- Email: [email protected]
The inhibitory effect of uncouplers of oxidative phosphorylation on mitochondrial respiration
PRELIMINARY NOTES
407
to glucose addition; i.e., those systems in which NAD+ is accumulating appear to be more prone to oscillate. It is suggested that this increase in the NAD + level is an early reflection of substrate limitation. Fig. 2 shows another experiment in which NAD +, ATP, and fructose 1,6-diphosphate were measured. These data show that the oscillations are not limited to the pyridine nucleotide. We were not able to show oscillations in 02 consumption using the Gilson vibrating platinum electrode and more dilute cell suspensions. When phase-plane plots 5 of fructose 1,6-diphosphate vs. ATP concentration are made from the data of Fig. 2, it is found that ATP is out of phase with fructose 1,6-diphosphate throughout the experiment. This is consistent with the concept that fructose 1,6-diphosphate levels change in response to changes in the activity of phosphofructokinase which in turn is controlled b y the level of the various adenine nucleotides 14. Furthermore, these changes appear to be readily and continuously reversed. We are presently investigating the responses of additional intermediates and are trying to elucidate more definitively the conditions necessary for oscillations. This investigation was supported by Public Health Service Research Grant No. CA-o7883-o2 from the National Cancer Institute and by the Rebecca Payne Livingston Foundation.
Deflartment of Biochemistry, California College of Medicine, University of California, Los Angeles, Calif. (U.S.A.)
K. H. IBSEN K. W. SCHILLER
CHANCE AND B. HESS, J. Biol. Chem., 234 (1959) 2421. L. COE, Biochim. Biophys. Acid, 118 (1966) 495. ]7[. IBSEN, E. L. COE AND R. \¥. MCKEE, Biochim. Biophys. Acta, 30 (1958) 384 . \Vu, J. Biol. Chem., 24o (1965) 2827. BETZ AND B. CHANCE, Arch. Biochem. Biophys., lO9 (1965) 585 . 6 B . CHANCE, R. \V. ESTABROOK AND A. GOSH, Natl. Acad. Sci. U.S., 51 (1964) 1244. 7 B . CHANCE, B. SCHOENER AND S. ELSAESSER, J. Biol. Chem., 24o (1965) 317 o. 8 1~. FRENKEL, Biochem. Biophys. Res. Commun., 21 (1965) 4979 J. HIGGENS, Natl. Acad. Sci. U.S., 51 (1964) 989. IO F. A. HOMMES AND F . M . A . H . SCHUURMANS STEKHOVEN,Biochim. Biophys. Acta, 86 (1964) 427 . I I K. H. IBSEN AND J. P. F o x , Arch. Biochem. Biophys., 112 (i965) 580. iz R. W. MCKEE, K. LONBERG-HOLM AND J. JEHL, Cancer Res., I i (1951) 855. 13 R. W. ESTABROOK AND P. K. MAITRA, Anal. Biochem., 7 (1964) 474. 14 D. GARFINKLE AND B. HESS, J. Biochem., 239 (1964) 971.
1 2 3 4 5
B. t~. K. R. A.
Received December 28th, 1966 Biochim. Biophys. Acta, 131 (1967) 405-407
BBA 41 IO1
The inhibitory effect of uncouplers of oxidative phosphorylation on mitochondrial respiration It has been known for some time that excessive concentrations of uncouplers of oxidative phosphorylation will inhibit mitochondrial respiration. For the case of Abbreviations: FCCP, carbonyl cyanide p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e ; T T F B , tetrachlorotrifluoromethylbenzimidazole; TMPD, t e t r a m e t h y l - p - p h e n y l e n e d i a m i n e ; ETP, electront r a n s p o r t particle.
