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Chemical studies of the glucose metabolism of Pseudomonas aeruginosa A.T.C. 9027 Idler, David Richard 1950

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CHEMICAL STUDIES OF THE GLUCOSE METABOLISM OP PSEUDOMONAS AERUGINOSA A.T.C. 9027 - b y -D a v i d R i c h a r d I d l e r 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 A T h e s i s S u b m i t t e d f o r t h e D e g r e e o f MASTER OF -ARTS i n t h e DEPARTMENT OF CHEMISTRY THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A u g u s t , 19^0. ABSTRACT The f i r s t p a r t o f t h i s w o r k w a s u n d e r t a k e n i n a n e f -f o r t t o d e v i s e a s a t i s f a c t o r y f r a c t i o n a t i o n p r o c e d u r e f o r s t u d y i n g t h e 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 i n t h e g l u c o s e m e t a b o l i s m o f P. A e r u g i n o s a . B y c h e m i c a l a n d c h r o m a t o g r a p h i c a n a l y s i s o f t h e s e f r a c t i o n s i t was h o p e d t o o b t a i n more i n -f o r m a t i o n o n t h e c h e m i c a l s e q u e n c e o f t h e b r e a k d o w n . A m o d i f i e d b a r i u m f r a . c t i o n a t i o n p r o c e d u r e i s p r e s e n t e d f o r i s o l a t i n g t h e t r i c h l o r o a c e t i c a c i d s o l u b l e p h o s p h a t e e s t e r s f r o m t h e c e l l s o f P. A e r u g i n o s a . S e v e r a l c o n c l u s i o n s a r e d r a w n f r o m t h e a n a l y s i s o f t h e b a r i u m f r a c t i o n s . The a d e n o s i n e p h o s p h a t e coenzyme s y s t e m i s shown t o b e p r e s e n t . P h o s p h o g l y c e r i c a c i d and h e x o s e - d i - p h o s p h a t e a r e absent,. E v i d e n c e i s p r e s e n t e d t o show t h a t g l u c o s e - 6 - p h o s p h a t e i s p r o b a b l y n o t p r e s e n t a n d n e i t h e r p h o s p h o p y r u v i c - a c i d n o r t h e t r i o s e p h o s p h a t e s o c c u r i n d e t e c t a b l e c o n c e n t r a t i o n s . T h e s e d a t a a l l p o i n t t o a p h o s p h o r y l a t i o n m e c h a n i s m w h i c h d o e s n o t i n v o l v e a n E m b d e n - M e y e r h o f s y s t e m . O t h e r t h a n t h e coenzyme s y s t e m s t h e r e a r e no n i t r o g e n o u s compounds p r e s e n t . A l l f r a c -t i o n s c o n t a i n p h o s p h a t e e s t e r s w h i c h h a v e r e d u c i n g v a l u e s b e f o r e h y d r o l y s i s . The r e d u c i n g v a l u e s , o n h y d r o l y s i s w i t h n o r m a l h y d r o c h l o r i c a c i d , p a r a l l e l t h e r e l e a s e o f i n o r g a n i c p h o s p h a t e . E v e r y b a r i u m f r a c t i o n c o n t a i n s a l a r g e p e r c e n t a g e o f n o n - p h o s p h o r y l a t e d compounds. T h e s e compounds a r e n o t r e d u c i n g t o t h e F o l i n - M a l m r o s r e a g e n t . I n t h e c h r o m a t o g r a p h y s t u d i e s s e v e r a l s o l v e n t s h a v e b e e n f o u n d s a t i s f a c t o r y f o r t h e r e s o l u t i o n o f s u g a r s , p a r -t i c u l a r l y p e n t o s e s . The p r e s e n c e o f a n a r a b i n o s e p h o s p h a t e i s e s t a b l i s h e d b y p a p e r c h r o m a t o g r a p h y . A r a b o n i c a c i d i s a l s o i d e n t i f i e d b u t t h e c h r o m a t o g r a p h i c e v i d e n c e i s n o t c o n -s i d e r e d c o n c l u s i v e . The i m p o r t a n c e o f t h e s e two compounds i s d i s c u s s e d i n t h e l i g h t o f t h e p r e s e n t k n o w l e d g e o f g l u c o s e b r e a k d o w n b y P. A e r u g i n o s a . A new d e v e l o p e r i s p r e s e n t e d f o r u s e i n t h e c h r o m a -t o g r a p h y o f c e r t a i n p h o s p h a t e e s t e r s . The s e c o n d p a r t o f t h i s w ork was u n d e r t a k e n i n a n e f f o r t t o e s t a b l i s h t h e c h e m i c a l c o m p o s i t i o n o f t h e w a t e r a n d a c i d i n s o l u b l e gum p r o d u c e d when P. A e r u g i n o s a i s g r o w n o n a g l u c o s e - m i n e r a l s a l t s m e d i a . The amino a c i d s p r e s e n t i n t h e gum a r e a l a n i n e , g l u -t a m i c a c i d a n d s e r i n e . T h e s e w e r e i d e n t i f i e d b y p a p e r c h r o -m a t o g r a p h y . Amino a c i d c a r b o n d i o x i d e a c c o u n t s f o r 9« 25% o f t h e d r y gum w e i g h t . The u r o n i c a c i d c o n t e n t o f t h e gum i s 31.4%. The p h o s p h o r u s c o n t e n t ( a s PO^) i s 13.2%. F r u c t o s e i s p r e s e n t . The gum i s v e r y r e s i s t a n t t o h y d r o l y s i s b y f o u r n o r m a l h y d r o c h l o r i c a c i d . ' T e n h o u r s a r e r e q u i r e d t o l i b e r -a t e a l l c a r b o n d i o x i d e f r o m t h e u r o n i c a c i d c o m p o n e n t . The i o d i n e r e a c t i o n i s n e g a t i v e . No s u g a r s c o u l d b e i d e n t i f i e d b y p a p e r c h r o m a t o g r a p h y " . ACKNOWLEDGMEMT. I w i s h t o e x p r e s s my s i n c e r e t h a n l c s t o D r . J . J . E . C a m p b e l l f o r h i s k e e n i n t e r e s t a n d i n v a l u a b l e a d v i c e . TABLE OF CONTENTS PART I . - STUDIES ON INTERMEDIATE METABOLISM I n t r o d u c t i o n 1 E x p e r i m e n t a l B a c t e r i o l o g i c a l 4 C h e m i c a l F r a c t i o n a t i o n 4 P u r i f i c a t i o n o f t h e b a r i u m s a l t s 8 A n a l y t i c a l m e t h o d s 10 C h r o m a t o g r a p h y 10 R e s u l t s a n d d i s c u s s i o n 14 Summary 29-a PART I I . THE CHEMICAL COMPOSITION OF THE GUM: I n t r o d u c t i o n 30 E x p e r i m e n t a l 31 R e s u l t s a n d d i s c u s s i o n 32 Summary 37 1 INTRODUCTION I t i s now g e n e r a l l y c o n c e d e d t h a t a n a e r o b i c m i c r o -o r g a n i s m s b r e a k down g l u c o s e a s f a r a.s p y r u v i c a c i d b y t h e E m b d e n - M e y e r h o f ( 2 2 ) ( 3 0 ) p h o s p h o r y l a t i o n scheme. The p y r u v i c a c i d may t h e n b e d i s s i m i l a t e d b y a n y one o f a number o f m e c h a n i s m s . I n t i s s u e m e t a b o l i s m p y r u v i c a c i d i s o x i -d i z e d t o c a r b o n d i o x i d e a n d w a t e r v i a t h e K r e b s (15) ( l 6 ) t r i c a r b o x y l i c a c i d c y c l e . H o w e v e r , v e r y l i t t l e w o r k h a s b e e n done o n t h e g l u c o s e m e t a b o l i s m o f a e r o b e s . D o u d o r o f f ( 9 ) 5 i n s t u d y i n g t h e p h o s p h o r o l y s i s o f s u c r o s e b y P. P u t r e -f a c i e n s , h a s shown t h a t g l u c o s e - 1 - p h o s p h a t e , g l u c o s e - 6 -p h o s p h a t e a n d f r u c t o s e - 6 - p h o s p h a t e w e r e f o r m e d . W o r k i n g w i t h y e a s t a n d m u s c l e , D i c k e n s ( 7 ) p o s t u l a t e d a scheme f o r t h e d i r e c t o x i d a t i o n o f g l u c o s e . The f i r s t p r o d u c t was a p h o s p h o r y l a t e d h e x o n i c a c i d , w h i c h was d e h y d r o g e n a t e d t o a p h o s p h o k e t o h e x o n i c a c i d w h i c h i s t h e n d e c a r b o x y l a t e d t o a p h o s p h o p e n t o n i c a c i d . R e c e n t l y C o hen ( 5 ) h a s p o s t u l a t e d a r a b i n o s e - 5 - p h o s p h a t e a s t h e f i r s t p r o d u c t f r o m t h e b r e a k -down o f 6 - p h o s p h o g l u c o n i c a c i d b y y e a s t e x t r a c t s . L o c k w o o d , T a b e r i k i n a n d Ward ( 1 8 ) , e m p l o y i n g c o n d i t i o n s o f i n t e n s e a e r a t i o n , o b t a i n e d h i g h y i e l d s o f g l u c o n i c a n d 2 - k e t o g l u -c o n i c a c i d s f r o m t h e b r e a k d o w n o f g l u c o s e b y c u l t u r e s o f P s e u d o m o n a s a n d P h y t o m o n