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The effect of monovalent cations on the dinitrophenol-induced ATPase of rat-liver mitochondria
45 2
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
COMMUNICATIONS
BBA 432O5
The effect of monovalent cations on the dinitrophenol-induced ATPase of rat-liver mitochondria Several years ago LARDY AND WELLMANI showed that KC1 and NaC1 enhance the dinitrophenol-induced ATPase of rat-liver mitochondria, measured in a medium containing only neutralized ATP and sucrose, and this was confirmed by MYERS AND SLATER2. The nature of this stimulation has now been further examined. With 6 mM ATP the dinitrophenol-induced ATPase was stimulated about 60 % when sucrose was replaced by an isomolar concentration of KC1 or when KC1 was added at a constant sucrose concentration (Fig. IA). Varying the sucrose concentration had little effect (Fig. IB). Thus the stimulation by KC1 is not an osmotic effect. No stimulation by KC1 was obtained in the presence of IO mM MgC12. ~0"51
T,
'
' A
'
'
'B
E o.3oV
.
.
.
.
~o 20 40 60 80 1oo o ~KCl (raM)
IGo 2Go sucrose(raM)
Fig. I. Effect of KCI and sucrose concentrations on the dinitrophenol-induced ATPase of rat-liver m i t o c h o n d r i a (prepared as in ref. 2). The reaction m i x t u r e contained 6 mM ATP (disodium salt b r o u g h t to p H 7.5 with Tris), o.I mM 2,4-dinitrophenol, 0.5 mM E D T A (free acid b r o u g h t to p H 7.5 w i t h Tris), o.I 8 m g / m l mitochondrial protein 1°, and the concentrations of KC1 and sucrose indicated. The reaction was s t a r t e d b y adding the mitochondria. After 16 min at 25 ° the reaction was stopped with trichloroacetic acid to 5% and inorganic p h o s p h a t e m e a s u r e d 11. The values are corrected for Pt present in ATP and in mitochondria. A. O, the osmolarity was kept practically c o n s t a n t b y replacing sucrose b y KC1, a s s u m i n g 25 ° mM sucrose is isoosmolar with 15o mM KC1; × , the sucrose concentration was kept at 75 mM. B. No added KCI.
The maximum effect of KC1 was obtained with about 5o raM. KC1 could be replaced with NaC1 (cf. ref. I), LiC1, RbC1, CsC1, NH4C1, triethylammonium chloride and Tris chloride. KBr, KI and KNOa stimulated similarly, whereas KF was inhibitory above 6 mM (cf. refs. I, 3). K~SO4 (and K2SeOa) stimulated much less than KC1, and inhibited in the presence of KC1. This inhibition was reversible by sedimenting the mitochondria by centrifugation and washing. The potassium salts of monocarboxylic acids (formate and acetate) and of dicarboxylic acids (oxalate, malonate, D-tartrate, L-tartrate, mesotartrate and maleate) stimulated to a smaller extent than KC1. Inhibition by anions of carboxylic acids, measured in the presence of KC1, have been reported by VELDSEMA-CURRIEAND SLATER4. In these experiments with 6 mM ATP, stimulation by added KC1 or other salts Biochim. Biophys. Acta, 162 (1968) 452-454
453
SHORT COMMUNICATIONS
amounted to only about 6o %. However, even in the absence of added salts, approx. 24 mM Na ÷ and Tris + are present to neutralize the ATP and about 1.5 mM to neutralize the EDTA. With lower concentrations of ATP, and consequently lower concentrations of cations, the stimulation by added cations was greater. Indeed when the ATPase activity is plotted against the total cation concentration, the lines extrapolated to zero cation concentration indicate an absolute requirement for cation for the dinitrophenol-induced ATPase, m a x i m u m activity being obtained with about 60 mM KC1 (Fig. 2). This experiment shows clearly that it is the cation and not the anion that is responsible for the stimulation by added salts. The requirement for a monovalent cation for the dinitrophenol-induced ATPase is rather unexpected, since monovalent ions (except NH4+ ) are only sluggishly permeable to the mitochondrial inner membrane, in the absence of an agent such as valinomycin 5. It seems likely that the requirement is related to the difference in charge between ATP and ADP (cf. KLINGENBERGAND PFAFF 6 and see also ref. 7). In all experiments described to date, a concentration of o.I mM 2,4-dinitrophenol was used. Fig. 3 shows the effect of varying both the KC1 and the dinitrophenol concentration. The optimal concentration of dinitrophenol (cf. ref. 8) tends to increase with increasing KC1 concentration. .'~ 0.5 2 o.
