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Enzymic synthesis of guanosine and cytidine triphosphates: A note on the nucleotide specificity of the pyruvate phosphokinase reaction
616
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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.
VOL. 16 (I955)
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CDP, p r e p a r e d by t r a n s p h o s p h o r y l a t i o n b e t w e e n A T P a n d C M P *1° a n d isolating t h e p r o d u c t s b y anion e x c h a n g e c h r o m a t o g r a p h y . E a c h of t h e nucleoside d i p h o s p h a t e s e m p l o y e d served as a p h o s p h a t e acceptor in t h e spectrop h o t o m e t r i c test (Fig. i). T h e relative rates of reaction with A D P , GDP, I D P , U D P a n d C D P (each at a c o n c e n t r a t i o n of a b o u t 4' 1°-~ M a n d w i t h p y r u v a t e p h o s p h o k i n a s e limiting) were ioo, 19, 12, q.O0~ 3 a n d 2 respectively. 0 90~
Fig. I. R e a c t i o n of p h o s p h o p y r u v a t e w i t h ADP, GDP and CDP catalyzed by pyruvate phosphokinase. Each cuvette contained the nucleoside dip h o s p h a t e indicated, 0.2 p.'lI p h o s p h o p y r u v a t e , 0 . 0 7 5 / x M D P N H , a n d lactic d e h y d r o g e n a s e in 0. 5 ml. o.I M p o t a s s i u m p h o s p h a t e , 0.004 M MgC12, p H 7.0. A t A, p y r u v a t e p h o s p h o k i n a s e was a d d e d in t h e relative a m o u n t s of i (ADP), 2. 5 (GDP), 12. 5 (CDP) a n d 12.5 (no nucleotide). At B, a n excess of p y r u v a t e p h o s p h o k i n a s e was a d d e d to t h e l a t t e r three. T h e a m o u n t s of nucleoside dip h o s p h a t e a d d e d a n d D P N H oxidized were as follows: 0.05 2 /Z2~r A D P , 0.049/z31 D P N H oxidized; 0.043 ftM G D P , 0.039 113l D P N H oxidized; 0.o53 /tM CDP, o.o49 lzM D P N H oxidized.
0.80~
0.70~ ~a60C "~0.50C
ADp~'I
~ 0.40C
~0.306 0.2000
f s
J 1o
I 15
I
20
I 25
I 30
J 35
P 40
1,5
t'linotes
I n order to d e m o n s t r a t e t h a t t h e c o r r e s p o n d i n g t r i p h o s p h a t e s were f o r m e d in t h e reaction, i n c u b a t i o n m i x t u r e s were electrophoresed in 0.02 M c i t r a t e buffer, p H 5.111. I n each case a nucleotide w a s f o r m e d w h i c h h a d a g r e a t e r m o b i l i t y t h a n t h e nucleoside d i p h o s p h a t e (relative mobilities a b o u t I . i : I). T h e c o m p o u n d s f o r m e d w i t h A D P , I D P , U D P a n d C D P h a d t h e s a m e electrophoretic mobilities as t h e c o r r e s p o n d i n g t r i p h o s p h a t e s * . No s a m p l e of G T P was available for c o m p a r i s o n . I n order to isolate GTP, a larger scale i n c u b a t i o n was carried o u t with 4 8 / z M G D P (Sigma), 8 5 / z M p h o s p h o p y r u v a t e a n d e n z y m e in 30 inl 0.004 M p o t a s s i u m p h o s p h a t e , 0.003 M MgC12, p H 6.9. T h e course of t h e r e a c t i o n w a s followed b y r e m o v i n g aliquots a n d a s s a y i n g for p y r u v a t e w i t h D P N H a n d lactic d e h y d r o g e n a s e . A t t h e c o m p l e t i o n of t h e reaction (3 ° m i n u t e s ) t h e i n c u b a t i o n m i x t u r e was r u n o n t