a s . L o c k w o o d a n d S t o d o l a . ( 1 7 ) i s o l a t e d <>c - k e t o g l u t a r i c a c i d f r o m s u b m e r g e d c u l t u r e s o f P. A e r u g i n o s a 9 0 2 7 . E m p l o y i n g p a p e r c h r o m a t o g r a p h y , N o r r i s a n d C a m p b e l l ( 2 5 ) w e r e a b l e t o show t h e p r e s e n c e o f g l u c o n i c 2. a n d 2 - k e t o g l u c o n i c : a c i d s a s n o r m a l i n t e r m e d i a t e s i n t h e g l u c o s e m e t a b o l i s m o f P. A e r u g i n o s a 9 0 2 7 . C a m p b e l l a n d N o r r i s (3) h a v e p r e s e n t e d e v i d e n c e t o r u l e o u t t h e p o s s i -b i l i t y o f a n E m b d e n - M e y e r h o f i n P. A e r u g i n o s a . T h e " - e v i d e n c e i s b a s e d o n p h o s p h o r u s d i s t r i b u t i o n s t u d i e s . No f r u c t o s e was f o u n d i n a n y f r a c t i o n n o r w e r e t h e r e a n y r e d u c i n g com-p o u n d s e i t h e r b e f o r e o r a f t e r h y d r o l y s i s o f t h e p h o s p h a t e e s t e r s . T h e s e d a t a r u l e d o u t f r u c t o s e 1-6 d i p h o s p h a t e , f r u c t o s e 6- p h o s p h a t e , g l u c o s e - 1 - p h o s p h a t e a n d g l u c o s e - 6 - p h o s p h a t e a s p o s s i b l e I n t e r m e d i a t e s . C a m p b e l l a n d N o r r i s (4) h a v e i d e n t i f i e d a c e t i c a c i d a s a n o r m a l i n t e r m e d i a t e . W a r b u r t o n a n d C a m p b e l l (34-)? e m p l o y i n g p a p e r c h r o m a t o g r a p h y , h a v e i d e n t i f i e d p y r u v i c a c i d a s a n i n t e r m e d i a t e i n g l u c o s e b r e a k d o w n b y P. A e r u g i n o s a . The p r e s e n t s t u d y was u n d e r t a k e n i n an e f f o r t t o f r a c t i o n a t e a n d i d e n t i f y 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 f o r m e d d u r i n g t h e b r e a k d o w n o f g l u c o s e b y P. A e r u g i n o s a . The p r o -c e d u r e s o f D u b o i s a n d P o t t e r ( 8 ) , U m b r e i t a n d L e P a g e (32) a n d S a c k s ( 2 7 ) , w e r e a l l d e v i s e d f o r i s o l a t i n g p h o s p h a t e e s t e r s f r o m o r g a n i s m s known t o p o s s e s s a n E m b d e n - M e y e r h o f s y s t e m . The p r o c e d u r e o f U m b r e i t a n d L e P a g e was s e l e c t e d a n d m o d i f i e d t o m e e t t h e r e q u i r e m e n t s o f t h e p r e s e n t s t u d y . S o o n a f t e r t h e w o r k was b e g u n i t was f o u n d t h a t p h o s p h a t e e s t e r s , w h i c h showed r e d u c i n g v a l u e s t o t h e F o l i n -M a l m r o s r e a g e n t , w e r e p r e s e n t i n a l l b a r i u m f r a c t i o n s . F u r -t h e r i n v e s t i g a t i o n showed t h a t t h e S h a f f e r a n d S o m o g y i ( 2 8 ) r e a g e n t e m p l o y e d b y C a m p b e l l a n d N o r r i s (3) was not. s u f f i c i -e n t l y s e n s i t i v e t o d e t e c t t h e q u a n t i t i e s o f r e d u c i n g compounds w h i c h w e r e p r e s e n t . F o r t h i s r e a s o n i t v/as c o n s i d e r e d n e c e s -s a r y t o r e e x a m i n e t h e r e l a t i o n s h i p b e t w e e n t h e p h o s p h a t e e s t e r s a n d t h e i r r e d u c i n g c o m p o n e n t s . 4. EXPERIMEMTAL BACTERIOLOGICAL The c u l t u r e o f P. A e r u g i n o s a A.T.C. 9027 was a t y p i -c a l , s t r o n g l y p i g m e n t i n g s t r a i n . The s t o c k c u l t u r e s o f t h e o r g a n i s m w e r e m a i n t a i n e d o n a l i v e r e x t r a c t g e l a t i n a g a r 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 : 1.0% t r y p t o n e , 0.3% K^EPQ^? 0.1 g l u c o s e , 0.3% g l y c e r o l , 1 0 . 0 % o f l i v e r e x t r a c t , 0.5% a g a r , 2.0% g e l a t i n , pH 7«2. G r o w t h was f i r s t i n i t i a t e d a t 30°C a n d t h e n t h e c u l t u r e s w e r e r e f r i g e r a . t e d . When r e q u i r e d , t h e c u l t u r e was t r a n s f e r r e d 2 t o 3 t i m e s a t 24 h o u r i n t e r v a l s i n S u l l i v a n ' s (29) m e d i a . A v i g o r o u s u n d i s s o c i a t e d c u l t u r e was m a i n t a i n e d b y t a k i n g a f r e s h c u l t u r e s e m i - m o n t h l y f r o m a r e f r i g e r a t e d s t o c k . The m e d i a u s e d f o r g r o w i n g c e l l s f o r m e t a b o l i s m s t u d i e s v/as composed o f 0.3% ffii^I^PO^, 0.3% K 2 H P 0 4 , 0 . 1 % M g S 0 4 .7 H 2 0 , 0.3% g l u c o s e a n d 0 . 5 P.P.M. F e a s FeSO^. The c e l l s w e r e g r o w n a t 30°C f o r l6 h o u r s a n d t h e n b r o u g h t down i n a S h a r p i e s c e n t r i f u g e . Y i e l d : 46.7 gms. o f m o i s t c e l l s f r o m 10 l i t r e s o f m e d i a . CHEMICAL F r a c t i o n a t i o n : The f r a c t i o n a t i o n p r o c e d u r e s a v a i l a b l e w e r e a l l d e -v i s e d t o i d e n t i f y 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 i n t h e m e t a b o l i s m o f g l u c o s e b y b a c t e r i a w h i c h h a v e a n Embden-M e y e r h o f s y s t e m . I t h a s b e e n p o i n t e d o u t i n t h e i n t r o d u c -t i o n t h a t s u c h a s y s t e m i s b e l i e v e d t o b e a b s e n t i n 5. P. A e r u g i n o s a . F o r t h i s r e a s o n t h e U m b r e i t a n d L e P a g e ( 3 2 ) p r o c e d u r e was m o d i f i e d a s f o l l o w s : 1. The f i r s t p o s s i b i l i t y t o b e c o n s i d e r e d was t h a t com-p o u n d s w h i c h w e r e n o t p h o s p h o r y l a t e d b u t w h i c h w e r e r e d u c i n g ( i . e . u r o n i c a c i d s ) o r c o n t a i n e d n i t r o g e n ( i . e . a c i d s o f amino s u g a r s ) m i g h t f o r m i n s o l u b l e b a r i u m s a l t s . T h i s w o u l d g i v e a f a l s e r e l a t i o n s h i p o f r e d u c i n g a n d n i t r o g e n v a l u e s t o p h o s p h o r o u s . To a v o i d t h i s p o s s i b i l i t y a l l f r a c t i o n s w e r e p u r i f i e d a n d w e i g h e d i n s t e a d o f b e i n g l e f t a s t r i c h l o r o a c e t i c e x t r a c t s . A c a l c u l a t i o n c o u l d t h e n b e made t o d e t e r m i n e w h e t h e r o r n o t t h e w h o l e f r a c t i o n was pho s p h o r y l a t e d . 2 . A f t e r p r e c i p i t a t i o n s h a d b e e n made f r o m t h e o r i g i n a l t r i c h l o r o a c e t i c (T.C.A.) e x t r a c t t h e f r a c t i o n s w e r e f o u n d t o c o n t a i n a l a r g e e x c e s s o f n i t r o g e n . C h r o m a t o g r a p h y r e v e a l e d t h e n i t r o g e n t o b e p r o t e i n i n n a t u r e . T h e r e w e r e two p o s -s i b l e e x p l a n a t i o n s f o r t h i s ; e i t h e r t h e c o m p o n e n t s w e r e o f l o w m o l e c u l a r w e i g h t ( i . e . p e p t i d e s ) o r p r o t e i n was p r e s e n t a s a c o n t a m i n a n t . To d e t e r m i n e w h i c h was t h e c a s e t h e p r e -c i p i t a t e s w e r e d r i e d and r e e x t r a c t e d w i t h s m a l l v o l u m e s o f i c e - c o l d T.C.A. T h i s e s t a b l i s h e d t h a t t h e n i t r o g e n was due t o a n i n s o l u b l e p r o t e i n c o n t a m i n a n t . 3 « The A.T.P.-A.D.P. f r a c t i o n was r e m o v e d w i t h Lohman's r e a g e n t i n s t e a d o f b e i n g l e f t i n t h e b a r i u m i n s o l u b l e f r a c -t i o n " T l i i s was done t o d e t e r m i n e i f p h o s p h o g l y c e r i c a c i d o r h e x o s e - d i p h o s p h a t e ( H.D.P.) was p r e s e n t . P r e v i o u s e x p e r i -m e n t s h a d i n d i c a t e d t h e p r e s e n c e o f a q u i t e i n s o l u b l e a r a b i n o s e p h o s p h a t e . I t was p o s s i b l e t h a t t h e a r a b i n o s e 6 . w o u l d be i n t h i s f r a c t i o n a n d i t was c o n s i d e r e d d e s i r a b l e t o remove t h e r i b o s e w h i c h w o u l d b e p r e s e n t a s a. c o n s t i t u e n t o f t h e n u c l e o t i d e s . 