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Inhibition of oxidative mechanism by 3-fluoropyruvate
VOL. 2 0 ( I 9 5 6 )
PRELIMINARY NOTES
583
Inhibition of oxidative mechanism by 3-fluoropyruvate* Fluoropyruvate has been found to produce convulsions in rats without the concomitant rise in citric acid such as is observed in fluoroacetate poisoning, thereby indicating that in the body it did not undergo oxidative decarboxylation represented by the equation: FCH2COCOOH + H20 - - FCH2COOH + CO2 + 2 H + + 2e The mechanism of generating the fluoroacetyl fragment thus remained inoperative. However, fluoropyruvate inhibited the aerobic oxidation of pyruvate in vitro and of 14C-labeled acetate in vivoZ, n.
Using the pigeon breast muscle pyruvic oxidase preparation of SCHWEETs, as modified by SmqAD#, we have now shown t h a t fluoropyruvate inhibits the anaerobic oxidation of pyruvate when indophenol is used as electron acceptor (Table I). TABLE I I N H I B I T I O N OF PYRUVIC OXIDASE BY 3 - F L U O R O P Y R U V A T E
System
Enzyme Enzyme Enzyme Enzyme Enzyme
alone , + 0. 3 pmoles fluoropyruvate + i.o pmoles fluoropyruvate + 1.65/*moles pyruvate + 1.65/*moles pyruvate + o. 3 /*moles fluoropyruvate Enzyme + 1.65/*moles pyruvate + 1.o /*moles fluoropyruvate
Dye mgmoles ~'educed/a rain
% Inhibition
1.26 1.26 2.1o 11.25
-----
6.42
48°/0
3.84
83 %
The reaction mixture consisted of potassium phosphate buffer, pH 7.5 (4/*moles) MgC12 (6.6 /*moles), cocarboxylase (66/*g), 2-6 dichlorophenolindophenol to give an initial optical density of approximately o.6o0, and pyruvic oxidase (45o/*g of dry weight of protein), in 1 ml final volume. Amount of dye reduced was calculated from the decrement in optical density for the 2-minute period following the addition of the enzyme. Measurements were carried out on a Beckman DU Spectrophotometer at 6oo m~. Further evidence of inhibition of aerobic pyruvate oxidation has been obtained from experiments with liver mitochondria prepared in sucrose or mannitol according to the method modified after LSUTHARDT.AND MULLER5. Fluoropyruvate (1 /,mole) inhibited pyruvate (9/*moles) in both tris and phosphate buffers to 45-750/0 . The oxidation of fumarate (2o/*moles) was inhibited to 55-8o% and 35-5o% by fluoropyruvate, 3 and 1/*moles, respectively. Fluoropyruvate also inhibited the entry of fluoroacetate into the tricarboxylic acid cycle. This was judged both from its effect upon citrate accumulation in .presence of fluoroacetate (Table II) and also by enzymic estimation of the fluorocitrate formed. For instance, with kidney particles, using malate as substrate in presence of fluoroacetate (49/,moles) and fluoropyruvate (8. 3/*moles), there was a reduction of approximately 60 % in the amount of fluorocitrate formed, as judged by its effect on brain particles. Contrary to the in rive results6, the rat liver mitochondria of the fasted male rats appear to activate fluoroacetate, although results with neither rat 1 nor pigeon liver homogenateLs,0 would indicate the formation of fluoroacetylhydroxamate. In keeping with this difference between acetate and fluoroacetate, Table II shows that acetate has slightly increased the effect of fumarate on the mitochondrial respiration. However, it does not seem to be available for the cycle in presence of fluoropyruvate (3/*moles) even at a high acetate concentration (3°/,moles). Consequently, the inhibition is not released. At a lower inhibitory level of fluoropyruvate (1 /*mole), the addition of acetate (lO #,mole) significantly reversed the effect of the inhibitor. In other experiments, fluoropyruvate also effected an extensive dephosphorylation of ATP in an inorganic-phosphate free mitochondrial syster~ in presence of fumarate and pyruvate. * This work was supported by a grant, B-6o8, from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, U.S. Public Health Services.
