h e l e a s t s u p p o r t i n g e v i d e n c e . I n t h e f i r s t p l a c e , i t was n o t p o s s i b l e t o d e m o n s t r a t e g l u c o n o k i n a s e ; a n d s e c o n d l y , ATP d i d n o t h a l t t h e r a p i d o x i d a t i o n o f g l u c o n i c a c i d ( 3 1 ) . T h i s s e c o n d p o i n t , h o w e v e r , d o e s n o t r u l e o u t t h e p o s s i b i l i t y t h a t i n t h e p r e s e n c e o f ATP g l u c o n a t e i s o x i d i z e d a f t e r p h o s p h o r y l a t i o n . The t h i r d p a t h w a y , t h a t i s , t h e c o n v e r s i o n o f g l u c o s e t o 6 - p h o s p h o g l u c o n a t e v i a 2 - k e t o g l u c o n a t e i s p r o b a b l y t h e most i m p o r t a n t p a t h w a y a l t h o u g h t h e s u p p o r t i n g e v i d e n c e i s n o t c o n c l u s i v e . P o u r o f t h e f i v e enzymes r e q u i r e d f o r t h i s c o n v e r s i o n h a v e b e e n d e m o n s t r a t e d a n d t h e p r e s e n c e o f t h e 2 - k e t o - 6 - p h o s p h o g l u c o n a t e (2K6PG) 52 r e d u c t a s e r e p r e s e n t s t h e i m p o r t a n t l i n k i n t h i s p a t h w a y . A p p e a r a n c e o f t h i s enzyme i n e x t r a c t s o f g l u c o s e g r o w n c e l l s i s i n c o n t r a s t t o t h e e q u i v a l e n t enzyme i n L . m e s e n t e r o i d e s , w h i c h c o u l d be d e m o n s t r a t e d o n l y i h e x t r a c t s f r o m c e l l s grown on 2 - k e t o g l u c o n a t e (3). However, i n P. f l u o r e s c e n s ( 2 8 ) , A. s u b o x y d a n s ( 2 7 ) , a n d i n B. c e r e u s s p o r e s ( 2 1 ) , t h e r e d u c t a s e enzyme a p p e a r e d i n e x t r a c t s p r e p a r e d f r o m g l u c o s e grown c e l l s . T h i s enzyme i s n o t o n l y i m p o r t a n t s i m p l y a s a n e n z y m a t i c l i n k , b u t a l s o f r o m an e n e r g e t i c p o i n t o f v i e w i n p r o v i d i n g s u p p o r t f o r t h e n o n - p h o s p h o r y l a t e d p a t h w a y a s t h e m a j o r o x i d a t i v e r o u t e . D o i e t a l ( 2 1 ) f o u n d , i n e x t r a c t s o f B. c e r e u s s p o r e s , t h a t p y r u v a t e was i n v a r i a b l y f o r m e d f r o m two t o f o u r t i m e s f a s t e r f r o m 2K6PG t h a n f r o m 6 - p h o s p h o -g l u c o n a t e ( 6 P G ) . The i m m e d i a t e e x p l a n a t i o n w o u l d be a d i r e c t r o u t e f r o m 2K6PG n o t i n v o l v i n g 6PG. Wood ( 2 1 ) however, h a s s u g g e s t e d t h a t 2K6PG i s r e d u c e d t o 6PG, t h e n c l e a v e d t o g l y c e r a l d e h y d e - 3 - p h o s p h a t e (G3P). The o x i d a t i o n o f G3P t o p y r u v a t e t h e n - s u p p l i e s n e c e s s a r y h y d r o g e n atoms f o r t h e r e d u c t i o n o f 2K6PG b y t h e DPN l i n k e d r e d u c t a s e . A n o t h e r e x p l a n a t i o n i n v o l v e s t h e d i r e c t t r a n s f e r o f t h e h y d r o g e n f r o m t h e g l u c o s e o r g l u c o n i c a c i d d e h y d r o g e n a s e h y d r o g e n a c c e p t o r s t o t h e coenzyme (DPN) o f 2K6PG r e d u c t a s e . T h e r e f o r e , i n t h e p r e s e n c e o f a p h o s p h o r y l a t i n g s y s t e m t h e o x i d o - r e d u c t i o n c h a i n i s c o m p l e t e w i t h t h e r a p i d r e m o v a l o f 2K6PG d u r i n g t h e o x i d a t i o n o f g l u c o s e . T h i s m e chanism w o u l d p r o b a b l y be t h e more e f f i c i e n t o f t h e two s y s t e m s s u g g e s t e d . T h e r e i s 53 -'chromatographic evidence i n support of this third pathway. In this particular experiment two large Warburg vessels were set up as described in Materials and Methods. The contents were then chromatographed by applying the material in a long streak at the origin. After development i n a n-propanol, formic acid, water solvent system for 14- hours, the chromatograms