Biochim. Biophys. Acta, 131 (1967) 4o7-411
4o8
PRELIMINARY
NOTES
substituted nitrophenols a hypothesis was proposed by HULSMANN1 and HEMKER2 that explained the inhibitory effect on the basis of binding of the uncoupler with an unknown factor involved in the energy-conserving reactions of mitochondria. This mechanism could explain differences in inhibitory power between equally strong uncouplers. However, the observation by WENNER 3 that the inhibition could be overcome by higher substrate concentration did not seem compatible with this mechanism. Later work b y WILSON4 has shown that the inhibition of succinate oxidation is kinetically of the competitive type between substrate and uncoupler, i.e. the uncoupler increases the Km for the substrate. The present study was undertaken to locate more precisely the site of inhibition. Although the inhibitory effect does extend to uncoupler-activated ATPase 2, only experiments on mitochondrial respiration will be reported here. TABLE
1
UNCOUPLING AND INHIBITORY ACTIVITY OF VARIOUS AG]~NTS I n a v o l u m e of 3 nil w e r e i n c u b a t e d a t r o o m t e m p e r a t u r e a n d p H 7 . 4 : 5 ° m M KC1, 5o m M T r i s c h l o r i d e , 5 0 m M s u c r o s e , i o m M Pl, 5 m M MgC12, i m M E D T A , i / , g / m l r o t e n o n e , 4 m M s u c c i n a t e a n d 2. 3 m g / m l r a t - l i v e r m i t o c h o n d r i a . T h e r a t e of o x y g e n u p t a k e w a s d e t e r m i n e d a f t e r a d d i t i o n of v a r y i n g a m o u n t s of t h e i n d i c a t e d a g e n t s . I n t h e c a s e of g r a m i c i d i n B t h e m e d i u m c o n s i s t e d o f : 2 5 0 m M s u c r o s e , 2 0 m M T r i s c h l o r i d e , 5 m M N a C I , 3 m M s u c c i n a t e a n d 2.2 m g / m l m i t o c h o n d r i a .
Compound
Concentration (M) required for Maximal respiration
2,4-Dinitrophenol Dicumarol FCCP TTFB Gramicidin B
3" 5" 3" 7" 4 .t"
lO-4 lO-6 IO 7 lO-7 lO-7
5o % inhibited respiration 1.5. 1. 5. 4' 7" 9"
io 3 IO 5 IO-5 IO-~ io ~
The inhibition of respiration is not limited to uncoupling agents in the classical sense, like 2,4-dinitrophenol, dicumarol, carbonyl cyanide 39-trifluoromethoxyphenylhydrazone (FCCP) or tetrachlorotrifluoromethylbenzimidazole (TTFB) but is also observed with excessive concentrations of several agents inducing ion transport, like gramicidin B (see Table I). In the latter case tile inhibited state can be reached either by titrating into the solution excess sodium at a fixed gramicidin level or by adding excess gramicidin at a fixed sodium concentration. The inhibition is observed with most of the substrates tested, viz. succinate, ~-oxoglutarate, malate, glutamate, fl-hydroxybutyrate (and N A D H in submitochondrial particles), the exceptional substrates being tetramethyl-/b-phenylenediamine (TMPD) (plus ascorbate) and choline. The degree of inhibition in each case is dependent on the substrate concentration 4. Inhibition of respiration by the classical uncoupling agents is observed in mitochondria of different sources as well as in several non-phosphorylating submitochondrial particles, for instance heart-muscle preparation 2 and electron-transfer particle (ETP) s. In submitochondrial particles no inhibitory effect by agents inducing ion transport has yet been observed. Biochim. Biophys. Acta, 131 (1967) 4 o 7 - 4 1 1
409
PRELIMINARY NOTES
Both in intact mitochondria and in submitochondrial particles the inhibition is readily reversible, for instance by the addition of serum albumin which binds the uncouplers 6. With intact mitochondria the phosphorylating system is still completely intact after the treatment with uncoupler and serum albumin since a cycle of enhanced respiration can be shown upon addition of a limited amount of ADP (ref. 7). The inhibitory effect of any combination of uncouplers or agents inducing ion transport is additive, i.e. the addition of so-called "optimal" concentrations of two or more uncouplers together will result in a submaximal rate of respiration. An extreme example of this m a y be found in intact mitochondria, where even the lowest concentration of uncoupler will inhibit respiration in the presence of ADP and PI (ref. 4) (Fig. I).