584
PRELIMINARY NOTES
VOL. 2 0
(1956)
TABLE II I N H I B I T I O N OF T H E E N T R Y OF F L U O R O A C E T A T E AND ACETATE IN T H E TCA CYCLE BY 3 - F L U O R O P Y R U V A T E
Substrate
Inhibitors
(pmole)
Citrate (~*mole)
QO,
% Inhibition
o.65 2.22 0.27 o-53I
7 o.7 63 .8 45.7 34.5
-9.8 35.4 51.9
A. Fumarate Fumarate Fumarate Fumarate
-Fluoroacetate Fluoropyruvate Fluoroacetate + fluoropyruvate
-3.3 i.o 3.3 I .o
Fumarate Fumarate Fumarate Fumarate + acetate (io/zmoles) Fumarate + acetate (I o p m o l e s ) Fumarate + acetate (3 o p m o l e s ) Fumarate + acetate (3 ° / , m o l e s )
-Fluoropyruvate Fluoropyruvate --
--
--
IO1
1.o 3.0 --
----
62. 4 22.8 125.o
38.i 77.4 --
Fluoropyruvate
1.o
--
lO9.2
12.6
--
--
123. 4
--
3.0
--
36.9
7o.1
B.
-Fluoropyruvate
--
T h e vessels c o n t a i n e d : t r i s - ( h y d r o x y m e t h y l ) - a m i n o m e t h a n e buffer (o.o2M) a t p H 7.28; MgC12 6 p m o l e s ; KCI I2 o p m o l e s ; p o t a s s i u m f u m a r a t e 2o.o /~moles; N a 2 A T P 0. 5 p m o l e s . S odi um a c e t a t e , f l u o r o a c e t a t e a n d f l u o r o p y r u v a t e as i n d i c a t e d . The i n h i b i t o r s were a d d e d in t h e flask. All s o l u t i o n s were m a d e u p in tris-buffer. To t h e flasks, L o m l of m i t o c h o n d r i a l s u s p e n s i o n from t h e l i v e r of o v e r n i g h t - f a s t e d m a l e W i s t a r r a t s w a s added. I n e x p e r i m e n t s u n d e r A, m a n n i t o l (840 p m o l e s p er flask) w a s p r e s e n t ; u n d e r B, sucrose ( 7 4 4 / , m o l e s pe r flask) w a s present. W a r b u r g ' s d i r e c t m a n o m e t r i c t e c h n i q u e w a s used a t 37 ° C. The Q02 v a l u e s are e x p r e s s e d as p l O~/h/mg m i t o c h o n d r i a l n i t r o g e n . T h e c i t r a t e 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 b y a m e t h o d modified a f t e r BUFFA AND PETERSe. O u r results, therefore, w o u l d i n d i c a t e t h a t 3 - f l u o r o p y r u v a t e n o t o n l y i n h i b i t s t h e p y r u v i c o x i d a s e s y s t e m , b u t also h a s an i n h i b i t i n g effect a t sites c o n t r o l l i n g t h e e n t r y of a c e t a t e a n d f l u o r o a c e t a t e i n t o t h e o x i d a t i v e cycle. EMERY M. GAL Departments o] Pharmacology and Physiology, Medical Center, ALAN S. FAIRHUEST University o] Cali/ornia, Los Angeles, Call/. (U.S.A .) ROBERT E. SMITH
and Biochemistry Department, A.R.C. Institute o/Animal Physiology, Cambridge (England)
RUDOLPH A. PETERS
1 E. M. GAL, R. A. PETERS AND R. W. WAKELIN, Proc. Biochem. Soc., 58 (1954) xiii; Biochem. J., (in press) I956. 2 R. A. PETERS, Bull. Johns Hopkins Hosp., 97 (1955) 21. 3 V . J A G A N N A T H A N AND R. S. S C H W E E T , J. Biol. Chem., 1 9 6 ( 1 9 5 2 ) 5 5 I. 4 j . W. LITTLEFIELD AND D. R. SANADI , J. Biol. Chem., 199 (I952) 65. 5 F. LEUTHARDT AND A. F. MULLER, Experientia, 4 (1948) 478. e p. BUFFA AND R. A. P E T E R S , J. Physiol., i i o (1949) 488. v R. A. PETERS, Brit. Med. J., 2 (1952) 1165. s j . F. MORRISON AND R. A. P E T E R S , Biochem. J., 58 (1954) 47~. e R. O. BRADY, J. Biol. Chem., 217 (1955) 213. R e c e i v e d March 6th, 1956
PRELIMINARY NOTES
583
Inhibition of oxidative mechanism by 3-fluoropyruvate* Fluoropyruvate has been found to produce convulsions in rats without the concomitant rise in citric acid such as is observed in fluoroacetate poisoning, thereby indicating that in the body it did not undergo oxidative decarboxylation represented by the equation: FCH2COCOOH + H20 - - FCH2COOH + CO2 + 2 H + + 2e The mechanism of generating the fluoroacetyl fragment thus remained inoperative. However, fluoropyruvate inhibited the aerobic oxidation of pyruvate in vitro and of 14C-labeled acetate in vivoZ, n.
