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Glucose-6-phosphate and gluconate-6-phosphate dehydrogenase in worms
VOL. 1 6
(1955)
6z5
SHORT COMMUNICATIONS, PRELIMINARY NOTES
Glucose-6-phosphate and gluconate-6-phosphate dehydrogenase in worms F r o m r e c e n t i n v e s t i g a t i o n s it b e c o m e s clear t h a t t h e h e x o s e - m o n o p h o s p h a t e o x i d a t i v e r o u t e is an i m p o r t a n t a n d w i d e - s p r e a d p a t h w a y of c a r b o h y d r a t e m e t a b o l i s m . This p a t h w a y h a s been d e t e c t e d in s e v e r a l t i s s u e s : m a m m a l i a n o r g a n s 1, a n g i o s p e r m s ~, yeast3, 4 a n d s ome bacteriaS, 8. I t s pr esence in o t h e r p h y l a of t h e a n i m a l a n d p l a n t k i n g d o m ha s n o t y e t been s t u d i e d . Thi s w o u l d be of i n t e r e s t for c o m p a r a t i v e b i o c h e m i s t r y . We s t u d i e d t h e presence of g l u c o s e - 6 - p h o s p h a t e ( G 6 P ) - a n d g l u c o n a t e - 6 - p h o s p h a t e ( G A 6 P ) d e h y d r o g e n a s e in s e v e r a l worms, m o s t of t h e m p a r a s i t i c ones. The l a t t e r were i s o l a t e d from fre s hl y killed, i n f e c t e d a n i m a l s . All t h e w o r m s were w a s h e d in p h y s i o l o g i c a l s a l i ne or d i s t i l l e d w a t e r , susp e n d e d in t w i c e t h e i r w e i g h t o.o 5 M Tris buffer p H 7.46 a n d d i s r u p t e d in a n all-glass h o m o g e n i z e r in t h e cold r o o m for a few m i n u t e s u n t i l a u n i f o r m p a s t e w a s o b t a i n e d . Some species of w o r m s were too h a r d to be c o m p l e t e l y c r u s h e d b y t h i s p r o c e d u r e a n d were p r e v i o u s l y t r e a t e d in a m i c r o - b l e n d o r or c r u s h e d b y g r i n d i n g w i t h sand. E x t r a c t i o n p r o c e e d e d a l w a y s w i t h Tris buffer a t o ° for 3 ° m i n u t e s . The t u r b i d m a s s w a s c e n t r i f u g e d a t o ° a t 5ooo g for 1-2 h ours a n d t h e c l e a r or o p a l e s c e n t s u p e r n a t a n t was u sed a t once for t h e e n z y m e d e t e r m i n a t i o n s . The d e h y d r o g e n a s e a c t i v i t y d e t e r m i n a t i o n s were c a r r i e d o u t a t r o o m t e m p e r a t u r e (22 ° C) in the B e c k m a n s p e c t r o p h o t o l n e t e r m o d e l D U a t 34 ° m/~. The e n z y m e m i x t u r e c o n t a i n e d o.o 4 M Tris buffer p H 7.46, o.o2 M Mg ++ o.3o # m o l e TPN+, 2 / , m o l e s o d i u m g l u c o n a t e - 6 - p h o s p h a t e or 4.5 # m o l e s o d i u m g l u e o s e - 6 - p h o s p h a t e a n d o.i m l s u p e r n a t a n t ; final v o l u m e 3.1 ml. The r e s u l t s are collected in T a b l e I. TABLE I The a c t i v i t y of s o l u b l e g l u c o s e - 6 - p h o s p h a t e - a n d g l u c o n a t e - 6 - p h o s p h a t e - d e h y d r o g e n a s e s in s e v e r a l wor ms, e x p r e s s e d as e n z y m e u n i t s per g r a m l i v i n g m a t e r i a l (a u n i t is t h e a m o u n t of e n z y m e able to r e d u c e 1 # m o l e T P N + per m i n u t e in t h e c o n d i t i o n s of our e x p e r i m e n t s ) a n d c o r r e c t e d for t h e b l a n k value. TPN