60
L P "~
2o3°[,o
~ l ~-ADP
,
,
,
0 0
2
3 4 Dicumorol (M)
5
6xlO "~'
Fig. I. O x y g e n u p t a k e w a s d e t e r m i n e d w i t h a C l a r k o x y g e n e l e c t rode a t room t e m p e r a t u r e a n d p H 7.4 in a m e d i u m c o n t a i n i n g : 5o mM sucrose, 5o mM Tris chloride, 5o mM KC1, i o mM Pl, 5 mM MgC12, I mM E D T A , o. 5 / z g / m l rotenone, 4 m g / m l r a t - l i v e r m i t o c h o n d r i a a n d 3 mM succinate. The r a t e w a s m e a s u r e d a t different d i c u m a r o l c o n c e n t r a t i o n s in t h e presence or a b s e n c e of I mM A D P .
The degree of reduction of the cytochromes in the inhibited state was measured by recording difference spectra at liquid-nitrogen temperature between maximally respiring and partially inhibited mitochondria. In this way it was found that under conditions of inhibition there was always a complete oxidation of all cytochromes (Table I I ; see also ref. 7). By direct analysis in acid and alkali extracts it was also established that the inhibited state was accompanied by an almost complete oxidation of the endogenous nicotinamide nucleotides of the mitochondria, regardless of the substrate present. Therefore, inhibition of respiration by excessive concentrations of uncouplers is characterized by a virtually complete oxidation of all carriers in the respiratory chain. This observation militates against localization of the inhibitory effect anywhere in the respiratory chain or in the energy-conserving reactions. In that case we would expect a reduction of at least some of the carriers in the respiratory chain as is the case in State 4 (cf. ref. 7)Both the competitive nature of the inhibition and the highly oxidized state of the respiratory chain in the inhibited state point to an influence of the uncoupler at the level of the interaction of the substrate with the primary dehydrogenase. This is also borne out b y the fact that the ferrocyanide reduction by succinate in E T P is Biochim. Biophys. Acta, 131 (1967) 4o7-411
4IO
PRELIMINARY
NOTES
inhibited by uncouplers. Although some other dehydrogenases are known to be directly inhibited by uncouplers 8 this can not be the sole reason for the respiratory inhibition in intact mitochondria, since addition of A D P (which by itself is not inhibitory) lowers the concentration of uncoupler required to give half-maximal inhibition (Fig. I). TABLE
l1
PERCENTAGE
REDUCTION
OF E L E C T R O N
CARRIERS IN THE UNCOUPLED
AND THE INHIBITED
STATE
In a v o l u m e of 3 m l t h e f o l l o w i n g were i n c u b a t e d at r o o m t e m p e r a t u r e a n d p H 7 . 4 : 5 ° m M Tris chloride, 5 ° n l M KC1, 5 ° InM s u c r o s e , lO m M Pl, 4.2 n l M g l u t a m a t e , 4.2 m M m a l a t e , 4 . 7 m g / m l r a t - l i v e r i n i t o c h o n d r i a . After d e t e r m i n i n g t h e s t e a d y s t a t e rate" of o x y g e n u p t a k e , s a m p l e s w e r e t a k e n e i t h e r f o r c h e m i c a l d e t e r m i n a t i o n of n i c o t i n a n l i d e n u c l e o t i d e s or for m e a s u r e m e n t of c v t o c h r o m e spectra at l i q u i d - n i t r o g e n t e m p e r a t u r e . ] ' h e r e d u c t i o n of c y t o c h r o m e s o b t a i n e d upon a n a e r o b i o s i s is t a k e n a s i o o % . D a t a g i v e n are p e r c e n t a g e s of this v a l u e .
Dicumarol (M)
Rate of oxygen uptake (natoms/mg protein per rain)
Reduction (%) eyt aa 3 cyt c
cyt b
NAD
A~4DP
lo 5 1o 4
47 24
o o
33 o
4o o
98 5°
17 o
It seems very likely, then, that the inhibition is due to lack of penetration of the substrates through a barrier or membrane, a conclusion also reached by HARRIS, H6FER AND PRESSMAN9 on the basis of respiratory experiments. In this respect it m a y be significant that the oxidation of the only positively charged substrates TMPD and choline is not inhibited. Further direct evidence for this hypothesis comes from recent experiments in which it was shown that the accumulation of substrate anions by mitochondria is indeed inhibited by relatively high concentrations of uncouplers 1°. The picture emerges that regulation of substrate metabolism in intact mitochondria can be exerted through the availability of energy for penetration; decreasing the available energy below a certain level will result in inhibition of substrate entry and thereby of substrate utilization. The author is indebted to Dr. E. J. HARRIS for many fruitful discussions and to Dr. D. F. WILSON for allowing him to read his manuscript before publication. This work was performed by the author during the tenure of post-doctoral fellowship I Fo5-TW-963-oI from the U.S. Public Health Service.