i
E 0.4
1
i
-~ 0.5C
E
0.4C
~.0.3
8
~
.£ E
~ 0.3¢
0,2
E >, 0.20 .>_. 0.15
11_
i
15 3'o 4;
i
i
60 ~
9'o 1;5 12o
concentration cation (raM)
i
o.os O.lO o.fo
o.~o
1.o
2,4-dinit rophenol (raM)
Fig. 2. Effect of a d d e d KC1 on d i n i t r o p h e n o l - i n d u c e d A T P a s e m e a s u r e d w i t h different concent r a t i o n s of ATP. The p r o c e d u r e was t h e s a m e as in Fig. i. 0.08 m g / m l m i t o c h o n d r i a l p r o t e i n , o. i mM 2,4 -d initro phenol, 0.5 mM E D T A . R e a c t i o n time, 12 min. On t h e a bs c i s s a t h e t o t a l concent r a t i o n of m o n o v a l e n t c a t i o n is p l o t t e d . This is c a l c u l a t e d as 4" [ATP~ + 3" [KC1]. T h e concent r a t i o n of sucrose was 245, 237 a n d 225 mM w i t h 1.2, 3 a n d 6 mM ATP, r e s p e c t i v e l y , i n t h e a b s e n c e of a d d e d KC1, a n d t h e o s m o l a r i t y w a s k e p t c o n s t a n t b y r e p l a c i n g sucrose w i t h KCI as in Fig. i. Curv e i, 1.2 mM A T P ; Curve 2, 3 mM A T P ; C urve 3, 6 mM ATP. Fig. 3- Effect of d i n i t r o p h e n o l c o n c e n t r a t i o n on A T P a s e a c t i v i t y m e a s u r e d in t h e pre s e nc e of different c o n c e n t r a t i o n s of KC1. The p r o c e d u r e w a s t h e s a m e as in Fig. I. o.14 m g / m l m i t o c h o n d r i a l pr otein , 0.5 mM E D T A , 6 mM ATP. R e a c t i o n time, 15 rain. C urve i, 225 mM sucrose; C u r v e 2, 7.5 mM KC1, 212 mM sucrose; Curve 3, 15 mM KC1, 200 mM sucrose; C urve 4, 3 ° mM KCt, i75 mM sucrose.
R. K R A A I J E N H O F (unpublished observation) has shown that the inhibition of the ATPase b y high concentrations of dinitrophenol 8 is competitive with respect to the ATP concentration. These results and those depicted in Fig. 3 are understandable if Biochim. Biophys. Acta, 162 (1968) 452-454
454
SHORT COMMUNICATIONS
the inhibition by excess dinitrophenol is due to inhibition of the entry of ATP into the mitochondria (cf. ref. 9), and if K + promotes the entry of ATP, thereby allowing it to compete more effectively with the dinitrophenol. This work was supported in part by research grants from the Life Insurance Medical Research Fund and the U.S. Public Health Service (Grant No. AM 08690).
Laboratory of Biochemistry, B. C. P. Jansen Institute, University of Amsterdam, Amsterdam (The Netherlands) I 2 3 4 5 6 7
8 9 IO II
R. AMONS* S. G. VAN DEN BERGH** E . C. SEATER
H. A. LARDY AND H. WELLMAN, J. Biol. Chem., 2Ol (1953) 357. D. K. MYERS AND E. C. SEATER, Biochem. J., 67 (1957) 558. D. K. MYERS AND E. C. SEATER, Biochem. J., 67 (1957) 572. IR. D. VELDSEMA-CURRIE AND E. C. SEATER, Biochim. Biophys. Acta, 162 (1968) 31o. E. J. HARRIS, J. D. JUDAH AND K. AttMED, in D. R. SANADI, Current Topics in Bioenergetics, Voh i, Academic Press, New York, 1966, p. 255. M. KLINGENBERG AND E. PFAFF, in T. W. GOODWlN, Metabolic Roles of Citrate, Academic Press, London, 1968, p. lO 5. E. C. SEATER, R. D. VELDSEMA-CURRIE, R. I~RAAIJENHOF AND R. AMONS, in S. PAPA, J. M. TAGER, E. QUAGLIARIELLO AND E. C. SEATER, Energy Level and Metabolic Control in Mitochondria, Adriatica Editrice, Bari (Italy), in the press. H. C. HEMKER, Biochim. Biophys. Acta, 63 (I962) 46. K. VAN DAM AND ]~. C. SEATER, Proc. Natl. Acad. Sci. U.S., 58 (1967) 2Ol 5. K. V~7. CLELAND AND E. C. SEATER, Biochem. J., 53 (1953) 547. J. B. SUMNER, Science, IOO (1944) 413 •
Received June 24th, 1968 * Present address: L a b o r a t o r y of Physiological Chemistry, State University of Leiden, Leiden. The Netherlands. ** Present address: L a b o r a t o r y of Veterinary Biochemistry, State University of Utrecht, Utrecht, The Netherlands.