Using the pigeon breast muscle pyruvic oxidase preparation of SCHWEETs, as modified by SmqAD#, we have now shown t h a t fluoropyruvate inhibits the anaerobic oxidation of pyruvate when indophenol is used as electron acceptor (Table I). TABLE I I N H I B I T I O N OF PYRUVIC OXIDASE BY 3 - F L U O R O P Y R U V A T E
System
Enzyme Enzyme Enzyme Enzyme Enzyme
alone , + 0. 3 pmoles fluoropyruvate + i.o pmoles fluoropyruvate + 1.65/*moles pyruvate + 1.65/*moles pyruvate + o. 3 /*moles fluoropyruvate Enzyme + 1.65/*moles pyruvate + 1.o /*moles fluoropyruvate
Dye mgmoles ~'educed/a rain
% Inhibition
1.26 1.26 2.1o 11.25
-----
6.42
48°/0
3.84
83 %
The reaction mixture consisted of potassium phosphate buffer, pH 7.5 (4/*moles) MgC12 (6.6 /*moles), cocarboxylase (66/*g), 2-6 dichlorophenolindophenol to give an initial optical density of approximately o.6o0, and pyruvic oxidase (45o/*g of dry weight of protein), in 1 ml final volume. Amount of dye reduced was calculated from the decrement in optical density for the 2-minute period following the addition of the enzyme. Measurements were carried out on a Beckman DU Spectrophotometer at 6oo m~. Further evidence of inhibition of aerobic pyruvate oxidation has been obtained from experiments with liver mitochondria prepared in sucrose or mannitol according to the method modified after LSUTHARDT.AND MULLER5. Fluoropyruvate (1 /,mole) inhibited pyruvate (9/*moles) in both tris and phosphate buffers to 45-750/0 . The oxidation of fumarate (2o/*moles) was inhibited to 55-8o% and 35-5o% by fluoropyruvate, 3 and 1/*moles, respectively. Fluoropyruvate also inhibited the entry of fluoroacetate into the tricarboxylic acid cycle. This was judged both from its effect upon citrate accumulation in .presence of fluoroacetate (Table II) and also by enzymic estimation of the fluorocitrate formed. For instance, with kidney particles, using malate as substrate in presence of fluoroacetate (49/,moles) and fluoropyruvate (8. 3/*moles), there was a reduction of approximately 60 % in the amount of fluorocitrate formed, as judged by its effect on brain particles. Contrary to the in rive results6, the rat liver mitochondria of the fasted male rats appear to activate fluoroacetate, although results with neither rat 1 nor pigeon liver homogenateLs,0 would indicate the formation of fluoroacetylhydroxamate. In keeping with this difference between acetate and fluoroacetate, Table II shows that acetate has slightly increased the effect of fumarate on the mitochondrial respiration. However, it does not seem to be available for the cycle in presence of fluoropyruvate (3/*moles) even at a high acetate concentration (3°/,moles). Consequently, the inhibition is not released. At a lower inhibitory level of fluoropyruvate (1 /*mole), the addition of acetate (lO #,mole) significantly reversed the effect of the inhibitor. In other experiments, fluoropyruvate also effected an extensive dephosphorylation of ATP in an inorganic-phosphate free mitochondrial syster~ in presence of fumarate and pyruvate. * This work was supported by a grant, B-6o8, from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, U.S. Public Health Services.