Johnson ResearchFoundation, University of Pennsylvania, Philadelphia, Pa. (U.S.A.)
KAREL
VAN
DAM*
I W . C. HOLSMANN, Over het Mechanisme van de ddernhalingsketenphosphorylering, M . D . Thesis, Klein Offset D r u k k e r i j , A m s t e r d a m , 1 9 5 8 . 2 H . C. HEMKER, Biochim, Biophys. Acla, 81 (1964) i. 3 C. E. \ ¥ E N N E R , Federation Prec., 24 (1965) 5 5 4 , * P e r m a n e n t address: L a b o r a t o r y of B i o c h e m i s t r y , M u i d e r g r a c h t 12, A i n s t e r d a n l , T h e N e t h e r l a n d s .
Biochim. Biophys. dcta, 131 (1967) 4 o 7 - 4 1 1
B.C.P. Jansen
Institute,
Plantage
411
PRELIMINARY NOTES 4 5 6 7 8 9 io
D. F. E. ]3. V. E. E.
F. WILSON, Arch. Biochem. Biophys., in the press. L. CRANE, J. L. GLENN AND D. E. GREEN, Biochim. Biophys. Acta, 22 (1956) 475C. WEINBACH AND J. GARBUS, J. Biol. Chem., 241 (1966) 3708. CHANCE, G. R. WILLIAMS AND G. HOLLUNGER, J. Biol. Chem., 238 (1963) 439. JURTSHUK, JR., I. SEKUZU AND D. E. GREEN, J. Biol. Chem., 238 (1963) 3595. J. HARRIS, M. H6FER AND ]3. C. PRESSMAN, s u b m i t t e d for publication. J. HARRIS AND K. VAN DAM, Nature, in the press.
Received November I2th, 1966 Revised manuscript received January 4th, 1967 Biochim. Biophys. Acta, 131 (1967) 4 o 7 - 4 I I
407
to glucose addition; i.e., those systems in which NAD+ is accumulating appear to be more prone to oscillate. It is suggested that this increase in the NAD + level is an early reflection of substrate limitation. Fig. 2 shows another experiment in which NAD +, ATP, and fructose 1,6-diphosphate were measured. These data show that the oscillations are not limited to the pyridine nucleotide. We were not able to show oscillations in 02 consumption using the Gilson vibrating platinum electrode and more dilute cell suspensions. When phase-plane plots 5 of fructose 1,6-diphosphate vs. ATP concentration are made from the data of Fig. 2, it is found that ATP is out of phase with fructose 1,6-diphosphate throughout the experiment. This is consistent with the concept that fructose 1,6-diphosphate levels change in response to changes in the activity of phosphofructokinase which in turn is controlled b y the level of the various adenine nucleotides 14. Furthermore, these changes appear to be readily and continuously reversed. We are presently investigating the responses of additional intermediates and are trying to elucidate more definitively the conditions necessary for oscillations. This investigation was supported by Public Health Service Research Grant No. CA-o7883-o2 from the National Cancer Institute and by the Rebecca Payne Livingston Foundation.
Deflartment of Biochemistry, California College of Medicine, University of California, Los Angeles, Calif. (U.S.A.)
