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Inhibition of nuclear NAD nucleosidase and poly ADP-ribose polymerase activity from rat liver by nicotinamide and 5′-methyl nicotinamide
82
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96804
I N H I B I T I O N OF NUCLEAR NAD NUCLEOSIDASE AND POLY ADP-RIBOSE POLYM-ERASE ACTIVITY FROM RAT L I V E R BY NICOTINAMIDE AND 5'-METHYL NICOTINAMIDE J. B. C L A R K , G. M. F E R R I S AND S. P I N D E R *
Biochemistry Department, Pharmacology Department, St. Bartholomew's Hospital Medical College, University of London, Charterhouse Square, ECIM 6BQ, London (Great Britain) (Received N o v e m b e r 3oth, 197 o)
SUMMARY
The effectiveness of nicotinamide and 5'-methyl nicotinamide as inhibitors of nuclear NAD nucleosidase and poly ADP-ribose polymerase from rat liver has been investigated. Nicotinamide inhibited the NAD nucleosidase and the poly ADP-ribose polymerase with K{s of 5" IO-4M and 2. Io-SM, respectively, 5'-methyl nicotinamide with Ki's of 3.3 " lO-4 M and 2 • lO -4 M, respectively. As both nicotinamide and 5'-methyl nicotinamide are similarly effective inhibitors of these enzymes, inhibition at these sites cannot explain the different effects that nicotinamide and 5-methyl nicotinamide have on hepatic NAD synthesis (Clark and Pinder, 1969). It is suggested therefore that the poly ADP-ribose polymerase is more likely involved in the control of DNA synthesis than in NAD synthesis (as suggested elsewhere).
INTRODUCTION
The activity of NAD nucleosidase (NAD glycohydrolase) in rat liver homogenates has been found primarily in the microsomal fraction, with a much smaller, but detectable activity in the nuclear fraction ~. The microsomal enzyme shows two activities, catalysing the hydrolysis of NAD (P)+ and an exchange reaction in which other pyridine derivatives substitute for the nicotinamide moiety of NAD(P) +2,3. The nuclear NAD glycohydrolase activity is associated with three types of reaction, NAD+ hydrolysis, NAD + transglycosidase reaction and the synthesis of a polymer of ADP-ribose units derived from NAD +4'5, hereafter referred to as poly ADP-ribose polymerase activity. It seems likely that a single nuclear enzyme is responsible for the poly ADP-ribose polymerase activity in addition to both the NAD + hydrolysis and exchange reaction 5,e. It has been suggested that poly ADP-ribose polymerase activity may be important in vivo in the control of tissue concentrations of the nicotinamide nucleotides 4 and that poly ADP-ribose may be a reservoir of NAD in the homeostatic control of nicotinamide nucleotide concentrations v. The inhibition of the microsomal NAD (P)÷ glycohydrolase by nicotinamide has been invoked as a possible mechanism for the * P r e s e n t a d d r e s s : R e s e a r c h Labs., M a y a n d Baker, D a g e n h a m , E s s e x , G r e a t Britain.
Biochim. Biophys. dcta, 238 (1971) 82-85
NAn
NUCLEOSIDASE AND POLY ADP-RIBOSE POLYMERASE
83
rise in hepatic NAD concentration following nicotinamide injection s,9. It has been shown, however, t h a t following the intraperitoneal injection of 5'-methyl nicotinamide in rats, there is no increase in hepatic NAD concentration, in contrast to the rapid rise following nicotinamide injection 1°. In the light of the powerful inhibition of the microsomal NAD(P) + glycohydrolase derived from Ehrlich ascites cells ~1 and rat liver ~°, b y both nicotinamide and 5'-methyl nicotinamide, it was concluded that NAD glycohydrolase activity is not important in controlling the hepatic NAD concentration following nicotinamide injection 1°. The present investigation was therefore undertaken to estimate the effectiveness of nicotinamide and 5'-methyl nicotinamide as inhibitors of nuclear NAD glycohydrolase activity and poly ADP-ribose polymerase activity derived from rat liver, in order to clarify the role of these enzyme activities in the control of NAD metabolism. This work shows that both nicotinamide and 5'-methyl nicotinamide are effective inhibitors of nuclear NAD glycohydrolase activity and poly ADP-ribose polymerase activity. Thus it is suggested that the poly ADP-ribose polymerase is unlikely to play a significant role in the control of NAD synthesis.
