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Interaction between mitochondrial adenosine triphosphatase and troponin component, TN-I
ARCHIVES
OF BtOCHEMISTBY
Interaction
AND
between
BIOPHYSICS
170,
743-744
(1975)
Mitochondrial Adenosine Triphosphatase Component, TN-I
and Troponin
The ATPase activity of AS-particles obtained from beef heart mitochondria was strongly inhibited by the troponin component TN-I, which had been combined with ASparticle. From kinetic study on the inhibitory action of the ATPase activity by Component TN-I, it was found that Component TN-I acts as a noncompetitive inhibitor of the ATPase activity. Effect of metal ions or nucleotides was also tested on the inhibition of the ATPase activity by Comnonent TN-I, which is quite similar to that by the mitochondrial ATPase inhibitor (2). In a previous study (l), Tamaura et al. demonstrated that one of the troponin components, TN-I, strongly inhibits the ATPase activity of AS-particles obtained from beef heart mitochondria. Horstman and Backer (2) isolated an inhibitor of mitochondrial ATPase (F,) from bovine heart mitochondria, and van de Stadt et al. (3) pointed out the importance of the ATPase inhibitor in a directional regulation of respiratory chain-linked energy transfer. The present paper deals with the mode of the inhibitory action of the mitochondrial ATPase activity by Component TN-I in order to clarify the role of the ATPase inhibitor in the regulation mechanism of the energy transformation process of ATP. A&particles were isolated from heavy layer beef heart mitochondria by the method of Backer and Ho&man (4) and troponin was prepared from a rabbit skeletal muscle by the method of Greaser and Gergely (5). The troponin component, TN-I, was obtained by DEAE-Sephadex chromatography in 6 M urea (5). The protein solution containing Component TN-I was dialyzed against 2 mM Tris buffer (pH 7.5) with 0.1 mM dithiothreitol. The protein concentration was determined by the Lowry method (6), using bovine serum albumin as a standard. Before measuring the ATPase activity, AS-particles suspended in 0.5 mM MgSO,, 0.5 mM ATP, and 15 mM Tris-HEPES (pH 6.7) were preincubated with Component TN-I for 15 min at 30°C. After the incubation, an aliquot (25 ~1) was assayed for ATPase activity (7). The amount of inorganic phosphate lib erated from ATP was measured by the method of Martin-Doty (8). The inhibition of the mitochondrial ATPase activity by Component TN-I is shown by curve A in Fig. 1. An almost complete loss of the ATPase activity of AS-particles occurs at higher concentrations of Component TN-I (1). In order to clarify whether Component TN-I binds to AS-particles or not, the mixture of AS-particles and Component TN-I was centrifuged for 15 min, at 140,OOQgand at 14°C. Under this condition, AS-particles, free from and bound with 743 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
0
0.04
TN-I Concentration
0.08
(mg/ml)
FIG. 1. Inhibition of the ATPase activity of ASparticles by Component TN-I. The ATPase activity was measured after preincubation of AS-particles (0.4 mg protein/ml) and Component TN-I in the presence of 0.5 mM ATP and 0.5 mM MgSO, for 15 min. Curve A; with the mixture of AS-particles and Component TN-I. Curve B; with AS-particles bound with Component TN-I, which were qbtained by centrifugation of the preincubation mixture. Component TN-I, were completely sedimented. The precipitate was resuspended in the preincubation medium and then the suspension was assayed for ATPase activity under the same experimental conditions as above. The result is shown by curve B, Fig. 1. The inhibitory effect of the mitochondrial ATPase activity by Component TN-I is the same as shown by curve A. This indicates that Component TN-I binds strongly to the AS-particle and the complex of Component TN-I and AS-particle does not dissociate even when resuspended into the preincubation medium after centrifugation. The kinetics of the inhibitory action of the ATPase activity of AS-particles by Component TN-I were studied, Fig. 2 shows the double-reciprocal plot of the ATPase activity against the concentration of ATP as a substrate, where curves A, B, and C were