DPN
Organisms G6P
GA6P
G6P
GA6P
o. 185 o.o5o
o.o5o o.o25
o o
o o
o.o7o-o.17o o.ioo
o-0.035 0.065
o o
o o
o.3oo-1.53o o.o8o-o.lo 5 0.04 ° o.2oo
o.12o-o.24o 0-0.02o 0.025 0.075
o o
o o
o
o
o o.2oo
o o.Ioo
o
o
0.20o-0.270 o.5oo-1.6oo
o,o9o-o.loo o,12o-o.23o
o.o15 o
o o
o.690 o.83o o.7IO o.195 o.300 O.lO6 o.o65-O.lOO
0.240 0.230 0.430 o.13o o.125 0.034 o.o3o-o.o45
o.i i o O.Ol 5 o o o.020** o o
o o o o o o o
I. P l a t y h e l m i n t h e s Free-living: planarians
Polycelis nigra Euplanaria torva Parasitic : trematodes Fasciola hepatica (cattle)
Dicrocoelium dendriticurn (sheep) P a r a s i t i c : cesto des
Anoplocephala per[oliata (horse) Moniezia benedeni (sheep) Taenia saginata* (man) Taenia pisi[ormis (dog) Echinococcus granulosus ( h y d a t i d c y s t s from horse-liver)
Dipylidium caninum (dog) II. Parasitic nematodes
.4 scaris lumbricoides (pig) ( d e r m o - m u s c u l a r layer) (female r e p r o d u c t i v e s y s t e m ) Parascaris equorum (horse) ( d e r m o - m u s c u l a r layer) (female r e p r o d u c t i v e s y s t e m ) Toxacara canis (dog) Ascaridia galli (fowl) Ascaridia columbae (pigeon) Heterakis gallinae (fowl) Strongylus edentatus (horse) III. Annelida
Tubilex tubi/ex Lumbricus terrestris
0.260 0.580
0.055** o.19o
o o
o o
* o b t a i n e d from an i n d i v i d u a l , b y a n t h e l m i n t h i c a l t r e a t m e n t w i t h r i c i n u s oil a n d p u m p k i n seed. ** t h e r e a c t i o n s t o p p e d before c o m p l e t e r e d u c t i o n w a s o b t a i n e d .
616
SHORT COMMUNICATIONS, PRELIMINAR'.r NOTES
VOL. 16 (Iq55)
It a p p e a r s t h a t a G 6 P - d e h y d r o g e n a s e is p r e s e n t in all species, e x c e p t in t h e larval s t a g e ol
]:.chznococcus. This e n z y m e is T P N - l i n k e d . In t h e few cases where a weak reaction with I)PN ~ was obtained, this n l u s t p r o b a b l y be a t t r i b u t e d to t h e fact t h a t G 6 P is also deconlposed b y w a y of the E m b d e n - M e y e r h o f glycolytic p a t h w a y . G A 6 P - d e h y d r o g e n a s e is also p r e s e n t in all species, except in Echiuococcus-larvae. I t is specifically T P N - l i n k e d a n d always less active t h a n G()P-dehydrogenase. T h e a n l o u n t of both e n z y m e s is relatively lo~x in p l a n a r i a n s a n d t r e m a t o d e s a n d v e r y variable a m o n g cestodes. A mzelida a n d especially parasitic n e m a t o d e s h a v e a h i g h e r c o n t e n t of b o t h d e h y d r o g e n a s e s . T h e e n z y m e c o n t e n t in t h e latter g r o u p is c o m p a r a b l e to t h a t of t h e b a c t e r i u m Aerobacte~, cloacae, which is k n o w n to h a v e an active s y s t e m of t h e h e x o s e - m o n o p h o s p h a t e o x i d a t i v e routeS: these hacteria c o n t a i n a b o u t 1.2 u n i t s soluble ( ; 6 P - d e h y d r o g e n a s e a n d a b o u t o. 3 u n i t s of soluble G A 6 P d e h y d r o g e n a s e per granl living m a t e r i a P . I n a s y s t e m c o n t a i n i n g o.05 M Tris buffer p H 7.46, 35 # m o l e s o d i u m g l u c o n a t e - 6 - p h o s p h a t e , o . 