MATERIALS AND METHODS
Nuclei were isolated from the homogenates of livers of i5oog female Albino rats 12 and the activities of NAD glycohydrolase '3 and poly ADP-ribose polymerase 7 assayed. Eadenine-8-14ClNAD+ was prepared from [adenine-8-1'C~ATP (Radiochemical Centre, Amersham, Great Britain) and purified yeast NAD pyrophosphorylase (Boehringer und Soehne, Mannheim, Germany) li. The ['4CJNAD was purified b y separation from contaminants on a Dowex I-X8-4oo column and stored in frozen solution. 5'-Methyl nicotinamide was kindly donated by Eli Lilly Co., Indianapolis, U.S.A., and all other reagents were of the Analar grade where possible. Inhibition and Michaelis constants were derived graphically 15.
RESULTS AND DISCUSSION
Table I summarises the Ki's and Michaelis parameters derived from reciprocal plots of initial reaction velocity against inhibitor concentration at three different substrate concentrations 15. Figs. I and 2 compare calculated lines based on the parameters from Table I with experimentally derived values of enzyme activity at different inhibitor concentrations. Both compounds inhibited nuclear NAD glycohydrolase, nicotinamide competitively with a Ki of 5 " lO-4 IV[and 5'-methyl nicotinamide non-competitively with a K i of 3.3 • lO -4 M. This m a y be compared with a K i of 3 " lO-4 M for nicotinamide for nuclear NAD glycohydrolase from Ehrlich ascites cells x3. The mean K,~ for nuclear NAD glycohydrolase for NAD + was 8.3 • lO -4 M. The K m for NAD + for NAD glycohydrolase has been reported as 6.0 • Io -4 M for nuclear enzyme derived from Ehrlich ascites cells 13 and 2.3 • lO -5 N[ for microsomal enzyme derived from pig spleen le. Biochim. Biophys. Acta, 238 (i97 I) 82-85
84
J.B. CLARK 0-5
©
t.2 0"4
F --~ 0"!
©
0"3
~ o.4 0.2
I 0"1
0
I 0.2
I 0.3
I 0"4
I 0"5
0
0!1
I nhibitor Concentration(raM)
01"2
13
I 0.4
0!5
I nh~itor Concentration(rnM)
Fig. i. E f f e c t of v a r i o u s c o n c e n t r a t i o n s of n i c o t i n a m i d e ( O - Q ) a n d 5 ' - m e t h y l n i c o t i n a m i d e ( O - C ) ) on t h e a c t i v i t y of N A D + g l y c o h y d r o l a s e in r a t l i v e r nuclei. T h e t h e o r e t i c a l lines w e re c a l c u l a t e d from t h e d a t a in T a b l e I a n d t h e e x p e r i m e n t a l p o i n t s were d e t e r m i n e d a t a s u b s t r a t e ( N A D +) c o n c e n t r a t i o n of 2.1 • lO -8 M, p H 7.o, 37 ° a n d ionic s t r e n g t h (I) of o. 3, as d e s e r i b e d in t h e t e x t . S i m i l a r e x p e r i m e n t s were c a r r i e d o u t a t s u b s t r a t e c o n c e n t r a t i o n s of 7 ' l ° - 4 M a n d 1. 4 • lO -3 M N A D +. E n z y m e a c t i v i t y is e x p r e s s e d a s / , m o l e s N A D + h y d r o l y s e d p e r h pe r m g p r o t e i n . Fig. 2. E f f e c t s of v a r i o u s c o n c e n t r a t i o n s of n i c o t i n a m i d e ( Q - Q ) a n d 5 " - m e t h y l n i c o t i n a m i d e (C)-C)) on t h e a c t i v i t y of p o l y A D P - r i b o s e p o l y m e r a s e in r a t l i v e r nuclei. T h e t h e o r e t i c a l lines were c a l c u l a t e d f r o m t h e d a t a in T a b l e I a n d t h e e x p e r i m e n t a l p o i n t s w e re d e t e r m i n e d a t a subs t r a t e c o n c e n t r a t i o n of 3.1 • lO -4 M, p H 7.4, 37 ° a n d a n ionic s t r e n g t h (I) of 0. 5, as d e s c r i b e d in t h e t e x t . S i m i l a r e x p e r i m e n t s were c a r r i e d o u t a t s u b s t r a t e c o n c e n t r a t i o n s of 2 - lO -4 M a n d 4 " lO-4 M. E n z y m e a c t i v i t y is e x p r e s s e d as n m o l e s N A D + i n c o r p o r a t e d i n t o acid i n s o l u b l e m a t e r ial per h p e r m g p r o t e i n .