4. A c c o r d i n g t o C o r i ( 6 ) , t h e f r a c t i o n d e s i g n a t e d a s t h e d o u b l e s a l t f r a c t i o n b e l o n g s i n t h e b a r i u m s o l u b l e f r a c -t i o n . B e c a u s e o f t h e e v i d e n c e a g a i n s t a n E m b d e n - M e y e r h o f , t h i s f r a c t i o n was p u r i f i e d s e p a r a t e l y . 5« The f r a c t i o n d e s i g n a t e d a s t h e n o n - p h o s p h o r y l a t e d f r a c t i o n was a n a l y z e d s e p a r a t e l y f o r much t h e same r e a s o n a s 4. 6 . The b a r i u m s o l u b l e - a l c o h o l i n s o l u b l e f r a c t i o n was p r e c i p i t a t e d w i t h two v o l u m e s i n s t e a d o f f o u r v o l u m e s o f a l c o h o l a n d a l l o w e d t o r e a c h r o o m t e m p e r a t u r e i n s t e a d o f 5°C. T h i s was done t o p u t t h e c o e n z y m e s i n t o t h e b a r i u m s o l u b l e -a l c o h o l s o l u b l e f r a c t i o n so t h a t t h e l a r g e n i t r o g e n a n d r i b o s e c o n t e n t w o u l d n o t h i n d e r t h e i n t e r p r e t a t i o n o f t h e d a t a o f t h i s f r a c t i o n . The f r a c t i o n i s o f m a j o r i m p o r t a n c e i n s t u d y i n g a b a c t e r i u m w i t h a n E m b d e n - M e y e r h o f s y s t e m . P r e v i o u s i n v e s t i g a t i o n h a d shown t h e l o s s t o b e n o t more t h a n 10% o f t h e f r a c t i o n a n d t h i s 10% h a d a l a r g e r i b o s e - n i t r o g e n c o n t e n t . The c e l l s w e r e t h o r o u g h l y w a s h e d w i t h w a t e r t o remove a l l r e s i d u a l .sugar. T h e y w e r e t h e n c e n t r a . f u g e d a n d t h e c e l l w a l l s b r o k e n b y g r i n d i n g i n 1.5 m l . o f 50% KOH f o r 8 m i n u t e s a t 0°C. The s l u r r y was t h e n n e u t r a l i z e d w i t h a c o n c e n t r a t e d s o l u t i o n o f t r i c h l o r o a c e t i c a c i d (T.C .A.). 100 m l . o f 8 . 0 % a q u e o u s T.C.A. was a d d e d a n d t h e p h o s p h a t e e s t e r s w e r e e x -c t r a c t e d f o r 24 h o u r s a t 5 C. The c e l l d e b r i s was t h e n c e n t r i -f u g e d o u t a n d t h e T.C.A. e x t r a c t s a v e d f o r a n a l y s i s . T h e , c e l l d e b r i s was. t h e n t r e a t e d w i t h a f u r t h e r 90 n i l * o f 5% T.C.A. a n d e x t r a c t e d f o r 24 h o u r s . - The f i l t r a t e s w e r e c o m b i n e d a n d t h e c e l l d e b r i s d i s c a r d e d . : The T.C.A. e x t r a c t was b r o u g h t t o pH 8 . 2 w i t h 50% KOH. T w e n t y m l . o f a 25% s o l u t i o n o f B a ( 0 A C ) 2 was a d d e d a n d t h e s o l u t i o n r e f r i g e r a t e d f o r 30 m i n u t e s . The p r e c i p i t a t e was c e n t r i f u g e d . T h i s i s t h e BARIUM INSOLUBLE FRACTION. The s u p e r n a t e n t (pH7»9) was a d j u s t e d t o pH . 8.2, 2 v o l u m e s o f a l c o h o l a d d e d , and t h e s o l u t i o n c h i l l e d f o r 30 m i n u t e s . The p r e c i p i t a t e was c e n t r i f u g e d . T h i s i s t h e BARIUM SOLUBLE-ALCOHOL INSOLUBLE FRACTION. . The f i l t r a t e i s t h e BARIUM SOLUBLE-ALCOHOL SOLUBLE FRACTION. F u r t h e r f r a c t i o n a t i o n : BARIUM INSOLUBLE FRACTION: The p r e c i p i t a t e was washed- s u c c e s s i v e l y w i t h 9°% e t h y l a l c o h o l , a c e t o n e , a n d f i n a l l y e t h e r a n d was t h e n t h o r o u g h l y d r i e d i n a n o p e n o v e n a t 50°C The f r a c t i o n was t h e n t a k e n u p i n 20 m l . o f i c e -c o l d 5% T.C.A. The p r o t e i n r e s i d u e was d i s c a r d e d . The v o l u m e was a d j u s t e d t o 40 m l . a n d t h e b a r i u m s a l t s r e p r e -c i p i t a t e d a t pH 8.2 w i t h two d r o p s o f 25% B a ( 0 A C ) 2 . The f i l t r a t e now c o n t a i n s t h e DOUBLE SALT FRACTION. T h i s was p r e c i p i t a t e d w i t h 2 v o l u m e s o f a l c o h o l a n d k e p t f o r p u r i f i -c a t i o n . The t r u e b a r i u m i n s o l u b l e s a l t s w e r e t h e n t a k e n up 8. i n 3 0 n i l . o f 0 . 2 IT HITO^ and 1 m l . o f Lohman's r e a g e n t a d d e d . The p r e c i p i t a t e i s t h e A.T.P. - A.D.P. FRACTION. I t v/as p u r i f i e d i m m e d i a t e l y . The m e r c u r y v/as r e m o v e d f r o m t h e f i l -t r a t e w i t h I-IgS, t h e s o l u t i o n a d j u s t e d t o pH 8 . 2 , a n d 5 n i l ' o f 2 5 % B a ( 0 A C ) 2 a d d e d . The p r e c i p i t a t e was f o u n d t o b e b a r i u m p h o s p h a t e . BARIUM SO LUBLE - ALCOHOL INSOLUBLE FRACTION; T h i s f r a c t i o n was w a s h e d a n d d r i e d b y t h e p r o c e d u r e u s e d o n t h e b a r i u m i n s o l u b l e p r e c i p i t a t e . The d r i e d b a r i u m s a l t was t a k e n up i n i c e - c o l d $.0% T.C .A. a n d t h e p r o t e i n r e s i d u e d i s c a r d e d . The f i l t r a t e v/as a d j u s t e d t o pH 8 . 2 a n d 5 n i l ' o f 2 5 % B a ( 0 A C ) 2 a d d e d . The p r e c i p i t a t e I s t h e NOIT-PHOS- PHORYLATED FRACTION. The t r u e b a r i u m s o l u b l e f r a c t i o n was t h e n p r e c i p i t a t e d w i t h 2 v o l u m e s o f a l c o h o l a t 5°C • f o r 3 0 m i n u t e s . The p r e c i p i t a t e v/as a l l o w e d t o r e a c h r o o m t e m p e r a -t u r e b e f o r e c e n t r i f u g i n g a n d i s t h e BARIUM SO LUBLE-ALCOPIOL INSOLUBLE FRACTION. P u r i f i c a t i o n . > o f t h e b a r i u m s a l t s : A.T.P. - A.P.P. FRACTION: The p r e c i p i t a t e was imme-d i a t e l y t a k e n up i n 40 v o l u m e s o f w a t e r a n d t h e m e r c u r y r e m o v e d w i t h H 2 S . The H 2 S was e x p e l l e d f r o m t h e s u p e r n a t e n t b y a e r a t i o n . The. HgS was w a s h e d w i t h IT/ 100 HC1 a n d t h e w a s h i n g s a d d e d t o t h e s u p e r n a t e n t . The pH was a d j u s t e d t o 8 . 2 w i t h KOH. The l a s t t r a c e s o f HgS w e r e r e m o v e d b y a d d i n g 5 m l . o f 0.1 N B a ( 0 A C ) 2 t o t h e f i l t r a t e , p r e c i p i t a t i n g t h e b a r i u m w i t h O.l'lTHgSO/j., a n d c e n t r i f u g i n g . The i n o r g a n i c p h o s p h a t e v/as r e m o v e d w i t h m a g n e s i a m i x t u r e . The pH was r e -adjusted to 8.2 with acetic acid, 5 ml. of 25% Ba(OAC) 2 added and the solution r e f r i g e r a t e d f o r 15 minutes. Washing was done with alcohol i n concentrations of 65% to 95%' Washing and drying of the p r e c i p i t a t e was carried out as previously described. Y i e l d : 40.1 mg. of the barium s a l t . DOUBLE SALT FRACTION: The f r a c t i o n was taken up i n 20 ml. of i c e - c o l d 5% T.C.A. and the protein residue d i s -carded. The solution was adjusted to pH 2.0 and the barium removed with an excess of 0.05 N B^SO^. Inorganic phosphate was removed with magnesia mixture. The f i l t r a t e was adjusted to pH 2.0 and 5 ml. of Ba(0AC) 2 added. The BaSO^.was re-, moved by centrifuging. The f i l t r a t e was adjusted to pH 8.2, alcohol added, and the p r e c i p i t a t e was washed and dried. Y i e l d : 4 8 . 