8 / , n l o l e T P N + , 4 4 / t m o l e p y r u v a t e , 4 ° u n i t s r a b b i t - m u s c l e lactic d e h y d r o g e n a s e a n d o.2 u n i t s G A 6 P - d e h y d r o g e n a s e f r o m t h e female genital t r a c t of Ascaris lumb~'icoides, s e d o h e p t u l o s e was formed, a n d d e t e c t e d b y p a p e r c h r o n m t o g r a t ) h y . T h e s e r e s u l t s s h o w t h a t a n u n l b e r of worms, free living a n d parasitic, c o n t a i n t h e e n z y m e s y s t e m for t h e h e x o s e - n l o n o p h o s p h a t e o x i d a t i v e route, or at least a closely related one. One of us (J.D.L.) is i n d e b t e d to t h e N a t i o n a a l F o n d s voor \ ¥ e t e n s c h a p p e l i j k O n d e r z o e k for a grant. R. VERCRUYSSE iS i n d e b t e d to Prof. MASSARa" for w o r k i n g facilities. J. DE LEY Laboratory/or Physiological Chemistry and Parasitological Department, R. VERCRUYSSE
Veterinary College, Stale Umversity, Ghent (Belgium) I G. E. GLOCK AN> 1'. MCLEAN, Biochem. J., 56 (i954) ~71 • 2 B. AXELROD et al., J. Biol. Chem., 202 (1953) 619. a F. DICKENS, Biochem. J., 32 (1938) 1626, 1645. 4 B. L. HORECKER et al., J. Biol. Chem., 193 (1951) 383 . 5 S. S. COHEN in W. MCELROV AND B. GLASS, Phospho*'us 3letabolis,m, I, Baltimore, 1951 , p. 148. 6 j . DE LEY, Enzymologia, 16 (1953) [4, 00. 7 j . DE LEY, u n p u b l i s h e d results, Received J a n u a r y 28th, 1955
Enzymic synthesis of guanosine and cytidine triphosphates: A note on the nucleotide specificity of the pyruvate phosphokinase reaction T h e r e c e n t isolation of 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 s of uridine, c y t i d i n e a n d g u a n o s i n e raises t h e q u e s t i o n of t h e e x a c t nucleotide specificity of a n u m b e r of e n z y m e s w h i c h h a v e been s t u d i e d e m p l o y i n g u s u a l l y adenosine, a n d s o m e t i m e s inosine, nucleotides. P y r u v a t e p h o s p h o k i n a s e , t h e e n z y m e c a t a l y z i n g t h e t r a n s f e r of p h o s p h a t e f r o m p h o s p h o p y r u v a t e to a nucleoside d i p h o s p h a t e , h a s a l r e a d y been s h o w n to be active w i t h A D P * , I D P a n d U D P 1-3. I t h a s n o w been f o u n d t h a t p h o s p h o r y l a t i o n of G D P a n d C D P is also c a t a l y z e d b y this e n z y m e . I n t h i s s t u d y p y r u v a t e p h o s p h o k i n a s e was p r e p a r e d from r a b b i t m u s c l e as described b y KOm~BERG AND PRICER 4. T h e m a t e r i a l o b t a i n e d b y this p r e p a r a t i o n a p p r o x i m a t e d in p u r i t y t h e m a t e r i a l r e p o r t e d b y t h e m . A c t i v i t y was m e a s u r e d b y coupling t h e r e a c t i o n to lactic d e h y d r o g e n a s e 4. T h e s u i t a b i l i t y of a nucleotide as a n acceptor in this reaction was, therefore, o b s e r v e d as t h e o x i d a t i o n of r e d u c e d d i p h o s p h o p y r i d i n e nucleotide ( D P N H ) m e a s u r e d a t 34 ° m # , in t h e coupled reaction. P h o s p h o p y r