744
SHORT
COMMUNICATIONS
obtained in the presence of Component TN-I with concentrations of 0, 0.02, and 0.026 mg protein/ml, respectively. Each curve intersects at the same point of the horizontal axis. The results imply that Component TN-I acts as a noncompetitive inhibitor of the ATPase activity and that the site of the ASparticle bound to Component TN-I is different from the ATP-binding site participating in the enzymic function of the ATPase molecule. The value of K, was estimated to be 2 x lo-’ M. Table I represents effects of various divalent cat-
-0
-4
0
ions and nucleotides in preincubation on the inhibition of the ATPase activity by Component TN-I. The first series of experiment is the effect of metal ions on the inhibition; divalent cations such as Mn*+, of ComI%?+, and Ca*+ ions enhance the inhibition ponent TN-I on the ATPase activity in comparison with no metal ion. Pullman et al. (7) reported the requirement of these divalent cations for inducing the mitochondrial ATPase activity. The second series of experiments is on the effects of nucleotides such as ATP, GTP, CTP, and UTP on the inhibition; Component TN-I is more inhibitory in the presence of purine nucleotides than in the presence of pyrimidine. The effect of K+ ion on the inhibition of the mitochondrial ATPase activity by Component TN-I was also tested and the result is quite similar to that by the mitochondrial ATPase inhibitor (2, 9); the inhibition was virtually abolished at more than 40 mM KCl. It is noteworthy that the mode of the inhibitory action of the mitochondrial ATPase activity by Component TN-I seems to agree with that by the mitochondrial ATPase inhibitor (2). These studies may have a clue to clarification of the mechanism of energy transformation process of ATP.
12 I /[A&l
x,0’
(M-l)
FIG. 2. Double reciprocal plot of the ATPase activity of AS-particles against concentration of ATP as a substrate. Curves A, B, and C; in the presence of Component TN-I with concentration of 0, 0.02, and 0.026 mg protein/ ml, respectively. TABLE
I
EFFECT OF METAL IONS AND NUCLEOTIDES IN PREINCUBATION ON THE INHIBITION OF THE ATPase ACTIVITY BY COMPONENT TN-I (0.04 mg/ml)” Additions to AS-particles
ATPase activity @moles Pi/l0 min) -TN-I
+TN-I
Inhibition (%)
ATP ATP ATP ATP ATP
EDTA MgSOd MnS04 CaCl, CuSOe
0.420 0.356 0.270 0.320 0.218
0.372 0.118 0.088 0.160 0.209
11 67 67 50 4
MgSO, MgSO, MgSO, MgSO,
ATP GTP CTP UTP
0.279 0.279 0.296 0.281
0.037 0.060 0.152 0.144
87 78 49 49
a In the first series of experiments, 1 mM ATP, 0.5 mM EDTA, and the indicated salts (1 mM1 were added to the preincubation system instead of 0.5 rnsr ATP and 0.5 mM Mg2+. In the second, 0.1 mM nucleotides and 0.1 mM Mg*+ were added instead of 0.5 rnsr ATP and 0.5 mM Mg*+.
REFERENCES 1. TAMAURA, Y., YAMAZAKI, S., HIROSE, S., AND INADA, Y. (1973) Biochem. Biophys. Res. Commzm. 53, 673-679. 2. HORSTMAN, L. L. AND RACKER, E. (19701 J. Biol. Chem. 245, 1336-1344. 3. STADT, R. J. VAN DE, BOER, B. L. DE, AND DAM, K. VAN (1973) Biochim. Biophys. Actu 292, 338-349. 4. RACKER, E. AND HORSTMAN, L. L. (1967) J. Biol. Chem. 242, 2547-2551. 5. GREASER, M. L. AND GERGELY, J. (1973) J. Biol. Chem. 248, 2125-2133. 6. LOWRY, 0. H., ROSEBROUCH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. PULLMAN, M. E., PENEFSKY, H. S., DATTA, A., AND RACKER, E. (1960) J. Biol. Chem. 235, 3322-3329. 8. MARTIN, J. B. AND DOTY, D. M. (1949) Anal. Chem. 21, 965-967. 9. PULLMAN, M. E. AND MONROY, G. C. (1963) J. Biol. Chem. 238, 3762-3769. HARUHIKO TAKISAWA SHOJIRO YAMA~AKI Y UTAKA TAMAURA SHICEHISA HIROSE YUJI INADA Laboratoy of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo, Japan Received March 25, 1975