1 mg. of the barium s a l t . NON-PHO SPHORYLATED FRACTION: The p r e c i p i t a t e was taken up i n 70 volumes of 0.05 N HCL, 2 volumes of alcohol added, the solution c h i l l e d , the new p r e c i p i t a t e freed of inorganic phosphate and then washed and dried as previously described. The f i l t r a t e was found to contain only inorganic •phosphate. . Y i e l d : 64.0 mg. of the barium s a l t . BARIUM SOLUBLE-ALCOHOL INSOLUBLE FRACTION: The procedure employed f o r the double s a l t f r a c t i o n applies here with one exception. Greater care must be observed i n wash-ing with alcohol or some of the f r a c t i o n w i l l be l o s t . Seventy-five per cent alcohol was found.to be the most s a t i s -1 0 . f a c t o r y . S m a l l v o l u m e s w e r e u s e d a n d e a c h w a s h i n g was t e s t e d f o r b a r i u m w i t h H^SO^. T h r e e w a s h i n g s w i t h 2 v o l u m e s was s u f f i c i e n t t o remove B a C l g . Y i e l d : 233*0 mg. o f t h e b a r i u m s a l t . COENZYME FRACTION: Two v o l u m e s o f a l c o h o l w e r e a d d e d t o t h e b a r i u m s o l u b l e a l c o h o l f r a c t i o n a n d t h e s o l u t i o n ' r e -f r i g e r a t e d f o r 30 m i n u t e s . The p r e c i p i t a t e was w a s h e d w i t h a c e t o n e t h e n w i t h e t h e r a n d d r i e d . A n a l y t i c a l M e t h o d s : P h o s p h o r u s was d e t e r m i n e d b y t h e m e t h o d o f L o w r y a n d L o p e z ( 2 0 . R e d u c i n g v a l u e s w e r e d e t e r m i n e d a c c o r d i n g t o F o l i n a n d M a l m r o s (10) . F r u c t o s e was d e t e r m i n e d b y t h e p r o -c e d u r e o f Roe ( 2 6 ). N i t r o g e n was d e t e r m i n e d b y a m o d i f i e d N e s s l e r ' s (31) m e t h o d . P e n t o s e was d e t e r m i n e d a c c o r d i n g t o M e i j b a u m ( 2 1 ) . A l k a l i a n d i o d i n e l a b i l e p h o s p h o r u s w e r e m e a s u r e d b y t h e p r o c e d u r e o f Lohman and M e y e r h o f ( 1 9 ) . C h r o m a t o g r a p h y : B o t h a s c e n d i n g a n d d e s c e n d i n g c o l u m n s w e r e e m p l o y e d . The w a l k - i n i n c u b a t o r was m a i n t a i n e d a t 29°C. f o r a l l r u n s . Number 1 W h a t m a n f i l t e r p a p e r was u s e d a n d t h e p a p e r p u r i f i e d a s f o l l o w s : A l a r g e B u c h n e r t y p e f u n n e l w a s c o n s t r u c t e d f r o m a n g l e i r o n a n d 1/4 i n c h p l a t e g l a s s . P o r c e l a i n c ement was u s e d f o r s e a l i n g t h e j o i n t s . The cement i s s o l u b l e i n h y d r o -f l u o r i c a.cid b u t i n s o l u b l e i n a l l m i n e r a l a c i d s . One h u n d r e d s h e e t s o f p a p e r (22 i n . b y 18-1/4 i n . ) w e r e w a s h e d a t one t i m e . A 5% s o l u t i o n o f a c e t i c a c i d was u s e d t o remove t h e 1 1 . a l k a l i m e t a l s . " A n y r e m a i n i n g t r a c e s o f h e a v y m e t a l s w e r e • r e n d e r e d u n a v a i l a b l e b y s a t u r a t i n g t h e a t m o s p h e r e a r o u n d t h e p a p e r w i t h H 2 S . When a two p h a s e s y s t e m was e m p l o y e d i n a d e s c e n d i n g c o l u m n t h e a t m o s p h e r e was s a t u r a t e d f o r 24 h o u r s w i t h b o t h p h a s e s p r i o r t o commencing t h e r u n . F o r t h e a s c e n d i n g c o l u m n s . p y r e x c y l i n d e r s 5-1/2 i n . i n d i a m e t e r a n d 18 i n . deep w e r e u s e d . C o m p o s i t i o n o f t h e s o l v e n t s : P i c r i c a c i d 4 gm., t e r t . b u t a n o l 80 m l . , HgO 20 m l . ( 1 2 ) , e t h a n o l 500 m l . , g l a c i a l , a c e t i c a c i d 4 . 8 m l . , H 2 0 100 m l . (5), e t h a n o l 80%, b o r i c a c i d 0.64% (5) , m e t h a n o l 45 m l . , e t h a n o l 4J? m l . , HgO 10 m l . (25)5 w a t e r p o o r p h a s e f r o m m e t h y l e t h y l k e t o n e and H 2 0 (13)j w a t e r p o o r p h a s e f r o m t e r t . a m y l a l c o h o l 3° m l . , g l a c i a l a c e t i c a c i d 10 m l . , H 2 0 30 m l . (13). S p r a y s : The p h o s p h a t e e s t e r s , w i t h t h e e x c e p t i o n : o f 3 p h o s -p h o g l y c e r i c a n d p h o s p h o g l u c o n i c a c i d s , w e r e d e v e l o p e d w i t h t h e m o l y b d a t e r e a g e n t o f I s h e r w o o d (13). The s p r a y h a d t h e f o l l o w i n g c o m p o s i t i o n : 2 j m l . o f 4% w/v (M-I/^MoO^ i n w a t e r , 10 m l . o f 1.0 11 HC1, 5 m l . o f 60% H C 1 0 4 d i l u t e d t o 100 m l . w i t h w a t e r . The s h e e t was s p r a y e d , a l l o w e d t o d r y f o r 10 m i n u t e s t h e n h e a t e d i n a n o p e n o v e n a t 8o°C. f o r 7 m i n u t e s t o h y d r o l y s e ' t h e e s t e r s . The s h e e t was t h e n a l l o w e d t o t a k e o n m o i s t u r e f r o m t h e a i r f o r two h o u r s . The c o l o r was d e v e l -o p e d w i t h H 0 S . 1 2 . 3 - P h o s p h o g l y c e r i c a n d p h o s p h o g l u c o n i c a c i d s a r e v e r y r e s i s t a n t t o h y d r o l y s i s a n d a new t e c h n i q u e was d e v i s e d t o d e v e l o p t h e s e e s t e r s . The m e t h o d I s b a s e d o n t h e i n s o l u b i l i t y o f t h e i r s i l v e r s a l t s . The s h e e t was s p r a y e d w i t h a n 6 .5% w/v a q u e o u s s o l u t i o n o f AgNO^ a n d i m m e d i a t e l y s u b m e r g e d i n w a t e r 3 t i m e s . The p a p e r was t h o r o u g h l y d r a i n e d b e t w e e n e a c h s u b m e r s i o n . The p a p e r was a l l o w e d t o d r y f o r 30 m i n u t e s t h e n s a t u r a t e d w i t h R"2S t o d e v e l o p t h e i n s o l u b l e s i l v e r s a l t s . The s u g a r s i n T a b l e s 6 a n d 7 w e r e d e v e l o p e d . w i t h a n i l i n e - p h t h a i i c a c i d . 0.93 n i l * °± a n i l i n e a n d 1 .66 gm. o f p h t h a l i c a c i d w e r e d i s s o l v e d i n 100 m l . o f w a t e r - s a t u r a t e d n - b u t a n o l . The s p r a y e d s h e e t was h e a t e d f o r 10 t o 15 m i n u t e s a t 120°C. t o d e v e l o p t h e c o l o r . The compounds i n T a b l e 8 w e r e d e v e l o p e d w i t h 0.11T AgNO^ c o n t a i n i n g a n e x c e s s o f ^TI-I^OH. The s h e e t s w e r e s p r a y e d a n d d e v e l o p e d i n t h e d a r k f o r 24 h o u r s . P r e p a r a t i o n of' S o l u t i o n s f o r C h r o m o t o g r a g h y : S t a n d a r d s o l u t i o n s o f t h e p h o s p h a t e e s t e r s w e r e p r e -p a r e d b y d i s s o l v i n g 5 wg> o f t h e b a r i u m s a l t s i n 0 . 0 I - I N O ^ . The b a r i u m was r e m o v e d w i t h L O W H^SO^ a n d t h e v o l u m e a d j u s t -e d t o 1.0 m l . A p p r o x i m a t e l y 1/800 m l . v/as u s e d f o r e a c h c h r o m a t o g r a m . S u g a r s a n d o t h e r s t a n d a r d s o l u t i o n s w e r e made up i n t h e same c o n c e n t r a t i o n a s t h e p h o s p h a t e e s t e r s . The v a r i o u s b a r i u m f r a c t i o n s f r o m P. A e r u g i n o s a w e r e t a k e n up i n 0 . 0 H C 1 , t h e b a r i u m r e m o v e d w i t h H 2 S 0 , a n d a 3-hour h y d r o l y s i s v/as c a r r i e d o u t i n 1.0IT HC1. The h y d r o l y -13-s a t e s were then concentrated under vacuum and the pentoses i d e n t i f i e d . The barium soluble-alcohol soluble f r a c t i o n was reduced i n volume and the barium removed with H„SO.. 1 4 . RESULTS AND DISCUSSION The c h e m i c a l c o m p o s i t i o n o f t h e f r a c t i o n s i s r e c o r d e d i n T a b l e s 1, 2, 3? 4 a n d 5 and t h e r e l a t i o n s h i p b e t w e e n p h o s -p h o r u s r e l e a s e a n d r e d u c i n g v a l u e s o n h y d r o l y s i s i s g i v e n i n F i g u r e s 1, 2 a n d 3' I t was v e r y s u r p r i s i n g t o f i n d a r e d u c i n g compound o t h e r t h a n p e n t o s e i n t h e A.T.P.