TABLE
I
EFFECTS OF NICOTINAMIDE AND 5t-METHYLNICOTINAMIDE ON MICHAELIS PARAMETERS :~UCLEAR NAD NUCLEOSlDASE &ND POLY ADP-RIBOSE POLY~tERASE
OF RAT LIVER
C o n d i t i o n s of a s s a y as in Figs. I a n d 2.
Enzyme
NAD nucleosidase Poly ADP-ribose polymerase
I~m × 2o 4/or N A D + (M )
Vmax (nmoles /mg protein per h)
K l × I o 5 (M) Nicotinamide
5'-Methylnicotinamide
8. 3
68o
5°
33
8.5
5
2
2o
5 ' - M e t h y l n i c o t i n a m i d e was a less effective inhibitor of poly A D P - r i b o s e polymerase a c t i v i t y t h a n n ic o t in a m i d e , w i t h K i ' s of 2 • lO -4 M a n d 2 • lO _5 M, respectively. Grap h i cal d e t e r m i n a t i o n of the K m for N A D + for p o l y A D P - r i b o s e polymerase a c t i v i t y gave a v a l u e of 8.5 • lO -4 M. B o t h n i c o t i n a m i d e a n d 5 ' - m e t h y l n i c o t i n a m i d e Biochim. Biophys. Acta, 238 (1971) 82-85
N A D NUCLEOSIDASE AND POLY ADP-RIBOSE POLYMERASE
85
w e r e m o r e e f f e c t i v e i n h i b i t o r s of p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t y t h a n n u c l e a r NAD glycohydrolase. I t a p p e a r s , t h e r e f o r e , t h a t b o t h n i c o t i n a m i d e a n d 5 ' - m e t h y l n i c o t i n a m ; d e are e f f e c t i v e i n h i b i t o r s of t h e N A D g l y c o h y d r o l a s e a n d p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t i e s of r a t l i v e r n u c l e i in vitro. Since in vivo it h a s b e e n d e m o n s t r a t e d t h a t 5'm e t h y l n i c o t i n a m i d e does n o t e l e v a t e h e p a t i c N A D levels as d o e s n i c o t i n a m i d e 1° it s e e m s u n l i k e l y t h a t e i t h e r t h e n u c l e a r N A D g l y c o h y d r o l a s e or t h e p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t y p l a y s a s i g n i f i c a n t r e g u l a t o r y role in N A D b i o s y n t h e s i s (c[. refs. 4 a n d 7). T h i s w o u l d s u g g e s t t h a t in vivo t h e p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t y of r a t l i v e r n u c l e i m u s t h a v e a n a l t e r n a t i v e m e t a b o l i c f u n c t i o n , p o s s i b l y ass o c i a t e d w i t h t h e r e g u l a t i o n a n d c o n t r o l of D N A s y n t h e s i s , as h a s r e c e n t l y b e e n proposed by BORZIO AND KOIDE 17.
ACKNOWLEDGEMENT T h i s w o r k w a s s u p p o r t e d in p a r t b y t h e C a n c e r R e s e a r c h C a m p a i g n .
REFERENCES I S. C. SUNG AND J. N. WILLIAMS, JR., .]. Biol. Chem., 197 (1952) 175. 2 L. J. ZATMAN, N. O. KAPLAN AND S. P. COLOWICK, J. Biol. Chum,, 2o0 (1953) 197.