584
PRELIMINARY NOTES
VOL. 2 0
(1956)
TABLE II I N H I B I T I O N OF T H E E N T R Y OF F L U O R O A C E T A T E AND ACETATE IN T H E TCA CYCLE BY 3 - F L U O R O P Y R U V A T E
Substrate
Inhibitors
(pmole)
Citrate (~*mole)
QO,
% Inhibition
o.65 2.22 0.27 o-53I
7 o.7 63 .8 45.7 34.5
-9.8 35.4 51.9
A. Fumarate Fumarate Fumarate Fumarate
-Fluoroacetate Fluoropyruvate Fluoroacetate + fluoropyruvate
-3.3 i.o 3.3 I .o
Fumarate Fumarate Fumarate Fumarate + acetate (io/zmoles) Fumarate + acetate (I o p m o l e s ) Fumarate + acetate (3 o p m o l e s ) Fumarate + acetate (3 ° / , m o l e s )
-Fluoropyruvate Fluoropyruvate --
--
--
IO1
1.o 3.0 --
----
62. 4 22.8 125.o
38.i 77.4 --
Fluoropyruvate
1.o
--
lO9.2
12.6
--
--
123. 4
--
3.0
--
36.9
7o.1
B.
-Fluoropyruvate
--
T h e vessels c o n t a i n e d : t r i s - ( h y d r o x y m e t h y l ) - a m i n o m e t h a n e buffer (o.o2M) a t p H 7.28; MgC12 6 p m o l e s ; KCI I2 o p m o l e s ; p o t a s s i u m f u m a r a t e 2o.o /~moles; N a 2 A T P 0. 5 p m o l e s . S odi um a c e t a t e , f l u o r o a c e t a t e a n d f l u o r o p y r u v a t e as i n d i c a t e d . The i n h i b i t o r s were a d d e d in t h e flask. All s o l u t i o n s were m a d e u p in tris-buffer. To t h e flasks, L o m l of m i t o c h o n d r i a l s u s p e n s i o n from t h e l i v e r of o v e r n i g h t - f a s t e d m a l e W i s t a r r a t s w a s added. I n e x p e r i m e n t s u n d e r A, m a n n i t o l (840 p m o l e s p er flask) w a s p r e s e n t ; u n d e r B, sucrose ( 7 4 4 / , m o l e s pe r flask) w a s present. W a r b u r g ' s d i r e c t m a n o m e t r i c t e c h n i q u e w a s used a t 37 ° C. The Q02 v a l u e s are e x p r e s s e d as p l O~/h/mg m i t o c h o n d r i a l n i t r o g e n . T h e c i t r a t e 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 b y a m e t h o d modified a f t e r BUFFA AND PETERSe. O u r results, therefore, w o u l d i n d i c a t e t h a t 3 - f l u o r o p y r u v a t e n o t o n l y i n h i b i t s t h e p y r u v i c o x i d a s e s y s t e m , b u t also h a s an i n h i b i t i n g effect a t sites c o n t r o l l i n g t h e e n t r y of a c e t a t e a n d f l u o r o a c e t a t e i n t o t h e o x i d a t i v e cycle. EMERY M. GAL Departments o] Pharmacology and Physiology, Medical Center, ALAN S. FAIRHUEST University o] Cali/ornia, Los Angeles, Call/. (U.S.A .) ROBERT E. SMITH
and Biochemistry Department, A.R.C. Institute o/Animal Physiology, Cambridge (England)
RUDOLPH A. PETERS
1 E. M. GAL, R. A. PETERS AND R. W. WAKELIN, Proc. Biochem. Soc., 58 (1954) xiii; Biochem. J., (in press) I956. 2 R. A. PETERS, Bull. Johns Hopkins Hosp., 97 (1955) 21. 3 V . J A G A N N A T H A N AND R. S. S C H W E E T , J. Biol. Chem., 1 9 6 ( 1 9 5 2 ) 5 5 I. 4 j . W. LITTLEFIELD AND D. R. SANADI , J. Biol. Chem., 199 (I952) 65. 5 F. LEUTHARDT AND A. F. MULLER, Experientia, 4 (1948) 478. e p. BUFFA AND R. A. P E T E R S , J. Physiol., i i o (1949) 488. v R. A. PETERS, Brit. Med. J., 2 (1952) 1165. s j . F. MORRISON AND R. A. P E T E R S , Biochem. J., 58 (1954) 47~. e R. O. BRADY, J. Biol. Chem., 217 (1955) 213. R e c e i v e d March 6th, 1956