-A.D.P. f r a c t i o n . I n a b a c -t e r i u m , h a v i n g a n E m b d e n - M e y e r h o f s y s t e m , L o h m a n 1 s r e a g e n t i s s p e c i f i c f o r t h e n u c l e o t i d e s . The F o l i n - M a l m r o s r e a g e n t i s n o t r e d u c e d b y o f - h y d r o x y a c i d s a n d o n l y t o a v e r y s l i g h t d e g r e e b y c<- k e t o a c i d s . C h r o m a t o g r a p h y ( T a b l e s 6 a n d 7) showed o n l y r i b o s e t o b e p r e s e n t b u t i n t h i s r e g a r d i t m u s t be r e membered t h a t t h e a n i l i n e - p h t h a l i c a c i d d e v e l o p e r i s much more s e n s i t i v e t o w a r d s p e n t o s e s t h a n t o w a r d s h e x o s e s a n d t h e c o n c e n t r a t i o n o f a h e x o s e may h a v e b e e n t o o s m a l l t o d e t e c t . Time d i d n o t p e r m i t a more t h o r o u g h i n v e s t i g a t i o n a n d t h e i d e n t i t y o f t h e r e d u c i n g compound m u s t be l e f t t o some f u t u r e t i m e . The a b s e n c e o f a b a r i u m i n s o l u b l e f r a c t i o n , a f t e r r e m o v i n g t h e A.T.P.-A.D.P. f r a c t i o n , I s v e r y s i g n i f i c a n t . I f a n E m b d e n - M e y e r h o f s y s t e m was p r e s e n t , 3- p h o s p h o g l y c e r i c a c i d a n d h e x o s e - d i p h o s p h a t e w o u l d be i n t h i s f r a . c t i o n . T h i s T h i s i s t h e r e f o r e f u r t h e r c o n f i r m a t i o n o f t h e w o r k o f Camp-b e l l a n d N o r r i s (3) t h a t t h i s o r g a n i s m d o e s n o t h a v e a n E m b d e n-Meyerhof s y s t e m . The m e t h o d f o r d e t e r m i n i n g n i t r o g e n was f a r f r o m s a t i s f a c t o r y , b u t , b e c a u s e o f t h e l a r g e number o f d e t e r m i n a t i o n s 15. c a r r i e d o u t , t h e r e s u l t s a r e c o n s i d e r e d t o b e r e a s o n a b l y a c c u r a t e . The r a t i o o f p h o s p h o r u s t o p e n t o s e makes i t e x - ; t r e m e l y u n l i k e l y t h a t t h e r e i s a n y n i t r o g e n o u s compound o t h e r t h a n n u c l e o t i d e s p r e s e n t i n a n y f r a c t i o n . T h i s i s c o n s i d e r e d t o b e s u f f i c i e n t j u s t i f i c a t i o n f o r t h e e x t r a t i m e i n v o l v e d i n d r y i n g p r o c e d u r e s a n d r e e x t r a . c t i o n w i t h t r i c h l o r o a c e t i c a c i d . The i d e n t i f i c a t i o n o f a r a b i n o s e - p h o s p h a t e ( T a b l e s 6 a n d 7) i n t h e d o u b l e s a l t f r a c t i o n may be o f c o n s i d e r a b l e i m p o r t a n c e . I t h a s b e e n p o i n t e d o u t t h a t C a m p b e l l a nd M o r r i s (25) c o n s i d e r t h e b r e a k d o w n .of g l u c o s e t o p r o c e e d b y way o f 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 . F u r t h e r , t h e b a r i u m s o l u b l e - a l c o h o l s o l u b l e f r a c t i o n i s b e l i e v e d t o c o n -t a i n a r a b o n i c a c i d ( T a b l e 8 ) . A l l t h e e v i d e n c e p o i n t s t o a c a r b o n - b y - c a r b o n d e g r a d a t i o n r a t h e r t h a n a t h r e e c a r b o n s p l i t o f g l u c o s e . A l a r g e p e r c e n t a g e o f n o n - p h o s p h o r y l a t e d compounds a r e p r e s e n t i n a l l b a r i u m f r a c t i o n s a n d p a r t i c u l a r l y i n t h e b a r i u m s o l u b l e - a l c o h o l i n s o l u b l e f r a c t i o n . T h e s e compounds a r e n o t r e d u c i n g t o t h e F o l i n - M a l m r o s r e a g e n t . A l o g i c a l e x p l a n a t i o n f o r t h i s i s t h e p r e s e n c e o f an a c i d ( i . e . g l u -c o n i c , oi - k e t o g l u c o n i c , e t c . , ) w h i c h w o u l d f o r m a n i n s o l u b l e b a r i u m s a l t u n d e r t h e c o n d i t i o n s o f p r e c i p i t a t i o n . T h i s w o u l d b e an i n t e r e s t i n g p o i n t t o i n v e s t i g a t e f u r t h e r b e c a u s e g l u c o n i c a n d 2 - k e t o g l u c o n i c a c i d s h a v e n o t b e e n i s o l a t e d f r o m w i t h i n t h e c e l l . G l u c o s e was n o t i d e n t i f i e d a s a h y d r o l y s i s p r o d u c t i n -1 6 . t h e b a r i u m s o l u b l e - a l c o h o l i n s o l u b l e f r a c t i o n b u t i f a n E m b d e n - M e y e r h o f s y s t e m w w e r e p r e s e n t g l u c o s e - 6 - p h o s p h a t e w o u l d b e t h e p r e d o m i n a n t e s t e r . H o w e v e r , t h i s compound i s o n l y 10% h y d r o l y s e d w i t h : 1*1 h y d r o c h l o r i c a c i d i n 3 h o u r s , so no d e f i n i t e c o n c l u s i o n c a n b e d r a w n b e c a u s e g l u c o s e w o u l d b e p r e s e n t i n s u c h s m a l l a m o u n t s . The r a t i o o f t o t a l p h o s -p h o r u s t o t h e 3 h o u r r e d u c i n g v a l u e makes i t u n l i k e l y t h a t g l u c o s e - 6 - p h o s p h a t e c o u l d b e p r e s e n t i n a n y q u a n t i t y . The m o s t r e a s o n a b l e e x p l a n a t i o n f o r t h e r e d u c i n g v a l u e i s 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 e d u r o n i c a c i d . P a p e r c h r o m a t o g -r a p h y g a v e some p r e l i m i n a r y i n d i c a t i o n s t h a t t h i s w a s t h e c a s e . I n t h e s t u d i e s o n c h r o m a t o g r a p h y t h e p h o s p h a t e e s t e r s p r e s e n t e d a p a r t i c u l a r l y d i f f i c u l t p r o b l e m . A l l t h e s o l v e n t s o f . I s h e r w o o d (12) w e r e t r i e d w i t h v e r y l i t t l e s u c c e s s . The m o s t p r o m i s i n g s o l v e n t s w e r e t h e t e r t i a r y a l c o h o l s i n s t r o n g -a c i d s s u c h a s p i c r i c a n d p - t o l u e n e s u l f o n i c . T a b l e 9 g i v e s t h e d a t a f o r t h e p i c r i c a c i d s o l v e n t b u t no c o n c l u s i o n s c a n be d r a w n . The s o l v e n t s o f C o h e n (5) w e r e t r i e d r e p e a t e d l y a n d a l w a y s w i t h n e g a t i v e r e s u l t s . T h e s e f i n d i n g s w e r e i n a g r e e m e n t w i t h t h o s e o f I sherwood,. who f o u n d t h a t s t r o n g a c i d s w e r e r e q u i r e d t o move t h e p h o s p h a t e e s t e r s . The m a i n p r o b l e m seems t o b e t o f i n d a n a c i d s o l v e n t w h i c h i s s u f f i -c i e n t l y s t r o n g t o move t h e e s t e r s a n d y e t s u f f i c i e n t l y n e a r t h e a c i d s t r e n g t h o f t h e e s t e r s t o a l l o w a s e p a r a t i o n . The e x c e l l e n t m o l y b d a t e s p r a y o f I s h e r w o o d was n o t a v a i l a b l e when C o h e n ' s w o r k was done a n d t h i s may a c c o u n t f o r t h e d i s c r e p a n -17. c i e s b e t w e e n t h e two s e t s o f d a t a i n T a b l e 9 . I s u g g e s t , o n t h e b a s i s o f t h i s d a t a , t h a t C o h e n ' s c h r o m a t o g r a p h i c i d e n t i -f i c a t i o n o f a r a b i n o s e - 5 - p h o s p h a t e may h a v e b e e n i n e r r o r . The s o l v e n t s e m p l o y e d f o r t h e s u g a r s ( T a b l e s 6 a n d 7) w e r e o r i g i n a l l y u s e d b y I s h e r w o o d (13) t o s e p a r a t e t h e com-p o n e n t s o f t h e p o l y s a c c h a r i d e s i n t h e c e l l w a l l o f f r u i t s . T h e s e s o l v e n t s w e r e c h o s e n b e c a u s e p r e l i m i n a r y i n v e s t i g a t i o n i n d i c a t e d a n e x c e l l e n t s e p a r a t i o n o f t h e p e n t o s e s , p a r t i c u -l a r l y r i b o s e . The e t h a n o l - m e t h a n o l s o l v e n t u s e d i n t h e i d e n t i f i c a -t i o n o f a r a b o n i c a c i d ( T a b l e 7) v/as o r i g i n a l l y e m p l o y e d b y N o r r i s a n d C a m p b e l l (25) t o i d e n t i f y g l u c o n i c a n d 2 - k e t o -g l u c o n i c a c i d s i n t h e c e l l f r e e m e d i a . 18, TABLE 1 THE COMPOSITION O F THE A.T.P.-A.D.P . FRACTION T o t a l y i e l d o f b a r i u m s a l t s 4 0 . 1 mg. T a k e n f o r ' a n a l y s i s 12.0 mg. ugm P h o s p h o r u s ( 4 ) 7 m i n . 