3 N. I. SWlSLOCKI,M. I. KALISH, F. I. CHASALOWAND N. O. KAPLAN, J. Biol., Chem. 242 (1967) lO82. 4 Y. NISHIZUKA, K. UEDA, K. NAKAZAWAAND O. HAYAISHI, J. Biol. Chem., 242 (1967) 3164 . 5 Y. NISHIZUKA, I~. UEDA, K. ~AKAZAWA, R. H. REEDER, T. HONJO AND O. HAYAISHI, J. Vitaminol. Kyoto, 14 (1968) 143. 6 S. FUJIMURA, S. HASEGAWA,Y. SHIMIZUAND T. SUGIMURA,Biochim. Biophys. Acta, 145 (1967) 247. 7 M. E. HAINES, I. R. JOHNSTON, A. P. MATHIASAND D. RIDGE, Bioehem. J., 115 (1969) 881. 8 P. C*REENGARD, H. KALINSKY AND B. PETRACK, Biochim. Biophys. Acta, 52 (1961) 408. 9 S. GREEN AND O. BODANSKY, J. Biol. Chem., 238 (1963) 2119. io J. ]3. CLARKAND S. PINDER, Biochem. J., 114 (1969) 321. II E. KUN, ]3. LANGER, ~B. ULRICH, H. HOLZER AND H. G-RUNICKE, Proc. Natl. Acad. Sci. U,S.. 52 (1964) 15Ol. 12 C. C. WIDNELL AND J. R. TATA, Biochem. J., 92 (1964) 313. 13 K. W. BUCK, V. G~-NG, H. P. BEER, R. KRONAU AND H. GRUNICKE, European J. Biochem., 4 (1968) 357. 14 K. UEDA AND H. YAMAMURA, in S. P. COLOWICKAND N. O. KAPLAN, Methods in Enzymology, Vol. 1813, Academic Press, New York and London, 197 o, in the press. 15 M. DIXON, Biochem. J., 55 (1953) 17o. 16 H. W. DICKERMAN,A. SAN PIETRO AND ~. O. KAPLAN, Biochim. Biophys. Acta, 62 /I962) 23o. 17 L. BoRzlO ANI~ S. S. KOIDE, Biochem. Biophys. Res. Commun., 4 ° /197 o) lol 3.
Bioehim. Biophys. Acta, 238 (1971) 82-85
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96804
I N H I B I T I O N OF NUCLEAR NAD NUCLEOSIDASE AND POLY ADP-RIBOSE POLYM-ERASE ACTIVITY FROM RAT L I V E R BY NICOTINAMIDE AND 5'-METHYL NICOTINAMIDE J. B. C L A R K , G. M. F E R R I S AND S. P I N D E R *
Biochemistry Department, Pharmacology Department, St. Bartholomew's Hospital Medical College, University of London, Charterhouse Square, ECIM 6BQ, London (Great Britain) (Received N o v e m b e r 3oth, 197 o)
SUMMARY
The effectiveness of nicotinamide and 5'-methyl nicotinamide as inhibitors of nuclear NAD nucleosidase and poly ADP-ribose polymerase from rat liver has been investigated. Nicotinamide inhibited the NAD nucleosidase and the poly ADP-ribose polymerase with K{s of 5" IO-4M and 2. Io-SM, respectively, 5'-methyl nicotinamide with Ki's of 3.3 " lO-4 M and 2 • lO -4 M, respectively. As both nicotinamide and 5'-methyl nicotinamide are similarly effective inhibitors of these enzymes, inhibition at these sites cannot explain the different effects that nicotinamide and 5-methyl nicotinamide have on hepatic NAD synthesis (Clark and Pinder, 1969). It is suggested therefore that the poly ADP-ribose polymerase is more likely involved in the control of DNA synthesis than in NAD synthesis (as suggested elsewhere).
INTRODUCTION
The activity of NAD nucleosidase (NAD glycohydrolase) in rat liver homogenates has been found primarily in the microsomal fraction, with a much smaller, but detectable activity in the nuclear fraction ~. The microsomal enzyme shows two activities, catalysing the hydrolysis of NAD (P)+ and an exchange reaction in which other pyridine derivatives substitute for the nicotinamide moiety of NAD(P) +2,3. The nuclear NAD glycohydrolase activity is associated with three types of reaction, NAD+ hydrolysis, NAD + transglycosidase reaction and the synthesis of a polymer of ADP-ribose units derived from NAD +4'5, hereafter referred to as poly ADP-ribose polymerase activity. It seems likely that a single nuclear enzyme is responsible for the poly ADP-ribose polymerase activity in addition to both the NAD + hydrolysis and exchange reaction 5,e. It has been suggested that poly ADP-ribose polymerase activity may be important in vivo in the control of tissue concentrations of the nicotinamide nucleotides 4 and that poly ADP-ribose may be a reservoir of NAD in the homeostatic control of nicotinamide nucleotide concentrations v. The inhibition of the microsomal NAD (P)÷ glycohydrolase by nicotinamide has been invoked as a possible mechanism for the * P r e s e n t a d d r e s s : R e s e a r c h Labs., M a y a n d Baker, D a g e n h a m , E s s e x , G r e a t Britain.