Biochim. Biophys. Acta, 162 (1968) 452-454
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
COMMUNICATIONS
BBA 432O5
The effect of monovalent cations on the dinitrophenol-induced ATPase of rat-liver mitochondria Several years ago LARDY AND WELLMANI showed that KC1 and NaC1 enhance the dinitrophenol-induced ATPase of rat-liver mitochondria, measured in a medium containing only neutralized ATP and sucrose, and this was confirmed by MYERS AND SLATER2. The nature of this stimulation has now been further examined. With 6 mM ATP the dinitrophenol-induced ATPase was stimulated about 60 % when sucrose was replaced by an isomolar concentration of KC1 or when KC1 was added at a constant sucrose concentration (Fig. IA). Varying the sucrose concentration had little effect (Fig. IB). Thus the stimulation by KC1 is not an osmotic effect. No stimulation by KC1 was obtained in the presence of IO mM MgC12. ~0"51
T,
'
' A
'
'
'B
E o.3oV
.
.
.
.
~o 20 40 60 80 1oo o ~KCl (raM)
IGo 2Go sucrose(raM)
Fig. I. Effect of KCI and sucrose concentrations on the dinitrophenol-induced ATPase of rat-liver m i t o c h o n d r i a (prepared as in ref. 2). The reaction m i x t u r e contained 6 mM ATP (disodium salt b r o u g h t to p H 7.5 with Tris), o.I mM 2,4-dinitrophenol, 0.5 mM E D T A (free acid b r o u g h t to p H 7.5 w i t h Tris), o.I 8 m g / m l mitochondrial protein 1°, and the concentrations of KC1 and sucrose indicated. The reaction was s t a r t e d b y adding the mitochondria. After 16 min at 25 ° the reaction was stopped with trichloroacetic acid to 5% and inorganic p h o s p h a t e m e a s u r e d 11. The values are corrected for Pt present in ATP and in mitochondria. A. O, the osmolarity was kept practically c o n s t a n t b y replacing sucrose b y KC1, a s s u m i n g 25 ° mM sucrose is isoosmolar with 15o mM KC1; × , the sucrose concentration was kept at 75 mM. B. No added KCI.
The maximum effect of KC1 was obtained with about 5o raM. KC1 could be replaced with NaC1 (cf. ref. I), LiC1, RbC1, CsC1, NH4C1, triethylammonium chloride and Tris chloride. KBr, KI and KNOa stimulated similarly, whereas KF was inhibitory above 6 mM (cf. refs. I, 3). K~SO4 (and K2SeOa) stimulated much less than KC1, and inhibited in the presence of KC1. This inhibition was reversible by sedimenting the mitochondria by centrifugation and washing. The potassium salts of monocarboxylic acids (formate and acetate) and of dicarboxylic acids (oxalate, malonate, D-tartrate, L-tartrate, mesotartrate and maleate) stimulated to a smaller extent than KC1. Inhibition by anions of carboxylic acids, measured in the presence of KC1, have been reported by VELDSEMA-CURRIEAND SLATER4. In these experiments with 6 mM ATP, stimulation by added KC1 or other salts Biochim. Biophys. Acta, 162 (1968) 452-454
453
SHORT COMMUNICATIONS
amounted to only about 6o %. However, even in the absence of added salts, approx. 24 mM Na ÷ and Tris + are present to neutralize the ATP and about 1.5 mM to neutralize the EDTA. With lower concentrations of ATP, and consequently lower concentrations of cations, the stimulation by added cations was greater. Indeed when the ATPase activity is plotted against the total cation concentration, the lines extrapolated to zero cation concentration indicate an absolute requirement for cation for the dinitrophenol-induced ATPase, m a x i m u m activity being obtained with about 60 mM KC1 (Fig. 2). This experiment shows clearly that it is the cation and not the anion that is responsible for the stimulation by added salts. The requirement for a monovalent cation for the dinitrophenol-induced ATPase is rather unexpected, since monovalent ions (except NH4+ ) are only sluggishly permeable to the mitochondrial inner membrane, in the absence of an agent such as valinomycin 5. It seems likely that the requirement is related to the difference in charge between ATP and ADP (cf. KLINGENBERGAND PFAFF 6 and see also ref. 7). In all experiments described to date, a concentration of o.I mM 2,4-dinitrophenol was used. Fig. 3 shows the effect of varying both the KC1 and the dinitrophenol concentration. The optimal concentration of dinitrophenol (cf. ref. 8) tends to increase with increasing KC1 concentration. .'~ 0.5 2 o.
