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The Journal of Biochemistry
Vol. 88 (1980) No. 6 P 1629-1641

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In order to determine whether or not the main intermediate in the ATPase reaction catalyzed by one of the two heads of myosin molecule is identical with that catalyzed by the other head, we measured the amount of nucleotides bound to myosin and the size of P1 burst under various conditions. The following results were obtained: 1. In the presence of either Mg2+ or Mn2+ ions, myosin, HMM, and S-1 were each capable of binding 0.95±0.08 mol of nucleotides per mol of myosin head. The amounts of nucleotides bound to myosin, HMM, and S-1 in the steady state of ATP hydrolysis were estimated by measuring the ATP-dependent luminescence in the presence of both the luciferin-luciferase system and the pyruvate kinase system. 2. In the presence of Mg2+ ions, the size of the initial P1-burst was always larger than 0.5 mol/mol myosin head. For instance, it was 11 mol/mol myosin head at 1 μM Mg2+ ions, and decreased with increase in Mg2+, concentration, reaching 0.55-0.65 mol/mol myosin head at several mM Mg2+ ions. On the other hand, the P1-burst size in the presence of Mn2+ ions increased with increase in Mn2+ concentration, and it was 0.48±0.04 mol/mol myosin head at Mn2+ concentrations higher than 10 μM. These results support our view that the two heads of myosin are different from one another, and that the myosin-phosphate-ADP complex (MADPP) is produced only at one (burst head) of the two heads.
We also measured the rate of nucleotide release from myosin by two methods. 1. In the first method, nucleotides bound to myosin were released from myosin on addition of excess amounts of MgCTP. ADP thus released was immediately converted into ATP in the presence of excess amounts of pyruvate kinase and phosphoenol pyruvate. Thus, both ADP released and ATP released were measured by the luciferin-luciferase system. The rate of nucleotide release thus measured was found to obey first-order kinetics. The rate constant for nucleotide release was 0.90_??_min-1, slightly higher than the rate constant for ATPase reaction in the steady state, 0.5-0.7min-1 2. In the second method, the ATP released from myosin was rapidly converted into HMMADPP by adding excess amounts of HMM, and the amount of P1, associated with the formation of HMMADPP. was measured. The results thus obtained strongly suggested that the rate of ATP release from the myosin-ATP complex was higher than the rate of the initial P1-burst catalyzed by HMM. In other words, the rate of ATP release in high concentrations of nucleoside triphosphate (NTP) (in the first method) was about equal to the rate of ATP hydrolysis in the steady state, whereas the rate of ATP release in low concentrations of NTP (in the second method) was higher than the rate of ATP hydrolysis in the initial P1-burst which must be much higher than that in the steady state
. On the basis of these results, we concluded that release of ATP from the nonburst head was strongly inhibited by high concentrations of NTP.

Copyright © The Japanese Biochemical Society

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