K. H. IBSEN K. W. SCHILLER
CHANCE AND B. HESS, J. Biol. Chem., 234 (1959) 2421. L. COE, Biochim. Biophys. Acid, 118 (1966) 495. ]7[. IBSEN, E. L. COE AND R. \¥. MCKEE, Biochim. Biophys. Acta, 30 (1958) 384 . \Vu, J. Biol. Chem., 24o (1965) 2827. BETZ AND B. CHANCE, Arch. Biochem. Biophys., lO9 (1965) 585 . 6 B . CHANCE, R. \V. ESTABROOK AND A. GOSH, Natl. Acad. Sci. U.S., 51 (1964) 1244. 7 B . CHANCE, B. SCHOENER AND S. ELSAESSER, J. Biol. Chem., 24o (1965) 317 o. 8 1~. FRENKEL, Biochem. Biophys. Res. Commun., 21 (1965) 4979 J. HIGGENS, Natl. Acad. Sci. U.S., 51 (1964) 989. IO F. A. HOMMES AND F . M . A . H . SCHUURMANS STEKHOVEN,Biochim. Biophys. Acta, 86 (1964) 427 . I I K. H. IBSEN AND J. P. F o x , Arch. Biochem. Biophys., 112 (i965) 580. iz R. W. MCKEE, K. LONBERG-HOLM AND J. JEHL, Cancer Res., I i (1951) 855. 13 R. W. ESTABROOK AND P. K. MAITRA, Anal. Biochem., 7 (1964) 474. 14 D. GARFINKLE AND B. HESS, J. Biochem., 239 (1964) 971.
1 2 3 4 5
B. t~. K. R. A.
Received December 28th, 1966 Biochim. Biophys. Acta, 131 (1967) 405-407
BBA 41 IO1
The inhibitory effect of uncouplers of oxidative phosphorylation on mitochondrial respiration It has been known for some time that excessive concentrations of uncouplers of oxidative phosphorylation will inhibit mitochondrial respiration. For the case of Abbreviations: FCCP, carbonyl cyanide p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e ; T T F B , tetrachlorotrifluoromethylbenzimidazole; TMPD, t e t r a m e t h y l - p - p h e n y l e n e d i a m i n e ; ETP, electront r a n s p o r t particle.
Biochim. Biophys. Acta, 131 (1967) 4o7-411
4o8
PRELIMINARY
NOTES
substituted nitrophenols a hypothesis was proposed by HULSMANN1 and HEMKER2 that explained the inhibitory effect on the basis of binding of the uncoupler with an unknown factor involved in the energy-conserving reactions of mitochondria. This mechanism could explain differences in inhibitory power between equally strong uncouplers. However, the observation by WENNER 3 that the inhibition could be overcome by higher substrate concentration did not seem compatible with this mechanism. Later work b y WILSON4 has shown that the inhibition of succinate oxidation is kinetically of the competitive type between substrate and uncoupler, i.e. the uncoupler increases the Km for the substrate. The present study was undertaken to locate more precisely the site of inhibition. Although the inhibitory effect does extend to uncoupler-activated ATPase 2, only experiments on mitochondrial respiration will be reported here. TABLE
1
UNCOUPLING AND INHIBITORY ACTIVITY OF VARIOUS AG]~NTS I n a v o l u m e of 3 nil w e r e i n c u b a t e d a t r o o m t e m p e r a t u r e a n d p H 7 . 4 : 5 ° m M KC1, 5o m M T r i s c h l o r i d e , 5 0 m M s u c r o s e , i o m M Pl, 5 m M MgC12, i m M E D T A , i / , g / m l r o t e n o n e , 4 m M s u c c i n a t e a n d 2. 3 m g / m l r a t - l i v e r m i t o c h o n d r i a . T h e r a t e of o x y g e n u p t a k e w a s d e t e r m i n e d a f t e r a d d i t i o n of v a r y i n g a m o u n t s of t h e i n d i c a t e d a g e n t s . I n t h e c a s e of g r a m i c i d i n B t h e m e d i u m c o n s i s t e d o f : 2 5 0 m M s u c r o s e , 2 0 m M T r i s c h l o r i d e , 5 m M N a C I , 3 m M s u c c i n a t e a n d 2.2 m g / m l m i t o c h o n d r i a .