i
E 0.4
1
i
-~ 0.5C
E
0.4C
~.0.3
8
~
.£ E
~ 0.3¢
0,2
E >, 0.20 .>_. 0.15
11_
i
15 3'o 4;
i
i
60 ~
9'o 1;5 12o
concentration cation (raM)
i
o.os O.lO o.fo
o.~o
1.o
2,4-dinit rophenol (raM)
Fig. 2. Effect of a d d e d KC1 on d i n i t r o p h e n o l - i n d u c e d A T P a s e m e a s u r e d w i t h different concent r a t i o n s of ATP. The p r o c e d u r e was t h e s a m e as in Fig. i. 0.08 m g / m l m i t o c h o n d r i a l p r o t e i n , o. i mM 2,4 -d initro phenol, 0.5 mM E D T A . R e a c t i o n time, 12 min. On t h e a bs c i s s a t h e t o t a l concent r a t i o n of m o n o v a l e n t c a t i o n is p l o t t e d . This is c a l c u l a t e d as 4" [ATP~ + 3" [KC1]. T h e concent r a t i o n of sucrose was 245, 237 a n d 225 mM w i t h 1.2, 3 a n d 6 mM ATP, r e s p e c t i v e l y , i n t h e a b s e n c e of a d d e d KC1, a n d t h e o s m o l a r i t y w a s k e p t c o n s t a n t b y r e p l a c i n g sucrose w i t h KCI as in Fig. i. Curv e i, 1.2 mM A T P ; Curve 2, 3 mM A T P ; C urve 3, 6 mM ATP. Fig. 3- Effect of d i n i t r o p h e n o l c o n c e n t r a t i o n on A T P a s e a c t i v i t y m e a s u r e d in t h e pre s e nc e of different c o n c e n t r a t i o n s of KC1. The p r o c e d u r e w a s t h e s a m e as in Fig. I. o.14 m g / m l m i t o c h o n d r i a l pr otein , 0.5 mM E D T A , 6 mM ATP. R e a c t i o n time, 15 rain. C urve i, 225 mM sucrose; C u r v e 2, 7.5 mM KC1, 212 mM sucrose; Curve 3, 15 mM KC1, 200 mM sucrose; C urve 4, 3 ° mM KCt, i75 mM sucrose.
R. K R A A I J E N H O F (unpublished observation) has shown that the inhibition of the ATPase b y high concentrations of dinitrophenol 8 is competitive with respect to the ATP concentration. These results and those depicted in Fig. 3 are understandable if Biochim. Biophys. Acta, 162 (1968) 452-454
454
SHORT COMMUNICATIONS
the inhibition by excess dinitrophenol is due to inhibition of the entry of ATP into the mitochondria (cf. ref. 9), and if K + promotes the entry of ATP, thereby allowing it to compete more effectively with the dinitrophenol. This work was supported in part by research grants from the Life Insurance Medical Research Fund and the U.S. Public Health Service (Grant No. AM 08690).
Laboratory of Biochemistry, B. C. P. Jansen Institute, University of Amsterdam, Amsterdam (The Netherlands) I 2 3 4 5 6 7
8 9 IO II
R. AMONS* S. G. VAN DEN BERGH** E . C. SEATER
H. A. LARDY AND H. WELLMAN, J. Biol. Chem., 2Ol (1953) 357. D. K. MYERS AND E. C. SEATER, Biochem. J., 67 (1957) 558. D. K. MYERS AND E. C. SEATER, Biochem. J., 67 (1957) 572. IR. D. VELDSEMA-CURRIE AND E. C. SEATER, Biochim. Biophys. Acta, 162 (1968) 31o. E. J. HARRIS, J. D. JUDAH AND K. AttMED, in D. R. SANADI, Current Topics in Bioenergetics, Voh i, Academic Press, New York, 1966, p. 255. M. KLINGENBERG AND E. PFAFF, in T. W. GOODWlN, Metabolic Roles of Citrate, Academic Press, London, 1968, p. lO 5. E. C. SEATER, R. D. VELDSEMA-CURRIE, R. I~RAAIJENHOF AND R. AMONS, in S. PAPA, J. M. TAGER, E. QUAGLIARIELLO AND E. C. SEATER, Energy Level and Metabolic Control in Mitochondria, Adriatica Editrice, Bari (Italy), in the press. H. C. HEMKER, Biochim. Biophys. Acta, 63 (I962) 46. K. VAN DAM AND ]~. C. SEATER, Proc. Natl. Acad. Sci. U.S., 58 (1967) 2Ol 5. K. V~7. CLELAND AND E. C. SEATER, Biochem. J., 53 (1953) 547. J. B. SUMNER, Science, IOO (1944) 413 •
Received June 24th, 1968 * Present address: L a b o r a t o r y of Physiological Chemistry, State University of Leiden, Leiden. The Netherlands. ** Present address: L a b o r a t o r y of Veterinary Biochemistry, State University of Utrecht, Utrecht, The Netherlands.
Biochim. Biophys. Acta, 162 (1968) 452-454