were dried and each one cut into three lengthwise strips. One strip from each chromatogram was sprayed with a particular solution (Table XII). Table XII Chromatographic analysis of reaction mixtures with and without ATP. Reaction mixture Rf values and color reactions with various acid-molybdate O-phenylene diamine sprays orcinol .4-5(green spot, fluorescence under ultra-violet .4-5 (bright yellow spot) Reaction mixture +1<JUM ATP;25/ fig .86(fluorescent area under u l t r a -violet,no v i s i b l e spot) .4-5 (weak yellow spot); .86 (purple spot) Reaction mixture contains: Tris buffer; water; M/50 fluoride; lO^M glucose; c e l l extract (Hughes press). The spots with the Rf values of 0.45 are probably 2-ketogluconate since this compound reacts with the diamine reagent giving a 54 green, fluorescent compound. They also correspond roughly with the Rf value i n the literature of 0.39 (17). The purple orcinol spot, however, has not been identified, but since this reagent reacts with almost any ketose, such a compound may well be present since i t also reacted with the diamine spray, which i s known to react with almost any 1,2 dicarbonyl compound. The Rf value of 0.86 does not, however, correspond even closely to any values reported i n the literature, for example, 2K6PG with an Rf of 0.30 and 2K3D6PG with an Rf of 0.47 (17). 6PG and G6P are ruled out because they do not react with the reagents nor do they give appropriate Rf values. No spots were v i s i b l e with the molybdate spray for phosphate esters. This may have been a result of the rel a t i v e l y low concentrations of material applied to the paper. Both the O-phenylenediamihe: and the orcinol sprays are much more sensitive than the Hanes and Isherwood spray. With this i n mind a second chromatogram was run using material from the same reaction mixtures. This time, however, an ordinary spot chromatogram was prepared using more material. After spraying with the acid-molybdate solution, a distinct blue spot appeared that had migrated in a similar fashion to the fast moving compound described above. No such spot appeared on the chromatogram of the reaction mixture containing no ATP. One feature of these data i s the chromatographic ^evidence of a small amount of residual 2-ketogluconate present i n the reaction mixture 55 i w i t h ATP. T h i s w o u l d i n d i c a t e t h a t a t l e a s t p a r t o f t h e o x y g e n u p t a k e i s a c c o u n t e d f o r b y c o n v e r s i o n t o 2 - k e t o -g l u c o n a t e . A t t h i s p o i n t i t s h o u l d be p o i n t e d o u t t h a t w i t h e a c h m a n o m e t r i c e x p e r i m e n t a f r e s h e x t r a c t was made. W i t h i n t h e c o n f i n e s o f e a c h p r e p a r a t i o n r e s u l t s were c o n s i s t e n t b u t f r o m one p r e p a r a t i o n t o a n o t h e r , c e r t a i n i r r e g u l a r i t i e s were f o u n d ; f o r e x a m p l e , v a r i a b l e amounts o f o x y g e n were t a k e n up i n t h e p r e s e n c e o f g l u c o s e a n d ATP, a l s o a t t i m e s t h e r a t e o f e n d o g e n o u s r e s p i r a t i o n was r e d u c e d t o z e r o w h i l e a t o t h e r t i m e s i t was n o t a f f e c t e d . I t s h o u l d be e x p e c t e d t h a t some enzymes o r c o m p l e t e enzyme s y s t e m s may be p r e s e n t i n one e x t r a c t a n d a b s e n t i n a n o t h e r . T h i s w o u l d e x p l a i n how w i t h one p r e p a r a t i o n a h e x o s e p h o s p h a t e a p p e a r e d t o a c c u m u l a t e i n d i c a t i n g t h a t g l u c o s e was d i r e c t l y p h o s p h o r y l a t e d y e t i n a n o t h e r p r e p a r a t i o n a compound a c c u m u l a t e d w h i c h a p p e a r e d t o be more f u l l y o x i d i z e d t h a n a h e x o s e p h o s p h a t e . T h e r e f o r e , a n y d i s c u s s i o n o