o a c o l u m n of D o w e x - i CI (2 % cross-linked), a n d t h e p r o d u c t s eluted b y g r a d i e n t elution e m p l o y i n g m i x t u r e s of NaC1 in o.oi N tIC1. T w o u l t r a v i o l e t a b s o r b i n g p e a k s were observed. T h e first, eluted a t a b o u t 0.05 M NaC1 in o.oi N HC1, c o n t a i n e d 3 # M of nucleotide. This c o m p o u n d h a d t h e s a m e c h r o m a t o g r a p h i c a n d electrophoretic b e h a v i o r as GMP, a n d was also f o u n d as a n i m p u r i t y in t h e s t a r t i n g material. A second peak, eluted a t a b o u t 0.25 M NaC1 in o.oi N HC1, c o n t a i n e d 3 9 / z M nucleotide. T h e c o m p o u n d recovered f r o m t h i s p e a k b y charcoal a b s o r p t i o n a n d elution h a d a n R F in e t h a n o l - a m m o n i u m a c e t a t e - v e r s e n e 12 s m a l l e r t h a n G D P , a n d a m o b i l i t y in 0.o2 M citrate, p H 5.1, g r e a t e r t h a n G D P . I t c o n t a i n e d 2.8 moles of t o t a l p h o s p h o r u s per mole of guanosine, 1.9 moles of w h i c h were labile in i N HC1 in 15 m i n u t e s . G u a n o s i n e was calculated f r o m t h e 257 m/~ a b s o r p t i o n u s i n g 12,4oo as t h e m o l a r e x t i n c t i o n coefficient. U l t r a v i o l e t a b s o r p t i o n s p e c t r a b e t w e e n 22o a n d 31o m # in o.oi N HC1 a n d o.I N N a O H c o r r e s p o n d e d to t h e acid a n d alkaline s p e c t r a of guanosine. B y t h i s evidence t h e c o m p o u n d h a s been c h a r a c t e r i z e d as g u a n o s i n e t r i p h o s p h a t e * * . Nucleoside diphosphokinasen,14, is w a s a b s e n t f r o m t h e e n z y m e p r e p a r a t i o n s e m p l o y e d . I t is, therefore, believed t h a t t h e several p h o s p h o r y l a t i o n s c a t a l y z e d b y p y r u v a t e p h o s p h o k i n a s e are direct reactions, a n d do n o t involve t h e i n t e r m e d i a c y of c a t a l y t i c a m o u n t s of a d e n o s i n e nucleotides. R e c e n t l y several o t h e r r e a c t i o n s h a v e been f o u n d for p h o s p h o r y l a t i o n of U D P , CDP, I D P , a n d GDP~, 10, n , lS--19. I wish to e x p r e s s m y a p p r e c i a t i o n to Drs. H. M. KALCKAR, L. A. HEPPEL, a n d E. S. MAXWELL for m a n y s t i m u l a t i n g discussions d u r i n g tile p a s t y e a r a n d for a s s i s t a n c e with several of t h e preparations. JACK L. STROMTNGER
National Institute o[ Arthritis and Metabolic Diseases, National Institutes o] Health, Bethesda, Maryland (U.S.A.) * C M P a n d C T P were k i n d l y supplied b y Dr. A. FRIEDEN of t h e P a b s t Laboratories. ** T h e isolated G T P h a s also been f o u n d to r e a c t with A M P in t h e presence of an e n z y m e purified f r o m calf liver w i t h f o r m a t i o n of t w o nucleoside d i p h o s p h a t e s 13.
42
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LOHMANN AND O. MEYERHOF, Biochem. Z., 273 11034) 60. KLEINZELLER, Biochem. J., 36 (1942) 729. KORNBERG, Symposium on Phospllorous Metabolism, 1 (1951) 392. KORNBERG AND W . E. PRICER, Jr., J. Biol. Chem., 193 (I95t) 4,~1. 5 p. OHLMEYER, Biochem. Z., 297 119381 o0. 1 2 3 4
l{. :\. A. A.