Biochim. Biophys. dcta, 238 (1971) 82-85
NAn
NUCLEOSIDASE AND POLY ADP-RIBOSE POLYMERASE
83
rise in hepatic NAD concentration following nicotinamide injection s,9. It has been shown, however, t h a t following the intraperitoneal injection of 5'-methyl nicotinamide in rats, there is no increase in hepatic NAD concentration, in contrast to the rapid rise following nicotinamide injection 1°. In the light of the powerful inhibition of the microsomal NAD(P) + glycohydrolase derived from Ehrlich ascites cells ~1 and rat liver ~°, b y both nicotinamide and 5'-methyl nicotinamide, it was concluded that NAD glycohydrolase activity is not important in controlling the hepatic NAD concentration following nicotinamide injection 1°. The present investigation was therefore undertaken to estimate the effectiveness of nicotinamide and 5'-methyl nicotinamide as inhibitors of nuclear NAD glycohydrolase activity and poly ADP-ribose polymerase activity derived from rat liver, in order to clarify the role of these enzyme activities in the control of NAD metabolism. This work shows that both nicotinamide and 5'-methyl nicotinamide are effective inhibitors of nuclear NAD glycohydrolase activity and poly ADP-ribose polymerase activity. Thus it is suggested that the poly ADP-ribose polymerase is unlikely to play a significant role in the control of NAD synthesis.
MATERIALS AND METHODS
Nuclei were isolated from the homogenates of livers of i5oog female Albino rats 12 and the activities of NAD glycohydrolase '3 and poly ADP-ribose polymerase 7 assayed. Eadenine-8-14ClNAD+ was prepared from [adenine-8-1'C~ATP (Radiochemical Centre, Amersham, Great Britain) and purified yeast NAD pyrophosphorylase (Boehringer und Soehne, Mannheim, Germany) li. The ['4CJNAD was purified b y separation from contaminants on a Dowex I-X8-4oo column and stored in frozen solution. 5'-Methyl nicotinamide was kindly donated by Eli Lilly Co., Indianapolis, U.S.A., and all other reagents were of the Analar grade where possible. Inhibition and Michaelis constants were derived graphically 15.
RESULTS AND DISCUSSION
Table I summarises the Ki's and Michaelis parameters derived from reciprocal plots of initial reaction velocity against inhibitor concentration at three different substrate concentrations 15. Figs. I and 2 compare calculated lines based on the parameters from Table I with experimentally derived values of enzyme activity at different inhibitor concentrations. Both compounds inhibited nuclear NAD glycohydrolase, nicotinamide competitively with a Ki of 5 " lO-4 IV[and 5'-methyl nicotinamide non-competitively with a K i of 3.3 • lO -4 M. This m a y be compared with a K i of 3 " lO-4 M for nicotinamide for nuclear NAD glycohydrolase from Ehrlich ascites cells x3. The mean K,~ for nuclear NAD glycohydrolase for NAD + was 8.3 • lO -4 M. The K m for NAD + for NAD glycohydrolase has been reported as 6.0 • Io -4 M for nuclear enzyme derived from Ehrlich ascites cells 13 and 2.3 • lO -5 N[ for microsomal enzyme derived from pig spleen le. Biochim. Biophys. Acta, 238 (i97 I) 82-85
84
J.B. CLARK 0-5
©
t.2 0"4
F --~ 0"!
©
0"3
~ o.4 0.2
I 0"1
0
I 0.2
I 0.3
I 0"4
I 0"5
0
0!1
I nhibitor Concentration(raM)
01"2
13
I 0.4
0!5
I nh~itor Concentration(rnM)
Fig. i. E f f e c t of v a r i o u s c o n c e n t r a t i o n s of n i c o t i n a m i d e ( O - Q ) a n d 5 ' - m e t h y l n i c o t i n a m i d e ( O - C ) ) on t h e a c t i v i t y of N A D + g l y c o h y d r o l a s e in r a t l i v e r nuclei. T h e t h e o r e t i c a l lines w e re c a l c u l a t e d from t h e d a t a in T a b l e I a n d t h e e x p e r i m e n t a l p o i n t s were d e t e r m i n e d a t a s u b s t r a t e ( N A D +) c o n c e n t r a t i o n of 2.1 • lO -8 M, p H 7.o, 37 ° a n d ionic s t r e n g t h (I) of o. 3, as d e s e r i b e d in t h e t e x t . S i m i l a r e x p e r i m e n t s were c a r r i e d o u t a t s u b s t r a t e c o n c e n t r a t i o n s of 7 ' l ° - 4 M a n d 1. 4 • lO -3 M N A D +. E n z y m e a c t i v i t y is e x p r e s s e d a s / , m o l e s N A D + h y d r o l y s e d p e r h pe r m g p r o t e i n . Fig. 2. E f f e c t s of v a r i o u s c o n c e n t r a t i o n s of n i c o t i n a m i d e ( Q - Q ) a n d 5 " - m e t h y l n i c o t i n a m i d e (C)-C)) on t h e a c t i v i t y of p o l y A D P - r i b o s e p o l y m e r a s e in r a t l i v e r nuclei. T h e t h e o r e t i c a l lines were c a l c u l a t e d f r o m t h e d a t a in T a b l e I a n d t h e e x p e r i m e n t a l p o i n t s w e re d e t e r m i n e d a t a subs t r a t e c o n c e n t r a t i o n of 3.1 • lO -4 M, p H 7.4, 37 ° a n d a n ionic s t r e n g t h (I) of 0. 5, as d e s c r i b e d in t h e t e x t . S i m i l a r e x p e r i m e n t s were c a r r i e d o u t a t s u b s t r a t e c o n c e n t r a t i o n s of 2 - lO -4 M a n d 4 " lO-4 M. E n z y m e a c t i v i t y is e x p r e s s e d as n m o l e s N A D + i n c o r p o r a t e d i n t o acid i n s o l u b l e m a t e r ial per h p e r m g p r o t e i n .