OF BtOCHEMISTBY
Interaction
AND
between
BIOPHYSICS
170,
743-744
(1975)
Mitochondrial Adenosine Triphosphatase Component, TN-I
and Troponin
The ATPase activity of AS-particles obtained from beef heart mitochondria was strongly inhibited by the troponin component TN-I, which had been combined with ASparticle. From kinetic study on the inhibitory action of the ATPase activity by Component TN-I, it was found that Component TN-I acts as a noncompetitive inhibitor of the ATPase activity. Effect of metal ions or nucleotides was also tested on the inhibition of the ATPase activity by Comnonent TN-I, which is quite similar to that by the mitochondrial ATPase inhibitor (2). In a previous study (l), Tamaura et al. demonstrated that one of the troponin components, TN-I, strongly inhibits the ATPase activity of AS-particles obtained from beef heart mitochondria. Horstman and Backer (2) isolated an inhibitor of mitochondrial ATPase (F,) from bovine heart mitochondria, and van de Stadt et al. (3) pointed out the importance of the ATPase inhibitor in a directional regulation of respiratory chain-linked energy transfer. The present paper deals with the mode of the inhibitory action of the mitochondrial ATPase activity by Component TN-I in order to clarify the role of the ATPase inhibitor in the regulation mechanism of the energy transformation process of ATP. A&particles were isolated from heavy layer beef heart mitochondria by the method of Backer and Ho&man (4) and troponin was prepared from a rabbit skeletal muscle by the method of Greaser and Gergely (5). The troponin component, TN-I, was obtained by DEAE-Sephadex chromatography in 6 M urea (5). The protein solution containing Component TN-I was dialyzed against 2 mM Tris buffer (pH 7.5) with 0.1 mM dithiothreitol. The protein concentration was determined by the Lowry method (6), using bovine serum albumin as a standard. Before measuring the ATPase activity, AS-particles suspended in 0.5 mM MgSO,, 0.5 mM ATP, and 15 mM Tris-HEPES (pH 6.7) were preincubated with Component TN-I for 15 min at 30°C. After the incubation, an aliquot (25 ~1) was assayed for ATPase activity (7). The amount of inorganic phosphate lib erated from ATP was measured by the method of Martin-Doty (8). The inhibition of the mitochondrial ATPase activity by Component TN-I is shown by curve A in Fig. 1. An almost complete loss of the ATPase activity of AS-particles occurs at higher concentrations of Component TN-I (1). In order to clarify whether Component TN-I binds to AS-particles or not, the mixture of AS-particles and Component TN-I was centrifuged for 15 min, at 140,OOQgand at 14°C. Under this condition, AS-particles, free from and bound with 743 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
0
0.04
TN-I Concentration
0.08
(mg/ml)
FIG. 1. Inhibition of the ATPase activity of ASparticles by Component TN-I. The ATPase activity was measured after preincubation of AS-particles (0.4 mg protein/ml) and Component TN-I in the presence of 0.5 mM ATP and 0.5 mM MgSO, for 15 min. Curve A; with the mixture of AS-particles and Component TN-I. Curve B; with AS-particles bound with Component TN-I, which were qbtained by centrifugation of the preincubation mixture. Component TN-I, were completely sedimented. The precipitate was resuspended in the preincubation medium and then the suspension was assayed for ATPase activity under the same experimental conditions as above. The result is shown by curve B, Fig. 1. The inhibitory effect of the mitochondrial ATPase activity by Component TN-I is the same as shown by curve A. This indicates that Component TN-I binds strongly to the AS-particle and the complex of Component TN-I and AS-particle does not dissociate even when resuspended into the preincubation medium after centrifugation. The kinetics of the inhibitory action of the ATPase activity of AS-particles by Component TN-I were studied, Fig. 2 shows the double-reciprocal plot of the ATPase activity against the concentration of ATP as a substrate, where curves A, B, and C were