6 M. F. [JTTER Anl) K. KURAHASHI, .[. Biol. Chem., 207 (1954) 821. 7 S. H . LIPTON, S. A. MORRELL, A. FRIEDEN AND R . g{. BOCK, ol. Atn. Chem. Hoe., 75 11953) 5449. J. T. PARK, J. Biol. Chem., 194 (1952) 877, 885, 8979 J. L. STROM1NGER, Fed. Proc., 13 119541 307 . 10 J . L. STROMINGER, L. A. H E P P E L AND E. S. MAXWELL, Arch. Biochem. Biophys., 52 (I954) 488. 11 p . BERG AND \~7. K . JOKLIK, J. Biol. Chem., 2IO (1954) 657. z2 E. CABIB AND L. F. LELOIR, J. Biol. Chem., 2o6 (I954) 779. 13 j. L. STROMINGER, I,. A. HEPPEL AND 1'~. S. MAXWI,:LL, Fed. Proc., 14 (i955) ( ill press). ~ H. A. KREBS ANl) R. HEMS, Biochim. Biophys. Acta, 12 119531 17-'. 1.5 D. M. GIBSON, [). AYENGAP, AND D. t°t. SANADI, Abstracts, ~26th Meeting :Ira. Chem. Soc. (S@tember 1954), 4IC. 16 A. MUNC~t-PETERSEN, Acta Chem. Scand., 8 11954) ilO2. iv I. LIEBERMAN, A. KORNBERG AND E . S. SIMMS, J. A1~l. Clle~Jz. Soc., 76 (i9541 36oS. 18 D. R. SANADI, D. M. GIBSON AND P. AYENGAR, Biochim, Biophys. Acta, 14 119541 434. 19 E. IIERBERT, V. R. POTTER AND Y. TAKAGI, J. Biol. Chem., 11955) (in press.). Received J a n u a r y 24th, 1955
Increased liver glucose-6-phosphatase activity after cortisone administration* The behavior of liver glucose-6-phosphatase under physiological and pathological conditions has been u n d e r investigation in this laboratory. Studies of the glucose-O-phosphatase reaction ill normal fasting animals 1, and in precancerous, neoplastic ~-, regenerating, foetal and new born liver a have focussed our a t t e n t i o n on the regulation of this enzymic activity in tissues. I t has been k n o w n t h a t high doses of cortisone resulted in p e r m a n e n t diabetes in r a t '1, characterized by hyperglycemia and glycosuria, hlsulin resistant "cortisone diabetes" showing very high blood sugar levels and glycosuria was also described in h u m a n 5. The increase in tile q u a n t i t y of c a r b o h y d r a t e after cortisone a d m i n i s t r a t i o n has been a t t r i b u t e d to increased gluco-neogenesis and there is also evidence t h a t some phases of the c a r b o h y d r a t e utilization are inhibited 6. Recently however, it was d e m o n s t r a t e d t h a t cortisone a d m i n i s t r a t i o n caused a striking increase in the rate of glucose production v. Since glucose production from the liver is related to the rate of hydrolysis of the glucose-6-phosphate ester, it seemed of interest to s t u d y the glucose-6-phosphatase reactilm in the liver of animals treated with high doses of cortisone. The preliminary results of this investigation are reported here. Y o u n g male W i s t a r rats of zoo grains of weight were injected with 1 ml (25 mg) of cortisone acetate ( Cortone , Merck ) zntramuscularly, dally for five days and were killed by decapitation on the sixth day. Control animals received in lectIon of the vehicle , m which the cortisone is dissolved. Animals were m a i n t a i n e d on Purina F o x Chow and water ad libih¢m until sacrificed. Livers were pooled and the h o m o g e n a t e s were p r e p a r e d for enzymic studies as described in a previous c o m m u n i cation 2. The glucose-6-phosphatase activity was m e a s u r e d b y the m e t h o d of CORI AND CORI8 using a 15 m i n u t e s incubation time. Liver glycogen was determined b y the m e t h o d of GOOD, KRAMER AND SOMOGYI9 employing the NELSON'S a d a p t a t i o n of the SOMOGYI m e t h o d for glucose m. The enzymic activities are expressed per wet w e i g h t nitrogen, per average cell basis and also per liver weight/body x ~ i g h t ratio. Total nitrogen was determined by the micro-Kjeldahl procedure. Nuclear counts were done by the m e t h o d of PRICE AND I,AIRD 10 as modified by ALLARD et aI. 11. 131ood glucose was determined by the m e t h o d of FOLIN-Wu. The preliminary results are s h o w n in Figs. 1 and 2. The results are presented as percentage changes from the control values which are taken arbitrarily as ioo %. Fig. i shows t h a t cortisone injection increased the blood glucose level by 78 %. The well k n o w n glycogenic effect of cortisone on the liver is also illustrated in Fig. i. W h e n liver glycogen is expressed on per cell basis the increase in glycogen c o n t e n t after cortisone administration is higher t h a n when d a t a are expressed on the conventional g r a m per cent wet weight basis. * This project has been s u p p o r t e d b y grants from the Cancer Research Society, Montreal, National Cancer I n s t i t u t e of Canada and D a m o n R u n y o n Memorial F u n d for Cancer Research Inc., U.S.A. ** The cortisone acetate and vehicle was kindly supplied b y Dr. J. H. LAURIE of Merck and Co., Montreal.