u v i c acid ( b a r i u m salt) w a s t h e gift of Mr. W. E. PRICER, Jr., a n d D P N H was p r e p a r e d c h e m i c a l l y w i t h s o d i u m h y d r o s u l f i t e 5. It is believed t h a t each of t h e nucleotides e m p l o y e d w a s free of o t h e r nucleotides, e x c e p t where indicated. W h e r e possible, several i n d e p e n d e n t s a m p l e s of each nucleotide were e m p l o y e d . T h e sources of t h e nucleotides were as follows: A D P , o b t a i n e d from S i g m a Chemical Co., St. Louis; I D P , (i) f r o m S i g m a C h e m i c a l Co., a n d (2) k i n d l y supplied b y Drs. K. KORAblASHI AND M. UTTERS; U D P , (I) p r e p a r e d f r o m U T P ~ ( P a b s t L a b o r a t o r i e s , Milwaukee, Wisconsin) b y i n c u b a t i n g with a c r u d e y e a s t h e x o k i n a s e p r e p a r a t i o n a n d isolating t h e p r o d u c t s b y a n i o n e x c h a n g e c h r o m a t o g r a p h y * * , a n d (2) p r e p a r e d b y h y d r o l y s i s of U D P N - a c e t y l a m i n o s u g a r c o m p o u n d s from Staphylococcus aureusS; G D P , (i) b y h y d r o l y s i s of g u a n o s i n e d i p h o s p h a t e m a n n o s e from h e n ' s o v i d u c t 9, a n d (2) from S i g m a Chemical Co***; * AMP, A D P , ATP, IMP, IDP, ITI', UMP, t h e 5 ' - m o n o - , di- a n d t r i p h o s p h a t e s of adenosine, ** T h i s p r e p a r a t i o n of U D P c o n t a i n e d a b o u t *** B o t h p r e p a r a t i o n s of G D P c o n t a i n e d 5-IO
U D P , U T P , CMP, CDP, CTP, GMP, G D P , G T P are inosine, uridine, c y t i d i n e a n d g u a n o s i n e respectively. 5 % UMP. % GMP.
(1955)
6z5
SHORT COMMUNICATIONS, PRELIMINARY NOTES
Glucose-6-phosphate and gluconate-6-phosphate dehydrogenase in worms F r o m r e c e n t i n v e s t i g a t i o n s it b e c o m e s clear t h a t t h e h e x o s e - m o n o p h o s p h a t e o x i d a t i v e r o u t e is an i m p o r t a n t a n d w i d e - s p r e a d p a t h w a y of c a r b o h y d r a t e m e t a b o l i s m . This p a t h w a y h a s been d e t e c t e d in s e v e r a l t i s s u e s : m a m m a l i a n o r g a n s 1, a n g i o s p e r m s ~, yeast3, 4 a n d s ome bacteriaS, 8. I t s pr esence in o t h e r p h y l a of t h e a n i m a l a n d p l a n t k i n g d o m ha s n o t y e t been s t u d i e d . Thi s w o u l d be of i n t e r e s t for c o m p a r a t i v e b i o c h e m i s t r y . We s t u d i e d t h e presence of g l u c o s e - 6 - p h o s p h a t e ( G 6 P ) - a n d g l u c o n a t e - 6 - p h o s p h a t e ( G A 6 P ) d e h y d r o g e n a s e in s e v e r a l worms, m o s t of t h e m p a r a s i t i c ones. The l a t t e r were i s o l a t e d from fre s hl y killed, i n f e c t e d a n i m a l s . All t h e w o r m s were w a s h e d in p h y s i o l o g i c a l s a l i ne or d i s t i l l e d w a t e r , susp e n d e d in t w i c e t h e i r w e i g h t o.o 5 M Tris buffer p H 7.46 a n d d i s r u p t e d in a n all-glass h o m o g e n i z e r in t h e cold r o o m for a few m i n u t e s u n t i l a u n i f o r m p a s t e w a s o b t a i n e d . Some species of w o r