744
SHORT
COMMUNICATIONS
obtained in the presence of Component TN-I with concentrations of 0, 0.02, and 0.026 mg protein/ml, respectively. Each curve intersects at the same point of the horizontal axis. The results imply that Component TN-I acts as a noncompetitive inhibitor of the ATPase activity and that the site of the ASparticle bound to Component TN-I is different from the ATP-binding site participating in the enzymic function of the ATPase molecule. The value of K, was estimated to be 2 x lo-’ M. Table I represents effects of various divalent cat-
-0
-4
0
ions and nucleotides in preincubation on the inhibition of the ATPase activity by Component TN-I. The first series of experiment is the effect of metal ions on the inhibition; divalent cations such as Mn*+, of ComI%?+, and Ca*+ ions enhance the inhibition ponent TN-I on the ATPase activity in comparison with no metal ion. Pullman et al. (7) reported the requirement of these divalent cations for inducing the mitochondrial ATPase activity. The second series of experiments is on the effects of nucleotides such as ATP, GTP, CTP, and UTP on the inhibition; Component TN-I is more inhibitory in the presence of purine nucleotides than in the presence of pyrimidine. The effect of K+ ion on the inhibition of the mitochondrial ATPase activity by Component TN-I was also tested and the result is quite similar to that by the mitochondrial ATPase inhibitor (2, 9); the inhibition was virtually abolished at more than 40 mM KCl. It is noteworthy that the mode of the inhibitory action of the mitochondrial ATPase activity by Component TN-I seems to agree with that by the mitochondrial ATPase inhibitor (2). These studies may have a clue to clarification of the mechanism of energy transformation process of ATP.
12 I /[A&l
x,0’
(M-l)
FIG. 2. Double reciprocal plot of the ATPase activity of AS-particles against concentration of ATP as a substrate. Curves A, B, and C; in the presence of Component TN-I with concentration of 0, 0.02, and 0.026 mg protein/ ml, respectively. TABLE
I
EFFECT OF METAL IONS AND NUCLEOTIDES IN PREINCUBATION ON THE INHIBITION OF THE ATPase ACTIVITY BY COMPONENT TN-I (0.04 mg/ml)” Additions to AS-particles
ATPase activity @moles Pi/l0 min) -TN-I
+TN-I
Inhibition (%)
ATP ATP ATP ATP ATP
EDTA MgSOd MnS04 CaCl, CuSOe
0.420 0.356 0.270 0.320 0.218
0.372 0.118 0.088 0.160 0.209
11 67 67 50 4
MgSO, MgSO, MgSO, MgSO,
ATP GTP CTP UTP
0.279 0.279 0.296 0.281
0.037 0.060 0.152 0.144
87 78 49 49
a In the first series of experiments, 1 mM ATP, 0.5 mM EDTA, and the indicated salts (1 mM1 were added to the preincubation system instead of 0.5 rnsr ATP and 0.5 mM Mg2+. In the second, 0.1 mM nucleotides and 0.1 mM Mg*+ were added instead of 0.5 rnsr ATP and 0.5 mM Mg*+.
REFERENCES 1. TAMAURA, Y., YAMAZAKI, S., HIROSE, S., AND INADA, Y. (1973) Biochem. Biophys. Res. Commzm. 53, 673-679. 2. HORSTMAN, L. L. AND RACKER, E. (19701 J. Biol. Chem. 245, 1336-1344. 3. STADT, R. J. VAN DE, BOER, B. L. DE, AND DAM, K. VAN (1973) Biochim. Biophys. Actu 292, 338-349. 4. RACKER, E. AND HORSTMAN, L. L. (1967) J. Biol. Chem. 242, 2547-2551. 5. GREASER, M. L. AND GERGELY, J. (1973) J. Biol. Chem. 248, 2125-2133. 6. LOWRY, 0. H., ROSEBROUCH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. PULLMAN, M. E., PENEFSKY, H. S., DATTA, A., AND RACKER, E. (1960) J. Biol. Chem. 235, 3322-3329. 8. MARTIN, J. B. AND DOTY, D. M. (1949) Anal. Chem. 21, 965-967. 9. PULLMAN, M. E. AND MONROY, G. C. (1963) J. Biol. Chem. 238, 3762-3769. HARUHIKO TAKISAWA SHOJIRO YAMA~AKI Y UTAKA TAMAURA SHICEHISA HIROSE YUJI INADA Laboratoy of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo, Japan Received March 25, 1975