3 54 ugm 30 m i n . .; 4o0 ugrn 1 h r . 5 7 ° ugm T o t a l 936 ugm N u c l e o t i d e s (1) 486 ugm A l k a l i L a b i l e (3) • • None R e d u c i n g V a l u e 0 t i m e 3°0 ugm 7 m i n 1560 ugm 30 m i n 2040 ugm 1 h r 2400 ugm N i t r o g e n C o m p onents (1) 4 . 0 6 mg. A.T.P. 90% • A.D.P. 10% R e m a i n d e r , i n c l u d i n g r e d u c i n g compound 7 . 9 4 mg. P e n t o s e i d e n t i f i e d b y c h r o m a t o g r a p h y R i b o s e (1) C a l c u l a t e d f r o m n i t r o g e n : P e n t o s e r a t i o . . (2) M e a s u r e o f p h o s p h o p y r u v i c a c i d . (3) M e a s u r e o f t r i o s e p h o s p h a t e s . ( 4 ) H y d r o l y s i s i n I N h y d r o c h l o r i c a c i d . A r a b i n o s e was u s e d f o r a s t a n d a r d r e d u c i n g c u r v e . 19 FIGURE 1. H y d r o l y s i s o f t h e A.T.P.-A.D.P. f r a c t i o n i n l . O N h y d r o c h l o r i c a c i d . 20 4 0 ri I M U T E 5 1. R e d u c i n g v a l u e o f t h e h y d r o l y s a t e t o t h e F o l i n - M a l m r o s r e a g e n t e x p r e s s e d a s m i c r o g r a m s o f a r a b i n o s e . . 2. P h o s p h a t e r e l e a s e e x p r e s s e d a s m i c r o g r a m s o f P. 20. TABLE 2 THE COMPOSITION OF THE DOUBLE SALT FRACTION T o t a l y i e l d o f b a r i u m s a l t s . ...... 4 8 . 1 mg. T a k e n f o r a n a l y s i s 24 . 7 mg'-F r u c t o s e , None P h o s p h o r u s (2) 7 m i n 55 ugm 1 h o u r 242 ugm Q^ O "tjCft-la e • o © • 0 « o • « a ao 4 0 0 0 0 0 0 0 0 0 a o o • • » "^2 ^) UL^llT I o d i n e L a b i l e None A l k a l i L a b i l e None R e d u c i n g v a l u e (2) 0 "fclHTG 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ^0 0 Ul J^ IH 7 m i n . 875 ugm 1 h o u r 2200 ugm 3 h o u r s 3000 ugm E s t i m a t e d w e i g h t o f p h o s p h o r a t e e s t e r s i n t h i s f r a c t i o n (1) 10.52 mg. E s t i m a t e d w e i g h t o f non-pho s p h o r y l a t e d b a r i u m s a l t s i n t h i s f r a c t i o n 37«58 rag* P e n t o s e s i d e n t i f i e d b y c h r o m a t o g r a p h y A r a b i n o s e , R i b o s e ( s m a l l ) ( 1 ) C a l c u l a t e d f o r a h e x o s e m o n o p h o s p h a t e e s t e r M.W. 395. (2) H y d r o l y s i s i n 1.0N h y d r o c h l o r i c a c i d . A r a b i n o s e was u s e d f o r t h e s t a n d a r d r e d u c i n g c u r v e . 2 1 . FIGURE 2. Hydrolysis of the double s a l t f r a c t i o n i n 1.0H hydrochloric acid. eo 120 M I N U T E S 1. Reducing value of the hydrolysate to the Folin-Malmros reagent expressed as micro grains of arabinose. 2. Phosphate release expressed as micrograms of P. 22. TABLE 3 THE COMPOSITION OF THE BARIUM SOLUBLE-ALCOHOL INSOLUBLE FRACTION T o t a l y i e l d o f b a r i u m s a l t s 233'0 mg. T a k e n f o r a n a l y s i s 25.6 mg. P e n t o s e 1000 ugm N i t r o g e n 350 ugm F r u c t o s e None P h o s p h o r u s (2) 7 m i n . 30 ugm •3 h o u r s . . 255 ugm T o t a l 735 ugm I o d i n e L a b i l e None A l k a l i L a b i l e None R e d u c i n g V a l u e (2) 0 t i m e 588 ugm 7 m i n . 1875 ugm 3 h o u r s 3375 ugm E s t i m a t e d w e i g h t o f p h o s p h o r u s e s t e r s i n t h i s f r a c t i o n (1) 85.4 mg. E s t i m a t e d w e i g h t o f n o n - p h o s p h o r y l a t e d b a r i u m s a l t s i n t h i s f r a c t i o n 1 47 . 6 mg. P e n t o s e s i d e n t i f i e d b y c h r o m a t o g r a p h y . R i b o s e A r a b i n o s e ( s m a l l ) ( 1 ) C a l c u l a t e d f o r a h e x o s e m o n o p h o s p h a t e e s t e r M.W. 395-(2) H y d r o l y s i s i n I N h y d r o c h l o r i c a c i d . A r a b i n o s e was u s e d f o r t h e s t a n d a r d r e d u c i n g c u r v e . 2 3 -6 0 U O M I N U T E S 1 . R e d u c i n g v a l u e o f t h e h y d r o l y s a t e t o t h e F o l i n - M a l m r o s r e a g e n t e x p r e s s e d a s m i c r o g r a m s . o f a r a b i n o s e . 2 . P h o s p h a t e r e l e a s e e x p r e s s e d a s m i c r o g r a m s o f P . 2 4 . TABLE 4 THE COMPOSITION OF THE COENZYME' FRACTION T o t a l y i e l d o f b a r i u m s a l t s ............. 10 mg. T a k e n f o r a n a l y s i s 10 mg. N i t r o g e n . .• 450 ugm P e n t o s e . 320 ugm P h o s p h o r u s (1) 7 m i n 140 ugm 3 h o u r s 150 ugm T o t a l .......................... 300 ugm R e d u c i n g v a l u e (1) 0 t i m e 0 ugm 7 m i n 412 ugm • TABLE 5 THE-COMPOSITION OF THE HON-PHOSPHORYLATED FRACTION T o t a l y i e l d o f b a r i u m s a l t s 64..0 mg. T a k e n f o r a n a l y s i s . . 32 -5 mg. P e n t o s e 5°0 ugm N i t r o g e n N e g l i g i b l e P h o s p h o r u s (1) <5° ugm R e d u c i n g V a l u e (1) 0 "t»llTlG • • • o o o o a * • 0 • © o o • • o « o » • e a o • o 0 ^00 Ul^JIl 7 m i n 575 ugm 3, h o u r s . 575 ugm (1) H y d r o l y s i s i n I N h y d r o c h l o r i c a c i d . A r a b i n o s e was u s e d f o r t h e s t a n d a r d r e d u c i n g c u r v e . 2 5 . TABLE 6 The c h r o m a t o g r a p h i c i d e n t i f i c a t i o n o f t h e p e n t o s e s u g a r s ' • . r e l e a s e d , d u r i n g 3 h o u r h y d r o l y s i s o f t h e p h o s p h a t e e s t e r s , i n 1 .0N h y d r o c h l o r i c a c i d . Compound E t O H / B o r i c EtOH/HAC Me . E t . K e t o n e T.Amyl/HAC R i b o s e .680 .675 .202 .447 A r a b i n o s e .527 .590 .0815 .311 X y l o s e .563 .647' .111 .375 G l u c o s e .510 .538 .0600 .266 Mannose .552 .601 G a l a c t o s e .468 .542 . . . .296 A.T.P.-A.D.P. F r a c t i o n .680 .676 .202 B a S o l u b l e - .680 . . . .202 A l c o h o l i n s o l u b l e F r a c t i o n .528 . . . .082 D o u b l e S a l t F r a c t i o n .527 .590 .082 .311 A s c e n d i n g c o l u m n s r u n a t 29°C . 2 6 . TABLE 7 F u r t h e r c o n f i r m a t i o n ( s e e T a b l e 6) i n t h e d o u b l e s a l t f r a c t i o n . o f a r a b i n o s e Compound D i s t a n c e m cm. C o l o r A r a b o n i c Ho s p o t • • • G l u c o s e 13.6 G r e e n A r a b i n o s e 16.7 R e d R I b o s e 2 2 . 1 R e d X y l o s e 2 0 . 0 R e d G a l a c t o s e 135.6 G r e e n S o r b o s e 16.2 G r e e n Mannose 1 8 . 0 G r e e n Rhamnose 2 5 . 6 G r e e n R a f f i n o s e . No spot. • • • L y x o s e 1 9 . 6 R e d 2- K e t o G l u c o n i c 9 . 0 2 Deep R e d D o u b l e S a l t F r a c t i o n R e d R e d * D e s c e n d i n g c o l u m n w i t h t e r t . a m y l a l c o h o l -a c e t i c a c i d s o l v e n t . R u n f o r 36 h o u r s a t 29 C. ^ R i b o s e s p o t v e r y weak. 2 7 . TABLE 8 The c h r o m a t o g r a p h i c i d e n t i f i c a t i o n o f a r a b o n i c a c i d . , l a n o l E t h a n o l - A c e t i c . 2 1 , . 3 1 • 3 1 Compound M e t h a n o l - 3 L a c t i c . 6 5 A r a b o n i c (1) . 4 6 O x a l o a c e t i c . 7 7 A r a b i n o s e . 6 3 G l u c o s e • 5 7 G l u c o n i c . . 2 6 2 - k e t o g l u c o n i c • 3 9 G l u c u r o n i c • 5 9 P y r u v i c . 6 6 ©<-ketoglutaric . 5 0 ^ - k e t o g l u c o n i c .24 M a l i c a • • A c o n i t i c • • • C i t r i c .41 N a O x a l a t e • • » R i b o s e . 6 9 X y l o s e . 6 8 L y x o s e . 7 2 G l y c e r i c • 5 2 B a r i u m s o l u b l e -A l c o h o l s o l u b l e . 4 6 F r a c t i o n ' ' . 5 5 . 2 1 , . 3 1 R u n a s t h e s o d i u m s a l t . A r a b o n o l a c t o n e ( i . e . a c i d s o l u -t i o n ) g i v e s a s p o t w i t h a n R/F o f . 7 5 i n e t h a n o l -m e t h a n o l . Two s p o t s a r e u n a v o i d a b l e i n t h e a c i d s o l v e n t . 