m s were too h a r d to be c o m p l e t e l y c r u s h e d b y t h i s p r o c e d u r e a n d were p r e v i o u s l y t r e a t e d in a m i c r o - b l e n d o r or c r u s h e d b y g r i n d i n g w i t h sand. E x t r a c t i o n p r o c e e d e d a l w a y s w i t h Tris buffer a t o ° for 3 ° m i n u t e s . The t u r b i d m a s s w a s c e n t r i f u g e d a t o ° a t 5ooo g for 1-2 h ours a n d t h e c l e a r or o p a l e s c e n t s u p e r n a t a n t was u sed a t once for t h e e n z y m e d e t e r m i n a t i o n s . The d e h y d r o g e n a s e a c t i v i t y d e t e r m i n a t i o n s were c a r r i e d o u t a t r o o m t e m p e r a t u r e (22 ° C) in the B e c k m a n s p e c t r o p h o t o l n e t e r m o d e l D U a t 34 ° m/~. The e n z y m e m i x t u r e c o n t a i n e d o.o 4 M Tris buffer p H 7.46, o.o2 M Mg ++ o.3o # m o l e TPN+, 2 / , m o l e s o d i u m g l u c o n a t e - 6 - p h o s p h a t e or 4.5 # m o l e s o d i u m g l u e o s e - 6 - p h o s p h a t e a n d o.i m l s u p e r n a t a n t ; final v o l u m e 3.1 ml. The r e s u l t s are collected in T a b l e I. TABLE I The a c t i v i t y of s o l u b l e g l u c o s e - 6 - p h o s p h a t e - a n d g l u c o n a t e - 6 - p h o s p h a t e - d e h y d r o g e n a s e s in s e v e r a l wor ms, e x p r e s s e d as e n z y m e u n i t s per g r a m l i v i n g m a t e r i a l (a u n i t is t h e a m o u n t of e n z y m e able to r e d u c e 1 # m o l e T P N + per m i n u t e in t h e c o n d i t i o n s of our e x p e r i m e n t s ) a n d c o r r e c t e d for t h e b l a n k value. TPN
DPN
Organisms G6P
GA6P
G6P
GA6P
o. 185 o.o5o
o.o5o o.o25
o o
o o
o.o7o-o.17o o.ioo
o-0.035 0.065
o o
o o
o.3oo-1.53o o.o8o-o.lo 5 0.04 ° o.2oo
o.12o-o.24o 0-0.02o 0.025 0.075
o o
o o
o
o
o o.2oo
o o.Ioo
o
o
0.20o-0.270 o.5oo-1.6oo
o,o9o-o.loo o,12o-o.23o
o.o15 o
o o
o.690 o.83o o.7IO o.195 o.300 O.lO6 o.o65-O.lOO
0.240 0.230 0.430 o.13o o.125 0.034 o.o3o-o.o45
o.i i o O.Ol 5 o o o.020** o o
o o o o o o o
I. P l a t y h e l m i n t h e s Free-living: planarians
Polycelis nigra Euplanaria torva Parasitic : trematodes Fasciola hepatica (cattle)
Dicrocoelium dendriticurn (sheep) P a r a s i t i c : cesto des
Anoplocephala per[oliata (horse) Moniezia benedeni (sheep) Taenia saginata* (man) Taenia pisi[ormis (dog) Echinococcus granulosus ( h y d a t i d c y s t s from horse-liver)
Dipylidium caninum (dog) II. Parasitic nematodes
.4 scaris lumbricoides (pig) ( d e r m o - m u s c u l a r layer) (female r e p r o d u c t i v e s y s t e m ) Parascaris equorum (horse) ( d e r m o - m u s c u l a r layer) (female r e p r o d u c t i v e s y s t e m ) Toxacara canis (dog) Ascaridia galli (fowl) Ascaridia columbae (pigeon) Heterakis gallinae (fowl) Strongylus edentatus (horse) III. Annelida
Tubilex tubi/ex Lumbricus terrestris
0.260 0.580
0.055** o.19o
o o
o o
* o b t a i n e d from an i n d i v i d u a l , b y a n t h e l m i n t h i c a l t r e a t m e n t w i t h r i c i n u s oil a n d p u m p k i n seed. ** t h e r e a c t i o n s t o p p e d before c o m p l e t e r e d u c t i o n w a s o b t a i n e d .