f p a t h w a y s o f g l u c o s e o x i d a t i o n i n c e l l f r e e e x t r a c t s must be b a s e d on t h e e v i d e n c e g a i n e d f r o m a s i n g l e p r e p a r a t i o n , s i n c e t h e c o n d i t i o n s p r e s e n t i n one may n o t be p r e s e n t i n a n o t h e r . I n s p i t e o f t h e s e l i m i t a t i o n s some g e n e r a l i z a t i o n s a r e s t i l l p o s s i b l e . Of t h e t h r e e p o s s i b l e p a t h w a y s o f g l u c o s e o x i d a t i o n t o 6 - p h o s p h o g l u c o n a t e , t h e r e a r e two w h i c h seem t o be t h e 56 most l i k e l y a t p r e s e n t . I n c o n s i d e r i n g t h e d i r e c t p h o s p h o r y l a t i o n o f g l u c o s e f o l l o w e d b y o x i d a t i o n t o 6PG i t s h o u l d b e p o i n t e d o u t t h a t i n t h e p r e s e n c e o f h i g h ATP c o n c e n t r a t i o n s , t h e a p p a r e n t s h i f t f r o m one p a t h w a y t o a n o t h e r may be a n i n v i t r o phenomenon b a s e d e n t i r e l y o n f r e e e n e r g y c h a n g e s d u r i n g e n z y m a t i c r e a c t i o n s ; t h a t i s , t h e p a t h w a y o f g l u c o s e o x i d a t i o n i s p r o b a b l y g e a r e d t o t h e e n e r g y n e e d s o f t h e m e t a b o l i z i n g o r g a n i s m a n d t h e t e n d e n c y t o w a r d s t h e r m o d y n a m i c e q u i l i b r i u m , c h a r a c t e r i s t i c o f i n v i t r o r e a c t i o n s , d o e s n o t o c c u r i n t h e i n t a c t c e l l s i n c e t h e r e i s a c o n t i n u a l r e m o v a l o f m e t a b o l i c i n t e r m e d i a t e s . When t h e c e l l i s b r o k e n , t h e l o s s o f i n t r a c e l l u l a r i n t e g r i t y i s m a n i f e s t i n t h e d i s r u p t i o n o f v a r i o u s s y n t h e t i c a b i l i t i e s , f o r e x a m p l e , p r o t e i n s y n t h e s i s a n d s u c h e n d e r g o n i c r e a c t i o n s a s t h e s y n t h e s i s o f ATP w h i c h i s c o u p l e d w i t h t h e h i g h l y e x e r g o n i c h y d r o g e n t r a n s p o r t s y s t e m . I f , a s d e s c r i b e d b e f o r e , a l i n k o c c u r s b e t w e e n t h e h y d r o g e n t r a n s p o r t s y s t e m o f g l u c o s e o r g l u c o n a t e d e h y d r o g e n a s e a n d t h a t o f 2K6PG r e d u c t a s e t h e n b r e a k a g e o f t h e c e l l may w e l l r e s u l t i n t h e d e s t r u c t i o n o f t h i s i n t e g r a t e d t r a n s p o r t s y s t e m . I n s u c h a c a s e , a l t h o u g h p r o p e r c a t a l y s i s may be p r e s e n t i n t h e f o r m o f ATP, t h e c o m p e t i t i o n b e t w e e n p h o s p h o r y l a t i o n o f g l u c o s e a n d 2-k e t o g l u c o n a t e i s w e i g h e d i n f a v o r o f g l u c o s e b e c a u s e o f e q u i l i b r i u m c o n d i t i o n s w h i c h a r e no l o n g e r c o n t r o l l e d b y t h e c e l l s ' e n e r g y r e q u i r e m e n t s . The s e n s i t i v i t y o f G6P d e h y d r o g e n a s e t o ATP may a l s o be p r i m a r i l y a n i n v i t r o o c c u r r e n c e , t h a t i s , i n t h e m e t a b o l i z i n g o r g a n i s m ATP p r o b a b l y d o e s n o t e x i s t e i t h e r i n f r e e s o l u t i o n o r i n s u c h a b u n d a n t q u a n t i t i e s as i t d o e s i n t h e r e a c t i o n t u b e . I t i s more l i k e l y t h a t ATP as w e l l a s TPN a r e e a c h i n t i m a t e l y a s s o c i a t e d w i t h i t s own p a r t i c u l a r enzyme and a r e n o t , i n t h e s e n s e , f r e e t o i n t e r f e r e i n t h e manner o b s e r v e d i n t h e t e s t t u b e . I t i s p r o b a b l y u n w i s e t h e r e f o r e t o e x c l u d e a p a t h w a y i n v o l v i n g G6P d e h y d r o g e n a s e s o l e l y on t h e g r o u n d s t h a t t h i s enzyme i s s e n s i t i v e t o ATP. D i c k e n s a