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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.
VOL. 16 (I955)
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CDP, p r e p a r e d by t r a n s p h o s p h o r y l a t i o n b e t w e e n A T P a n d C M P *1° a n d isolating t h e p r o d u c t s b y anion e x c h a n g e c h r o m a t o g r a p h y . E a c h of t h e nucleoside d i p h o s p h a t e s e m p l o y e d served as a p h o s p h a t e acceptor in t h e spectrop h o t o m e t r i c test (Fig. i). T h e relative rates of reaction with A D P , GDP, I D P , U D P a n d C D P (each at a c o n c e n t r a t i o n of a b o u t 4' 1°-~ M a n d w i t h p y r u v a t e p h o s p h o k i n a s e limiting) were ioo, 19, 12, q.O0~ 3 a n d 2 respectively. 0 90~
Fig. I. R e a c t i o n of p h o s p h o p y r u v a t e w i t h ADP, GDP and CDP catalyzed by pyruvate phosphokinase. Each cuvette contained the nucleoside dip h o s p h a t e indicated, 0.2 p.'lI p h o s p h o p y r u v a t e , 0 . 0 7 5 / x M D P N H , a n d lactic d e h y d r o g e n a s e in 0. 5 ml. o.I M p o t a s s i u m p h o s p h a t e , 0.004 M MgC12, p H 7.0. A t A, p y r u v a t e p h o s p h o k i n a s e was a d d e d in t h e relative a m o u n t s of i (ADP), 2. 5 (GDP), 12. 5 (CDP) a n d 12.5 (no nucleotide). At B, a n excess of p y r u v a t e p h o s p h o k i n a s e was a d d e d to t h e l a t t e r three. T h e a m o u n t s of nucleoside dip h o s p h a t e a d d e d a n d D P N H oxidized were as follows: 0.05 2 /Z2~r A D P , 0.049/z31 D P N H oxidized; 0.043 ftM G D P , 0.039 113l D P N H oxidized; 0.o53 /tM CDP, o.o49 lzM D P N H oxidized.
0.80~
0.70~ ~a60C "~0.50C
ADp~'I
~ 0.40C
~0.306 0.2000
f s
J 1o
I 15
I
20
I 25
I 30
J 35
P 40
1,5
t'linotes
I n order to d e m o n s t r a t e t h a t t h e c o r r e s p o n d i n g t r i p h o s p h a t e s were f o r m e d in t h e reaction, i n c u b a t i o n m i x t u r e s were electrophoresed in 0.02 M c i t r a t e buffer, p H 5.111. I n each case a nucleotide w a s f o r m e d w h i c h h a d a g r e a t e r m o b i l i t y t h a n t h e nucleoside d i p h o s p h a t e (relative mobilities a b o u t I . i : I). T h e c o m p o u n d s f o r m e d w i t h A D P , I D P , U D P a n d C D P h a d t h e s a m e electrophoretic mobilities as t h e c o r r e s p o n d i n g t r i p h o s p h a t e s * . No s a m p l e of G T P was available for c o m p a r i s o n . I n order to isolate GTP, a larger scale i n c u b a t i o n was carried o u t with 4 8 / z M G D P (Sigma), 8 5 / z M p h o s p h o p y r u v a t e a n d e n z y m e in 30 inl 0.004 M p o t a s s i u m p h o s p h a t e , 0.003 M MgC12, p H 6.9. T h e course of t h e r e a c t i o n w a s followed b y r e m o v i n g aliquots a n d a s s a y i n g for p y r u v a t e w i t h D P N H a n d lactic d e h y d r o g e n a s e . A t t h e c o m p l e t i o n of t h e reaction (3 ° m i n u t e s ) t h e i n c u b a t i o n m i x t u r e was r u n o n t o a c o l u m n of D o w e x - i CI (2 % cross-linked), a n d t h e p r o d u c t s eluted b y