Compound
Concentration (M) required for Maximal respiration
2,4-Dinitrophenol Dicumarol FCCP TTFB Gramicidin B
3" 5" 3" 7" 4 .t"
lO-4 lO-6 IO 7 lO-7 lO-7
5o % inhibited respiration 1.5. 1. 5. 4' 7" 9"
io 3 IO 5 IO-5 IO-~ io ~
The inhibition of respiration is not limited to uncoupling agents in the classical sense, like 2,4-dinitrophenol, dicumarol, carbonyl cyanide 39-trifluoromethoxyphenylhydrazone (FCCP) or tetrachlorotrifluoromethylbenzimidazole (TTFB) but is also observed with excessive concentrations of several agents inducing ion transport, like gramicidin B (see Table I). In the latter case tile inhibited state can be reached either by titrating into the solution excess sodium at a fixed gramicidin level or by adding excess gramicidin at a fixed sodium concentration. The inhibition is observed with most of the substrates tested, viz. succinate, ~-oxoglutarate, malate, glutamate, fl-hydroxybutyrate (and N A D H in submitochondrial particles), the exceptional substrates being tetramethyl-/b-phenylenediamine (TMPD) (plus ascorbate) and choline. The degree of inhibition in each case is dependent on the substrate concentration 4. Inhibition of respiration by the classical uncoupling agents is observed in mitochondria of different sources as well as in several non-phosphorylating submitochondrial particles, for instance heart-muscle preparation 2 and electron-transfer particle (ETP) s. In submitochondrial particles no inhibitory effect by agents inducing ion transport has yet been observed. Biochim. Biophys. Acta, 131 (1967) 4 o 7 - 4 1 1
409
PRELIMINARY NOTES
Both in intact mitochondria and in submitochondrial particles the inhibition is readily reversible, for instance by the addition of serum albumin which binds the uncouplers 6. With intact mitochondria the phosphorylating system is still completely intact after the treatment with uncoupler and serum albumin since a cycle of enhanced respiration can be shown upon addition of a limited amount of ADP (ref. 7). The inhibitory effect of any combination of uncouplers or agents inducing ion transport is additive, i.e. the addition of so-called "optimal" concentrations of two or more uncouplers together will result in a submaximal rate of respiration. An extreme example of this m a y be found in intact mitochondria, where even the lowest concentration of uncoupler will inhibit respiration in the presence of ADP and PI (ref. 4) (Fig. I).
60
L P "~
2o3°[,o
~ l ~-ADP
,
,
,
0 0
2
3 4 Dicumorol (M)
5
6xlO "~'
Fig. I. O x y g e n u p t a k e w a s d e t e r m i n e d w i t h a C l a r k o x y g e n e l e c t rode a t room t e m p e r a t u r e a n d p H 7.4 in a m e d i u m c o n t a i n i n g : 5o mM sucrose, 5o mM Tris chloride, 5o mM KC1, i o mM Pl, 5 mM MgC12, I mM E D T A , o. 5 / z g / m l rotenone, 4 m g / m l r a t - l i v e r m i t o c h o n d r i a a n d 3 mM succinate. The r a t e w a s m e a s u r e d a t different d i c u m a r o l c o n c e n t r a t i o n s in t h e presence or a b s e n c e of I mM A D P .
The degree of reduction of the cytochromes in the inhibited state was measured by recording difference spectra at liquid-nitrogen temperature between maximally respiring and partially inhibited mitochondria. In this way it was found that under conditions of inhibition there was always a complete oxidation of all cytochromes (Table I I ; see also ref. 7). By direct analysis in acid and alkali extracts it was also established that the inhibited state was accompanied by an almost complete oxidation of the endogenous nicotinamide nucleotides of the mitochondria, regardless of the substrate present. Therefore, inhibition of respiration by excessive concentrations of uncouplers is characterized by a virtually complete oxidation of all carriers in the respiratory chain. This observation militates against localization of the inhibitory effect anywhere in the respiratory chain or in the energy-conserving reactions. In that case we would expect a reduction of at least some of the carriers in the respiratory chain as is the case in State 4 (cf. ref. 7)Both the competitive nature of the inhibition and the highly oxidized state of the respiratory chain in the inhibited state point to an influence of the uncoupler at the level of the interaction of the substrate with the primary dehydrogenase. This is also borne out b y the fact that the ferrocyanide reduction by succinate in E T P is Biochim. Biophys. Acta, 131 (1967) 4o7-411
4IO
PRELIMINARY
NOTES
inhibited by uncouplers. Although some other dehydrogenases are known to be directly inhibited by uncouplers 8 this can not be the sole reason for the respiratory inhibition in intact mitochondria, since addition of A D P (which by itself is not inhibitory) lowers the concentration of uncoupler required to give half-maximal inhibition (Fig. I). TABLE
l1
PERCENTAGE
REDUCTION
OF E L E C T R O N
CARRIERS IN THE UNCOUPLED
AND THE INHIBITED
STATE
In a v o l u m e of 3 m l t h e f o l l o w i n g were i n c u b a t e d at r o o m t e m p e r a t u r e a n d p H 7 . 4 : 5 ° m M Tris chloride, 5 ° n l M KC1, 5 ° InM s u c r o s e , lO m M Pl, 4.2 n l M g l u t a m a t e , 4.2 m M m a l a t e , 4 . 7 m g / m l r a t - l i v e r i n i t o c h o n d r i a . After d e t e r m i n i n g t h e s t e a d y s t a t e rate" of o x y g e n u p t a k e , s a m p l e s w e r e t a k e n e i t h e r f o r c h e m i c a l d e t e r m i n a t i o n of n i c o t i n a n l i d e n u c l e o t i d e s or for m e a s u r e m e n t of c v t o c h r o m e spectra at l i q u i d - n i t r o g e n t e m p e r a t u r e . ] ' h e r e d u c t i o n of c y t o c h r o m e s o b t a i n e d upon a n a e r o b i o s i s is t a k e n a s i o o % . D a t a g i v e n are p e r c e n t a g e s of this v a l u e .