n d G l o c k (20) h a v e r e p o r t e d t h a t r a b b i t l i v e r G6P d e h y d r o g e n a s e i s s e n s i t i v e t o ATP, y e t i t i s known t h a t a h e x o s e m o n o p h o s p h a t e s h u n t m e c h a n i s m o p e r a t e s i n t h e s e t i s s u e s . From t h e d a t a p r e s e n t e d i n t h i s t h e s i s c o m b i n e d w i t h t h e h i s t o r i c a l e v i d e n c e i t seems p r o b a b l e t h a t t h e m a j o r p a t h w a y o f g l u c o s e o x i d a t i o n i n Pseudomonas a e r u g i n o s a (ATCC 9027) i n v o l v e s t h e n o n - p h o s p h o r y l a t e d p a t h w a y t o 2 - k e t o g l u c o n a t e f o l l o w e d b y p h o s p h o r y l a t i o n t o 2 - k e t o - 6 -p h o s p h o g l u c o n a t e and r e d u c t i o n t o 6 - p h o s p h o g l u c o n a t e . The b r e a k d o w n o f t h i s i n t e r m e d i a t e i s t h e n m a i n l y b y way o f t h e E n t n e r - D o u d o r o f f p a t h w a y . F o r c o n v e n i e n c e , a summary o f t h e r e a c t i o n s d i s c u s s e d i n t h i s t h e s i s a r e s c h e m a t i c a l l y p r e s e n t e d i n F i g . 4. ATP Glucose ATP Glucose-6-0P0 Mg' -2H £-gluconolactone TPM 1 6-phosphoglucono-lactone ± H 2 0 Gluconate — 2H \ |r ± H 2 ° 6-phosphogluconate { DPN Mannose-6-OPO- — - — Mannose 6 Mg + + Fructose-l,6-(OPOj) 2 2-ketogluconate — 2 - k e t o - 6 - p h o s p h o -gluconate Fructose ' ^ Fructose-l-OPO-roxyacetone -OPO^ Glyceraldehyde Glyceroldehyde - 3- OP0 3 Pyruvate Rg. 4 Pathways of hexose dissimilation in P aeruginosa SUMMARY 1. Evidence i s presented for a hexokinase i n c e l l free extracts of Pseudomonas aeruginosa (ATCC 9027) which i s capable of phosphorylating glucose and probably fructose but not mannose. The presence of two phosphohexoisomerases was also demonstrated. 2. Glucose-;6-phosphate dehydrogenase was found to have a requirement for magnesium but an excess of this element was found to be inhibitory. 3. Nucleotides inhibited glucose-6;-phosphate dehydrogenase ++ by competing with TPN for the Mg. ATP was found to be the most effective inhibitor. Excess magnesium would p a r t i a l l y reverse ATP inhibition only when added after the complete reaction mixture, including ATP, was present. Inhibition by other nucleotides was reversed more readily. 4-. Glucose-6-phosphate dehydrogenase could be separated from hexokinase by an ethanol or alkaline ammonium sulfate fractionation. Hexokinase, however, could only be separated from the dehydrogenase by the latt e r method. 5. Cell free extracts of P. aeruginosa contain an alternate pathway of glucose oxidation involving glucose-6-phosphate and 6-phosphogluconate. In the whole c e l l , however, the main pathway of glucose oxidation i s probably the non-p h o s p h o r y l a t e d p a t h w a y i n v o l v i n g g l u c o n a t e , 2 k e t o g l u c o n a t e , t h e p h o s p h o r y l a t e d d e r i v a t i v e o f 2 - k e t o g l u c o n a t e a n d 6 - p h o s p h o g l u c o n a t e « 60 BIBLIOGRAPHY 1. Barron, E.S.G. and Friedemann, T.E. Studies on biological oxidations. XIV. 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The catabolism of glucose and gluconate i n Pseudomonas species. Arch. Biochem. and Biophys., S i 489 (1959). ~~ 62. Warburton, R.H., Eagles, B.A. and Campbell, J.J.R. The intermediate metabolism of Pseudomonas  aeruginosa, v. The identification of pyruvate as an intermediate i n glucose oxidation. Can. J. Botany, 29, 143 (1951). 63. Wood, W.A. and Schwerdt, R.P. Carbohydrate oxidation by Pseudomonas fluorescens. I. The mechanism of glucose and gluconate oxidation. J. B i o l . Chem., 201, 501 (1953). 64. Wood, W.A. and Schwerdt, R.P. Pathways of hexose oxidation in extracts of Pseudomonas fluorescens. J. Bio l . Chem., 206, 625 (1954). 

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