2 8 . TABLE 9 A s c e n d i n g c h r o m a t o g r a m u s i n g t e r t i a r y b u t a n o l -p i e r i c a c i d . , s o l v e n t . Compound R/F V a l u e S p r a y G l u c o s e - l - P .403, M o l y b d a t e F r u c t o s e - 6 - P .498 M o l y b d a t e 3-Pho s p h o g l y c e r i c .409 A g N 0 3/Ei 2S H e x o s e - d i - P .400 M o l y b d a t e I n o r g a n i c P ( 1 ) .697 M o l y b d a t e ' P h o s p h o g l u c o n i c .806 A g N 0 3 / H 2 S G l u c o s e .406 A.P.A. (2) G l u c u r o n i c • 554 A.P.A. (2) R i b o s e - 1 - P .401 M o l y b d a t e R i b o s e - 5 - P .400 M o l y b d a t e B a r i u m S o l u b l e -A l c o h o l I n s o l u b l e .390 M o l y b d a t e B a r i u m I n s o l u b l e .400 M o l y b d a t e ( 1 ) A s 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 . (2) A n i l i n e - p h t h a l i c a c i d d e v e l o p e r . 29. TABLE 10 A s c e n d i n g c h r o m a t o g r a m s u s i n g t h e s o l v e n t s o f C o h e n ( 5 ) • Compound G l u c o s e - l - P 3 - P h o s p h o g l y c e r i c P h o s p h o g l u c o n i c H e x o s e - d i - P I n o r g a n i c P F r u c t o s e - 6 - P G l u c o s e - 6 - P R i b o s e - 1 - P G l u c o s e G l u c u r o n i c B a r i u m s o l u b l e -A l c o h o l i n s o l u b l e B a r i u m i n s o l u b l e R i b o s e - 5 - P A r a b i n o s e - 5 - P 89 E t h a n o 1 - A c e t i c C o h e n I d l e r .118 0 0 .O^O .187 0 0 0 .517 .701 0 0 54 0 55 38 35 E t h a n o l - B o r i c C o h e n I d l e r .119 0 0 0 0 0 0 0 0 0 .519 .520 0 0 0 0 .25 29-a. SUIMARY. 1 . A m o d i f i e d b a r i u m f r a c t i o n a t i o n p r o c e d u r e i s p r e s e n t -e d f o r i s o l a t i n g t h e t r i c h l o r o a c e t i c a c i d s o l u b l e p h o s p h a t e e s t e r s f r o m t h e c e l l s , o f P. A e r u g i n o s a . 2. The a d e n o s i n e p h o s p h a t e s y s t e m i s shown t o b e p r e s e n t . 3 . P h o s p h o g l y c e r i c a c i d a nd h e x o r s e - d i - p h o s p h a t e a r e ab-s e n t . E v i d e n c e i s p r e s e n t e d t o show t h a t g l u c o s e - 6 - p h o s -h a t e i s p r o b a b l y n o t p r e s e n t a n d n e i t h e r p h o s p h o p y r u v i c a c i d n o r t h e o t h e r t r i o s e p h o s p h a t e s o c c u r i n d e t e c t a b l e c o n c e n t r a t i o n s . T h e s e d a t a a l l p o i n t t o a p h o s p h o r y l a t i o n m e c h a n i s m w h i c h d o e s n o t i n v o l v e a n I m b d e n - M e y e r h o f s y s t e m . 4. O t h e r t h a n t h e coenzyme s y s t e m s t h e r e a r e no n i t r o -g e n o u s compounds p r e s e n t . 5. A l l f r a c t i o n s c o n t a i n p h o s p h a t e e s t e r s w h i c h h a v e r e -d u c i n g v a l u e s b e f o r e h y d r o l y s i s . The r e d u c i n g v a l u e s , o n h y d r o l y s i s w i t h n o r m a l h y d r o c h l o r i c a c i d , p a r a l l e l t h e r e l e a s e o f i n o r g a n i c p h o s p h a t e . 6. E v e r y b a r i u m f r a c t i o n c o n t a i n s a l a r g e p e r c e n t a g e o f x n o n - pho s p h o r y l a t e d compounds. T h e s e compounds a r e not. ' r e d u c i n g t o t h e F o l i n - M a l m r o s r e a g e n t . 7. S e v e r a l s o l v e n t s h a v e b e e n f o u n d s a t i s f a c t o r y f o r t h e c h r o m a t o g r a p h y o f s u g a r s , p a r t i c u l a r l y p e n t o s e s . 8. The p r e s e n c e o f an a r a b i n o s e p h o s p h a t e i s e s t a b l i s h e d t»y p a p e r c h r o m a t o g r a p h y . A r a b o n i c a c i d i s a l s o i d e n t i f i e d b u t t h e c h r o m a t o g r a p h i c e v i d e n c e i s n o t c o n s i d e r e d t o b e c o n c l u s i v e . The i m p o r t a n c e o f t h e s e two compounds i s d i s -c u s s e d i n t h e l i g h t o f t h e p r e s e n t k n o w l e d g e o f g l u c o s e 29-t>. "breakdown b y P. A e r u g i n o s a . A - n e w d e v e l o p e r i s p r e s e n t e d f o r u s e i n t h e c h r o m a -t o g r a p h y o f c e r t a i n p h o s p h a t e e s t e r s . 30. INTRODUCTION I t h a s l o n g b e e n o b s e r v e d i n t h i s l a b o r a t o r y t h a t a n i n s o l u b l e gum i s f o r m e d d u r i n g t h e e a r l y s t a g e s o f t h e g r o w t h o f P. A e r u g i n o s a o n a g l u c o s e - m i n e r a l s a l t s m e d i a . The gum i s n o t p r e s e n t i n o l d e r c u l t u r e s a n d i t w o u l d a p p e a r t h a t t h e o r g a n i s m h a s t h e a b i l i t y t o u s e t h i s m a t e r i a l a s a c a r b o n s o u r c e a f t e r t h e g l u c o s e h a s b e e n d e p l e t e d . Ney (23) n o t e d t h e p r e s e n c e o f a gum when t h e o r g a n -i s m was g r o w n o n a g l u c o s e - ammonium s u c c i n a t e m e d i a . She f o u n d t h a t t h e s u b s t i t u t i o n o f ammonium c h l o r i d e f o r ammonium s u c c i n a t e r e d u c e d * t h e amount o f gum t o a c e r t a i n e x t e n t , a n d a l s o t h a t gum p r o d u c t i o n was e n h a n c e d b y p h e n y l a l a n i n e . K i n g ( 1 4 ) n o t e d t h a t a s u l f a t e c o n c e n t r a t i o n g r e a t e r t h a n 0.5% c o n t r i b u t e d t o t h e f o r m a t i o n o f a gum when c e l l s w e r e g r o w n o n ammonium s u c c i n a t e a n d g l y c e r o l . H yde ( 1 1 ) a l s o f o u n d t h a t s u l f a t e v/as n e c e s s a r y f o r gum f o r m a t i o n . N o r r i s ( 2 4 ) h a s f o u n d t h a t t h e w e i g h t o f gum p r o d u c e d p e r u n i t w e i g h t o f c e l l s d e c r e a s e d w i t h i n c r e a s i n g s u l f a t e f r o m 0 . 0 0 1 % t o 0.5% s u l f a t e . I n t e r m s o f gum w e i g h t p e r l i t r e o f m e d i a t h e y i e l d o f gum was f o u n d t o i n c r e a s e w i t h i n c r e a s i n g s u l f a t e o v e r t h e r a n g e i n d i c a t e d . F r o m t h e s e d a t a i t w o u l d a p p e a r t h a t t h e p r o b a b l e e f f e c t o f i n c r e a s e d s u l f a t e i s t o s t i m u l a t e g r o w t h a n d n o t gum p r o d u c t i o n a s p r e v i o u s l y s u g g e s t e d . The p r e s e n t w o r k v/as u n d e r t a k e n i n an e f f o r t t o e s -t a b l i s h t h e c h e m i c a l c o m p o s i t i o n o f t h e gum. 3 1 . EXPERIMENTAL The m e d i a was p r e p a r e d a s d e s c r i b e d i n P a r t 1. The c e l l s w e r e g r o w n - a t 3 0 ° C f o r 22 h o u r s . The gum s e t t l e d t o t h e b o t t o m o f t h e R o u x f l a s k s a n d t h e c e l l s w e r e d e c a n t e d . C e l l s s t i l l t r a p p e d i n t h e gum w e r e r e m o v e d b y r e p e a t e d f i l t r a t i o n t h r o u g h No. 1 Whatman p a p e r , f o l l o w e d b y l o w s p e e d c e n t r i f u g a t i o n . To r e m o v e i n o r g a n i c s a l t s , t h e gum was s u s p e n d e d i n 0.5N H C I , g r o u n d i n a m o r t a r , t h e n f i l t e r e d . T h e s e p r o c e d u r e s w e r e r e p e a t e d u n t i l m i c r o -s c o p i c e x a m i n a t i o n i n d i c a t e d t h e gum was f r e e o f b a c t e r i a l c o n t a m i n a t i o n . The gum was w a s h e d w i t h a c e t o n e a n d e t h e r t h e n d r i e d i n v a c u o a t 5Q°C. The y i e l d was 80 mg. o f d r y gum p e r 10 l i t r e s o f m e d i a . U r o n i c a c i d was d e t e r m i n e d b y t h e m i c r o m e t h o d o f B u r k -h a r t , e t a l ( 1 ). P h o s p h a t e was d e t e r m i n e d b y t h e m e t h o d o f L o w r y a n d L o p e z ( 2 0 ). R e d u c i n g v a l u e s w e r e d e t e r m i n e d a c c o r d i n g t o F o l i n a nd M a l m r o s ( 1 0 ) . F r u c t o s e was d e t e r m i n e d b y t h e p r o -c e d u r e o f Roe ( 2 6 ) . Amino a c i d COg was d e t e r m i n e d b y t h e n i n -h y d r i n m i c r o m e t h o d o f V a n S l y k e e t a l l (33) • The c o n s t i t u e n t amino a c i d s w e r e i d e n t i f i e d b y p a p e r c h r o m a t o g r a p h y , e m p l o y i n g b o t h a s c e n d i n g a n d d e s c e n d i n g c o l u m n s . P u r i f i c a t i o n o f t h e p a p e r a n d t h e c h r o m a t o g r a p h i c t e c h n i q u e s w e r e i d e n t i c a l t o t h o s e u s e d i n P a r t 1. C h r o m a t o -g r a m s w e r e d e v e l o p e d w i t h a 0.1% w/v/ s o l u t i o n o f n i n h y d r i n i n n - b u t y l a l c o h o l . 