Dicumarol (M)
Rate of oxygen uptake (natoms/mg protein per rain)
Reduction (%) eyt aa 3 cyt c
cyt b
NAD
A~4DP
lo 5 1o 4
47 24
o o
33 o
4o o
98 5°
17 o
It seems very likely, then, that the inhibition is due to lack of penetration of the substrates through a barrier or membrane, a conclusion also reached by HARRIS, H6FER AND PRESSMAN9 on the basis of respiratory experiments. In this respect it m a y be significant that the oxidation of the only positively charged substrates TMPD and choline is not inhibited. Further direct evidence for this hypothesis comes from recent experiments in which it was shown that the accumulation of substrate anions by mitochondria is indeed inhibited by relatively high concentrations of uncouplers 1°. The picture emerges that regulation of substrate metabolism in intact mitochondria can be exerted through the availability of energy for penetration; decreasing the available energy below a certain level will result in inhibition of substrate entry and thereby of substrate utilization. The author is indebted to Dr. E. J. HARRIS for many fruitful discussions and to Dr. D. F. WILSON for allowing him to read his manuscript before publication. This work was performed by the author during the tenure of post-doctoral fellowship I Fo5-TW-963-oI from the U.S. Public Health Service.
Johnson ResearchFoundation, University of Pennsylvania, Philadelphia, Pa. (U.S.A.)
KAREL
VAN
DAM*
I W . C. HOLSMANN, Over het Mechanisme van de ddernhalingsketenphosphorylering, M . D . Thesis, Klein Offset D r u k k e r i j , A m s t e r d a m , 1 9 5 8 . 2 H . C. HEMKER, Biochim, Biophys. Acla, 81 (1964) i. 3 C. E. \ ¥ E N N E R , Federation Prec., 24 (1965) 5 5 4 , * P e r m a n e n t address: L a b o r a t o r y of B i o c h e m i s t r y , M u i d e r g r a c h t 12, A i n s t e r d a n l , T h e N e t h e r l a n d s .
Biochim. Biophys. dcta, 131 (1967) 4 o 7 - 4 1 1
B.C.P. Jansen
Institute,
Plantage
411
PRELIMINARY NOTES 4 5 6 7 8 9 io
D. F. E. ]3. V. E. E.
F. WILSON, Arch. Biochem. Biophys., in the press. L. CRANE, J. L. GLENN AND D. E. GREEN, Biochim. Biophys. Acta, 22 (1956) 475C. WEINBACH AND J. GARBUS, J. Biol. Chem., 241 (1966) 3708. CHANCE, G. R. WILLIAMS AND G. HOLLUNGER, J. Biol. Chem., 238 (1963) 439. JURTSHUK, JR., I. SEKUZU AND D. E. GREEN, J. Biol. Chem., 238 (1963) 3595. J. HARRIS, M. H6FER AND ]3. C. PRESSMAN, s u b m i t t e d for publication. J. HARRIS AND K. VAN DAM, Nature, in the press.
Received November I2th, 1966 Revised manuscript received January 4th, 1967 Biochim. Biophys. Acta, 131 (1967) 4 o 7 - 4 I I