TABLE
I
EFFECTS OF NICOTINAMIDE AND 5t-METHYLNICOTINAMIDE ON MICHAELIS PARAMETERS :~UCLEAR NAD NUCLEOSlDASE &ND POLY ADP-RIBOSE POLY~tERASE
OF RAT LIVER
C o n d i t i o n s of a s s a y as in Figs. I a n d 2.
Enzyme
NAD nucleosidase Poly ADP-ribose polymerase
I~m × 2o 4/or N A D + (M )
Vmax (nmoles /mg protein per h)
K l × I o 5 (M) Nicotinamide
5'-Methylnicotinamide
8. 3
68o
5°
33
8.5
5
2
2o
5 ' - M e t h y l n i c o t i n a m i d e was a less effective inhibitor of poly A D P - r i b o s e polymerase a c t i v i t y t h a n n ic o t in a m i d e , w i t h K i ' s of 2 • lO -4 M a n d 2 • lO _5 M, respectively. Grap h i cal d e t e r m i n a t i o n of the K m for N A D + for p o l y A D P - r i b o s e polymerase a c t i v i t y gave a v a l u e of 8.5 • lO -4 M. B o t h n i c o t i n a m i d e a n d 5 ' - m e t h y l n i c o t i n a m i d e Biochim. Biophys. Acta, 238 (1971) 82-85
N A D NUCLEOSIDASE AND POLY ADP-RIBOSE POLYMERASE
85
w e r e m o r e e f f e c t i v e i n h i b i t o r s of p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t y t h a n n u c l e a r NAD glycohydrolase. I t a p p e a r s , t h e r e f o r e , t h a t b o t h n i c o t i n a m i d e a n d 5 ' - m e t h y l n i c o t i n a m ; d e are e f f e c t i v e i n h i b i t o r s of t h e N A D g l y c o h y d r o l a s e a n d p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t i e s of r a t l i v e r n u c l e i in vitro. Since in vivo it h a s b e e n d e m o n s t r a t e d t h a t 5'm e t h y l n i c o t i n a m i d e does n o t e l e v a t e h e p a t i c N A D levels as d o e s n i c o t i n a m i d e 1° it s e e m s u n l i k e l y t h a t e i t h e r t h e n u c l e a r N A D g l y c o h y d r o l a s e or t h e p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t y p l a y s a s i g n i f i c a n t r e g u l a t o r y role in N A D b i o s y n t h e s i s (c[. refs. 4 a n d 7). T h i s w o u l d s u g g e s t t h a t in vivo t h e p o l y A D P - r i b o s e p o l y m e r a s e a c t i v i t y of r a t l i v e r n u c l e i m u s t h a v e a n a l t e r n a t i v e m e t a b o l i c f u n c t i o n , p o s s i b l y ass o c i a t e d w i t h t h e r e g u l a t i o n a n d c o n t r o l of D N A s y n t h e s i s , as h a s r e c e n t l y b e e n proposed by BORZIO AND KOIDE 17.
ACKNOWLEDGEMENT T h i s w o r k w a s s u p p o r t e d in p a r t b y t h e C a n c e r R e s e a r c h C a m p a i g n .
REFERENCES I S. C. SUNG AND J. N. WILLIAMS, JR., .]. Biol. Chem., 197 (1952) 175. 2 L. J. ZATMAN, N. O. KAPLAN AND S. P. COLOWICK, J. Biol. Chum,, 2o0 (1953) 197.
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