3 2 . TABLE 1 C o n s t i t u e n t s o f t h e gum s y n t h e s i z e d b y P. A e r u g i n o s a Amino a c i d s a s CO2 9«25% U r o n i c A c i d 31.4% P h o s p h o r u s ( a s PO^) 13 .2% F r u c t o s e p o s . I o d i n e r e a c t i o n n e g . Amino A c i d s G l u t a m i c S e r i n e A l a n i n e 33-RESULTS AND DISCUSSION T a b l e 1 o u t l i n e s t h e c h e m i c a l c o n s t i t u e n t s o f t h e gum. The gum was e x t r e m e l y d i f f i c u l t t o h y d r o l y s e ( F i g . 1) a n d f o r t h i s r e a s o n no a t t e m p t v/as made t o q u a n t i t a t i v e l y d e t e r m i n e f r u c t o s e . The p o s i t i v e S e l e w i n o f f t e s t was o b -t a i n e d o n a 3-1/2 h o u r I N HC1 h y d r o l y s a t e . A t t e m p t s t o c o n -f i r m f r u c t o s e b y c h r o m a t o g r a p h y f a i l e d b u t t h i s v/as p r o b a b l y d u e to, t h e r a t e o f d e s t r u c t i o n o n t h e p r o l o n g e d h y d r o l y s i s . The r e l e a s e o f t h e component amino a c i d s v/as f o l l o w e d c h r o m a t o g r a p h i c a l l y . When no f u r t h e r c h a n g e i n t h e compon-e n t s was o b s e r v e d , t h e amino a c i d s w e r e i d e n t i f i e d a n d t h e V a n S l y k e d e t e r m i n a t i o n made. F i g u r e 1 g i v e s t h e h y d r o l y s i s d a t a o n t h e gum. I t w i l l be o b s e r v e d t h a t t h e u r o n i c a c i d d e c o m p o s i -t i o n p a r a l l e l s t h e r e l e a s e o f i n o r g a n i c p h o s p h a t e . The r e d u c i n g v a l u e o f t h e h y d r o l y s a t e , h o w e v e r , r e a c h e s a maximum w h e r e t h e p h o s p h a t e r e l e a s e a n d u r o n i c a c i d d e c o m p o s i t i o n c u r v e s b e g i n t o l e v e l out-. The e x p l a n a t i o n f o r t h i s l i e s i n t h e m i c r o r n e t h o d e m p l o y e d . The c o n d e n s o r o n t h e a p p a r a t u s v/as v e r y s m a l l a n d w h e r e a s t h e p e r c e n t a g e l o s s o f f u r f u r a l was n o t g r e a t i n t h e l a t e r s t a g e s , a p o i n t was r e a c h e d v/hen t h e a b s o l u t e l o s s o n p r o l o n g e d h y d r o l y s i s e q u a l l e d t h e s m a l l amount b e i n g r e l e a s e d . T a b l e 2 g i v e s t h e c h r o m a t o g r a p h i c d a t a o n t h e compon-e n t amino a c i d s . 34-. T h a t t h e amino a c i d s , s e r i n e , g l u t a m i c a n d a l a n i n e a r e a c t u a l c o m p o n e n t s o f t h e gum a n d n o t c e l l c o n t a m i n a n t s i s e v i d e n c e d b y t h e d a t a o f B i s s e t t ( 2 ) . She i d e n t i f i e d 20 amino a c i d s p r e s e n t a s n o r m a l c o n s t i t u e n t s o f t h e c e l l . 35-FIGURE 1 H y d r o l y s i s o f t h e gum i n 4.0 N h y d r o c h l o r i c a c i d . H O U R S 1. P h o s p h a t e r e l e a s e e x p r e s s e d a s P. 2. R e d u c i n g v a l u e o f t h e h y d r o l y s a t e t o t h e F o l i n -M a l m r o s r e a g e n t e x p r e s s e d a s a r a b i n o s e . 3. U r o n i c a c i d d e c o m p o s i t i o n m e a s u r e d a s c a r b o n d i o x i d e l i b e r a t e d . 3 6 . TABLE 2 The c h r o m a t o g r a p h i c i d e n t i f i c a t i o n o f g l u t a m i c a c i d , s e r i n e a n d a l a n i n e a s c o m p o n e n t s o f t h e gum, P h e n o l / H 2 0 B u t a n o l / H 2 0 Amino A c i d D e s c e n d i n g A s c e n d i n g S s p a r t i c . 11 A r g i n i n e .50 .10 A l a n i n e .52 .20 C y s t i n e .10 G l y c i n e .31 G l u t a m i c .17 H i s t i d i n e .62 I s o l e u c i n e .78 L y s i n e . 39 M e n t h i o n i n e .73 N o r l e u c i n e .81 P r o l i n e .84 P h e n y l a l a n i n e .83 S e r i n e .24 T y r o s i n e No s p o t T r y p t o p h a n e .68 V a l i n e .72 Gum h y d r o l y s a t e . 5 2 , .17, .24 .20 37. SUMMARY The i n s o l u b l e gum p r o d u c e d b y P. A e r u g i n o s a A.T.C. 9027 i s shown t o b e composed o f u r o n i c a c i d , p h o s p h o r u s , g l u t a m i c a c i d , s e r i n e , a l a n i n e a n d f r u c -t o s e . Q u a n t i t a t i v e d a t a i s p r e s e n t e d f o r a l l com-p o n e n t s e x c e p t f r u c t o s e . 3? REFERENCES (1) B u r k h a r t , B a u r a n d L i n k , J . B i o l . C h e m . 1 0 4 : 171 (1934). (2) B i s s e t t , T h e s i s f o r B.S.A. d e g r e e . D e p t . o f D a i r y i n g . U.B.C. 1950. (3) C a m p b e l l a n d N o r r i s , C an .J.Research.C T 2 8 : 203 (1950). (4) C a m p b e l l a n d N o r r i s , C a n . J . R e s e a r c h . C . 2 7 : 165 (1949). (5) C o h e n a n d S c o t t , S c i e n c e ^: 543 (1950). (6) C o r i a n d C o r i , J . B i o l . C h e m . 94: 561 (1932). (7) D i c k e n s , B i o c h e m . J . 3 2 : 1626 (1938). (8) D u b o i s a n d P o t t e r , J . B i o l . C h e m . 1 4 7 : 4 1 (1943). (9) D o u d o r o f f , Wiame a n d Wo l o chow, J . B a c t . 57: 423 (1949) (10) F o l i n a n d M a l m r o s , J . B i o l . C h e m . 8 3 : 115 (1929(-(11) H y d e , T h e s i s f o r B.S.A. d e g r e e . D e p t . o f D a i r y i n g . U.B.C. 1 9 4 8 . (12) I s h e r w o o d a n d H a n e s , N a t u r e 1 6 4 : 1107 (1949). (13) J e r m y n a n d I s h e r w o o d , B i o c h e m . J . 4 4 : 4 0 2 1949). ( 1 4 ) King;, T h e s i s f o r B.S.A. d e g r e e . D e p t . o f D a i r y i n g . . .. . U.B.C. 1948. (15) K r e b s a n d J o h n s o n , E n z y m o l o g l a 4 : 1 4 8 ' (1937) • (16) K r e b s , A dv. i n E n z . 3 : ' 191 (1943). (17) L o c k w o o d a n d S t o d o l a , U.S. P a t e n t 2 ,44^.919: (1948) (18) L o c k w o o d , T a b e n k i n a n d Ward, J . B a c t . 4 2 : 51 (1941). (19) Lohman a nd M e y e r h o f , B i o c h e m . Z . 2 7 3 : 60 (1934). (20) L o w r y a n d L o p e z , J . B i o l . C h e m . l 6 2 : 4 2 1 (1946). (21) M e i j b a u m , Z . P h y s i o l - C h e m . 258: 117 (1939). (22) M e y e r h o f , S y m p o sium o n R e s p i r a t o r y E n z y m e s . U n i v e r s i t y o f W i s c o n s i n P r e s s : 3 (1941) (23) Ney, T h e s i s f o r M.S.A. d e g r e e . D e p t . o f D a i r y i n g . U.B.C. (1948)". •37 REFERENCES ( C o n t i n u e d ) ( 2 4 ) N o r r i s , T h e s i s f o r B.S.A. d e g r e e . D e p t . o f D a i r y i n g . U.B.C. 1940. (25) N o r r i s a n d C a m p b e l l , C a n . J . R e s e a r c h . C . 2 7 : 253 (1949) • (26) R o e , J . B i o l . C h e m . 1 0 7 : 15 ( 1 9 3 4 ) . •(27) S a c k s , J . B i o l . C h e m . l 8 l ; 655 ( 1 9 4 9 ) . (28) S h a f f e r and S o m o g y i , J . B i o l . C h e m . 100: 695 ( 1 9 3 3 ) -(29) S u l l i v a n , J . M e d . R e s . 14: 109 ( 1 9 0 5 ) . (30) Sumner and S o m e r s , C h e m i s t r y a n d m e t h o d s o f enzymes. 2nd e d . New Y o r k A c a d e m i c P r e s s . ( 1 9 4 7 ) . (31) U m b r e i t , B u r r i s a n d S t a u f f e r , M a n o m e t r i c t e c h n i q u e s a n d r e l a t e d m e t h o d s f o r t h e s t u d y o f t i s s u e  m e t a b o l i s m . M i n n e a p o l i s . B u r g e s s P u b . C o . 194-5• (32) U m b r e i t a n d L e P a g e , J . B i o l . C h e m . 1 4 7 : 263 ( 1 9 4 3 ) . (33) V a n S l y k e , M a c F a d y e n and H a m i l t o n , J . B i o l . C h e m . 1 4 1 : 671 ( 1 9 4 1 ) . (34) W a r b u r t o n a n d C a m p b e l l , ( U n p u b l i s h e d d a t a ) . 

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