616
SHORT COMMUNICATIONS, PRELIMINAR'.r NOTES
VOL. 16 (Iq55)
It a p p e a r s t h a t a G 6 P - d e h y d r o g e n a s e is p r e s e n t in all species, e x c e p t in t h e larval s t a g e ol
]:.chznococcus. This e n z y m e is T P N - l i n k e d . In t h e few cases where a weak reaction with I)PN ~ was obtained, this n l u s t p r o b a b l y be a t t r i b u t e d to t h e fact t h a t G 6 P is also deconlposed b y w a y of the E m b d e n - M e y e r h o f glycolytic p a t h w a y . G A 6 P - d e h y d r o g e n a s e is also p r e s e n t in all species, except in Echiuococcus-larvae. I t is specifically T P N - l i n k e d a n d always less active t h a n G()P-dehydrogenase. T h e a n l o u n t of both e n z y m e s is relatively lo~x in p l a n a r i a n s a n d t r e m a t o d e s a n d v e r y variable a m o n g cestodes. A mzelida a n d especially parasitic n e m a t o d e s h a v e a h i g h e r c o n t e n t of b o t h d e h y d r o g e n a s e s . T h e e n z y m e c o n t e n t in t h e latter g r o u p is c o m p a r a b l e to t h a t of t h e b a c t e r i u m Aerobacte~, cloacae, which is k n o w n to h a v e an active s y s t e m of t h e h e x o s e - m o n o p h o s p h a t e o x i d a t i v e routeS: these hacteria c o n t a i n a b o u t 1.2 u n i t s soluble ( ; 6 P - d e h y d r o g e n a s e a n d a b o u t o. 3 u n i t s of soluble G A 6 P d e h y d r o g e n a s e per granl living m a t e r i a P . I n a s y s t e m c o n t a i n i n g o.05 M Tris buffer p H 7.46, 35 # m o l e s o d i u m g l u c o n a t e - 6 - p h o s p h a t e , o . 8 / , n l o l e T P N + , 4 4 / t m o l e p y r u v a t e , 4 ° u n i t s r a b b i t - m u s c l e lactic d e h y d r o g e n a s e a n d o.2 u n i t s G A 6 P - d e h y d r o g e n a s e f r o m t h e female genital t r a c t of Ascaris lumb~'icoides, s e d o h e p t u l o s e was formed, a n d d e t e c t e d b y p a p e r c h r o n m t o g r a t ) h y . T h e s e r e s u l t s s h o w t h a t a n u n l b e r of worms, free living a n d parasitic, c o n t a i n t h e e n z y m e s y s t e m for t h e h e x o s e - n l o n o p h o s p h a t e o x i d a t i v e route, or at least a closely related one. One of us (J.D.L.) is i n d e b t e d to t h e N a t i o n a a l F o n d s voor \ ¥ e t e n s c h a p p e l i j k O n d e r z o e k for a grant. R. VERCRUYSSE iS i n d e b t e d to Prof. MASSARa" for w o r k i n g facilities. J. DE LEY Laboratory/or Physiological Chemistry and Parasitological Department, R. VERCRUYSSE
Veterinary College, Stale Umversity, Ghent (Belgium) I G. E. GLOCK AN> 1'. MCLEAN, Biochem. J., 56 (i954) ~71 • 2 B. AXELROD et al., J. Biol. Chem., 202 (1953) 619. a F. DICKENS, Biochem. J., 32 (1938) 1626, 1645. 4 B. L. HORECKER et al., J. Biol. Chem., 193 (1951) 383 . 5 S. S. COHEN in W. MCELROV AND B. GLASS, Phospho*'us 3letabolis,m, I, Baltimore, 1951 , p. 148. 6 j . DE LEY, Enzymologia, 16 (1953) [4, 00. 7 j . DE LEY, u n p u b l i s h e d results, Received J a n u a r y 28th, 1955