g r a d i e n t elution e m p l o y i n g m i x t u r e s of NaC1 in o.oi N tIC1. T w o u l t r a v i o l e t a b s o r b i n g p e a k s were observed. T h e first, eluted a t a b o u t 0.05 M NaC1 in o.oi N HC1, c o n t a i n e d 3 # M of nucleotide. This c o m p o u n d h a d t h e s a m e c h r o m a t o g r a p h i c a n d electrophoretic b e h a v i o r as GMP, a n d was also f o u n d as a n i m p u r i t y in t h e s t a r t i n g material. A second peak, eluted a t a b o u t 0.25 M NaC1 in o.oi N HC1, c o n t a i n e d 3 9 / z M nucleotide. T h e c o m p o u n d recovered f r o m t h i s p e a k b y charcoal a b s o r p t i o n a n d elution h a d a n R F in e t h a n o l - a m m o n i u m a c e t a t e - v e r s e n e 12 s m a l l e r t h a n G D P , a n d a m o b i l i t y in 0.o2 M citrate, p H 5.1, g r e a t e r t h a n G D P . I t c o n t a i n e d 2.8 moles of t o t a l p h o s p h o r u s per mole of guanosine, 1.9 moles of w h i c h were labile in i N HC1 in 15 m i n u t e s . G u a n o s i n e was calculated f r o m t h e 257 m/~ a b s o r p t i o n u s i n g 12,4oo as t h e m o l a r e x t i n c t i o n coefficient. U l t r a v i o l e t a b s o r p t i o n s p e c t r a b e t w e e n 22o a n d 31o m # in o.oi N HC1 a n d o.I N N a O H c o r r e s p o n d e d to t h e acid a n d alkaline s p e c t r a of guanosine. B y t h i s evidence t h e c o m p o u n d h a s been c h a r a c t e r i z e d as g u a n o s i n e t r i p h o s p h a t e * * . Nucleoside diphosphokinasen,14, is w a s a b s e n t f r o m t h e e n z y m e p r e p a r a t i o n s e m p l o y e d . I t is, therefore, believed t h a t t h e several p h o s p h o r y l a t i o n s c a t a l y z e d b y p y r u v a t e p h o s p h o k i n a s e are direct reactions, a n d do n o t involve t h e i n t e r m e d i a c y of c a t a l y t i c a m o u n t s of a d e n o s i n e nucleotides. R e c e n t l y several o t h e r r e a c t i o n s h a v e been f o u n d for p h o s p h o r y l a t i o n of U D P , CDP, I D P , a n d GDP~, 10, n , lS--19. I wish to e x p r e s s m y a p p r e c i a t i o n to Drs. H. M. KALCKAR, L. A. HEPPEL, a n d E. S. MAXWELL for m a n y s t i m u l a t i n g discussions d u r i n g tile p a s t y e a r a n d for a s s i s t a n c e with several of t h e preparations. JACK L. STROMTNGER
National Institute o[ Arthritis and Metabolic Diseases, National Institutes o] Health, Bethesda, Maryland (U.S.A.) * C M P a n d C T P were k i n d l y supplied b y Dr. A. FRIEDEN of t h e P a b s t Laboratories. ** T h e isolated G T P h a s also been f o u n d to r e a c t with A M P in t h e presence of an e n z y m e purified f r o m calf liver w i t h f o r m a t i o n of t w o nucleoside d i p h o s p h a t e s 13.
42
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LOHMANN AND O. MEYERHOF, Biochem. Z., 273 11034) 60. KLEINZELLER, Biochem. J., 36 (1942) 729. KORNBERG, Symposium on Phospllorous Metabolism, 1 (1951) 392. KORNBERG AND W . E. PRICER, Jr., J. Biol. Chem., 193 (I95t) 4,~1. 5 p. OHLMEYER, Biochem. Z., 297 119381 o0. 1 2 3 4
l{. :\. A. A.
6 M. F. [JTTER Anl) K. KURAHASHI, .[. Biol. Chem., 207 (1954) 821. 7 S. H . LIPTON, S. A. MORRELL, A. FRIEDEN AND R . g{. BOCK, ol. Atn. Chem. Hoe., 75 11953) 5449. J. T. PARK, J. Biol. Chem., 194 (1952) 877, 885, 8979 J. L. STROM1NGER, Fed. Proc., 13 119541 307 . 10 J . L. STROMINGER, L. A. H E P P E L AND E. S. MAXWELL, Arch. Biochem. Biophys., 52 (I954) 488. 11 p . BERG AND \~7. K . JOKLIK, J. Biol. Chem., 2IO (1954) 657. z2 E. CABIB AND L. F. LELOIR, J. Biol. Chem., 2o6 (I954) 779. 13 j. L. STROMINGER, I,. A. HEPPEL AND 1'~. S. MAXWI,:LL, Fed. Proc., 14 (i955) ( ill press). ~ H. A. KREBS ANl) R. HEMS, Biochim. Biophys. Acta, 12 119531 17-'. 1.5 D. M. GIBSON, [). AYENGAP, AND D. t°t. SANADI, Abstracts, ~26th Meeting :Ira. Chem. Soc. (S@tember 1954), 4IC. 16 A. MUNC~t-PETERSEN, Acta Chem. Scand., 8 11954) ilO2. iv I. LIEBERMAN, A. KORNBERG AND E . S. SIMMS, J. A1~l. Clle~Jz. Soc., 76 (i9541 36oS. 18 D. R. SANADI, D. M. GIBSON AND P. AYENGAR, Biochim, Biophys. Acta, 14 119541 434. 19 E. IIERBERT, V. R. POTTER AND Y. TAKAGI, J. Biol. Chem., 11955) (in press.). Received J a n u a r y 24th, 1955
Increased liver glucose-6-phosphatase activity after cortisone administration* The behavior of liver glucose-6-phosphatase under physiological and pathological conditions has been u n d e r investigation in this laboratory. Studies of the glucose-O-phosphatase reaction ill normal fasting animals 1, and in precancerous, neoplastic ~-, regenerating, foetal and new born liver a have focussed our a t t e n t i o n on the regulation of this enzymic activity in tissues. I t has been k n o w n t h a t high doses of cortisone resulted in p e r m a n e n t diabetes in r a t '1, characterized by hyperglycemia and glycosuria, hlsulin resistant "cortisone diabetes" showing very high blood sugar levels and glycosuria was also described in h u m a n 5. The increase in tile q u a n t i t y of c a r b o h y d r a t e after cortisone a d m i n i s t r a t i o n has been a t t r i b u t e d to increased gluco-neogenesis and there is also evidence t h a t some phases of the c a r b o h y d r a t e utilization are inhibited 6. Recently however, it was d e m o n s t r a t e d t h a t cortisone a d m i n i s t r a t i o n caused a striking increase in the rate of glucose production v. Since glucose production from the liver is related to the rate of hydrolysis of the glucose-6-phosphate ester, it seemed of interest to s t u d y the glucose-6-phosphatase reactilm in the liver of animals treated with high doses of cortisone. The preliminary results of this investigation are reported here. Y o u n g male W i s t a r rats of zoo grains of weight were injected with 1 ml (25 mg) of cortisone acetate ( Cortone , Merck ) zntramuscularly, dally for five days and were killed by decapitation on the sixth day. Control animals received in lectIon of the vehicle , m which the cortisone is dissolved. Animals were m a i n t a i n e d on Purina F o x Chow and water ad libih¢m until sacrificed. Livers were pooled and the h o m o g e n a t e s were p r e p a r e d for enzymic studies as described in a previous c o m m u n i cation 2. The glucose-6-phosphatase activity was m e a s u r e d b y the m e t h o d of CORI AND CORI8 using a 15 m i n u t e s incubation time. Liver glycogen was determined b y the m e t h o d of GOOD, KRAMER AND SOMOGYI9 employing the NELSON'S a d a p t a t i o n of the SOMOGYI m e t h o d for glucose m. The enzymic activities are expressed per wet w e i g h t nitrogen, per average cell basis and also per liver weight/body x ~ i g h t ratio. Total nitrogen was determined by the micro-Kjeldahl procedure. Nuclear counts were done by the m e t h o d of PRICE AND I,AIRD 10 as modified by ALLARD et aI. 11. 131ood glucose was determined by the m e t h o d of FOLIN-Wu. The preliminary results are s h o w n in Figs. 1 and 2. The results are presented as percentage changes from the control values which are taken arbitrarily as ioo %. Fig. i shows t h a t cortisone injection increased the blood glucose level by 78 %. The well k n o w n glycogenic effect of cortisone on the liver is also illustrated in Fig. i. W h e n liver glycogen is expressed on per cell basis the increase in glycogen c o n t e n t after cortisone administration is higher t h a n when d a t a are expressed on the conventional g r a m per cent wet weight basis. * This project has been s u p p o r t e d b y grants from the Cancer Research Society, Montreal, National Cancer I n s t i t u t e of Canada and D a m o n R u n y o n Memorial F u n d for Cancer Research Inc., U.S.A. ** The cortisone acetate and vehicle was kindly supplied b y Dr. J. H. LAURIE of Merck and Co., Montreal.