Enzymic synthesis of guanosine and cytidine triphosphates: A note on the nucleotide specificity of the pyruvate phosphokinase reaction T h e r e c e n t isolation of 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 s of uridine, c y t i d i n e a n d g u a n o s i n e raises t h e q u e s t i o n of t h e e x a c t nucleotide specificity of a n u m b e r of e n z y m e s w h i c h h a v e been s t u d i e d e m p l o y i n g u s u a l l y adenosine, a n d s o m e t i m e s inosine, nucleotides. P y r u v a t e p h o s p h o k i n a s e , t h e e n z y m e c a t a l y z i n g t h e t r a n s f e r of p h o s p h a t e f r o m p h o s p h o p y r u v a t e to a nucleoside d i p h o s p h a t e , h a s a l r e a d y been s h o w n to be active w i t h A D P * , I D P a n d U D P 1-3. I t h a s n o w been f o u n d t h a t p h o s p h o r y l a t i o n of G D P a n d C D P is also c a t a l y z e d b y this e n z y m e . I n t h i s s t u d y p y r u v a t e p h o s p h o k i n a s e was p r e p a r e d from r a b b i t m u s c l e as described b y KOm~BERG AND PRICER 4. T h e m a t e r i a l o b t a i n e d b y this p r e p a r a t i o n a p p r o x i m a t e d in p u r i t y t h e m a t e r i a l r e p o r t e d b y t h e m . A c t i v i t y was m e a s u r e d b y coupling t h e r e a c t i o n to lactic d e h y d r o g e n a s e 4. T h e s u i t a b i l i t y of a nucleotide as a n acceptor in this reaction was, therefore, o b s e r v e d as t h e o x i d a t i o n of r e d u c e d d i p h o s p h o p y r i d i n e nucleotide ( D P N H ) m e a s u r e d a t 34 ° m # , in t h e coupled reaction. P h o s p h o p y r u v i c acid ( b a r i u m salt) w a s t h e gift of Mr. W. E. PRICER, Jr., a n d D P N H was p r e p a r e d c h e m i c a l l y w i t h s o d i u m h y d r o s u l f i t e 5. It is believed t h a t each of t h e nucleotides e m p l o y e d w a s free of o t h e r nucleotides, e x c e p t where indicated. W h e r e possible, several i n d e p e n d e n t s a m p l e s of each nucleotide were e m p l o y e d . T h e sources of t h e nucleotides were as follows: A D P , o b t a i n e d from S i g m a Chemical Co., St. Louis; I D P , (i) f r o m S i g m a C h e m i c a l Co., a n d (2) k i n d l y supplied b y Drs. K. KORAblASHI AND M. UTTERS; U D P , (I) p r e p a r e d f r o m U T P ~ ( P a b s t L a b o r a t o r i e s , Milwaukee, Wisconsin) b y i n c u b a t i n g with a c r u d e y e a s t h e x o k i n a s e p r e p a r a t i o n a n d isolating t h e p r o d u c t s b y a n i o n e x c h a n g e c h r o m a t o g r a p h y * * , a n d (2) p r e p a r e d b y h y d r o l y s i s of U D P N - a c e t y l a m i n o s u g a r c o m p o u n d s from Staphylococcus aureusS; G D P , (i) b y h y d r o l y s i s of g u a n o s i n e d i p h o s p h a t e m a n n o s e from h e n ' s o v i d u c t 9, a n d (2) from S i g m a Chemical Co***; * AMP, A D P , ATP, IMP, IDP, ITI', UMP, t h e 5 ' - m o n o - , di- a n d t r i p h o s p h a t e s of adenosine, ** T h i s p r e p a r a t i o n of U D P c o n t a i n e d a b o u t *** B o t h p r e p a r a t i o n s of G D P c o n t a i n e d 5-IO
U D P , U T P , CMP, CDP, CTP, GMP, G D P , G T P are inosine, uridine, c y t i d i n e a n d g u a n o s i n e respectively. 5 % UMP. % GMP.