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J. Biochem, 1974, Vol. 76, No. 4 755-764
© 1974 Japanese Biochemical Society

research-article

Binding of Adenosine Di- and Triphosphates to Myosin during the Hydrolysis of Adenosine Triphosphate*

Akio INOUE and Yuji TONOMURA

Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka

The amounts of ATP and ADP bound to myosin during the ATPase reaction [EC 3.6.1.3 [EC] ] were determined in the presence of 2 mM MgCl2 and 50 mM Tris-HCl at pH 7.8, using pyruvate kinase [EC 2.7.1.40 [EC] ] and phosphoenolpyruvate to regenerate ATP and 3H-labelled ATP as the substrate. The amounts of ADP and total nucleotides bound to myosin were measured, respectively, by thin layer chromatography after stopping the reaction with TCA and by a rappid-flow dialysis method. The following results were obtained; they were all consistent with our original reaction mechanism for myosin-ATPase but in conflict with the oversimplified variant proposed by Taylor et al.

  1. Binding of ATP to myosin was only observed when the amount of ATP added was more than about 0.6 mole/mole of myosin. In the presence of both 0.1 and 0.5 M KCl, the maximum amount of ATP bound was about 1 mole/mole of myosin, and the dissociation constant of binding (1 µM in 0.5 M KCl at 0°C) was equal to the Km value of myosin-ATPase in the steady state at high concentrations of ATP.
  2. When a sufficient amount of ATP was added to myosin, 1 mole of ADP bound rapidly to 1 mole of myosin during the initial phase of the reaction. Then, the amount of bound ADP decreased to the steady state level within a few minutes.
  3. The amount of bound ADP in the steady state increased almost linearly with increase in the amount of ATP added, and reached a constant value when the molar concentration of ATP added was higher than that of myosin. The maximum amount of bound ADP was 1 mole/mole of myosin at KCl concentrations above 1 M, and decreased with decrease in the KCl concentration. For example, in 0.125 M KCl at 20°C it was 0.4 mole/mole of myosin. The amount of bound ADP decreased slightly when the temperature was raised from 0 to 30°C.
  4. The rate of the ATPase reaction in the steady state was not proportional to the amount of ADP bound to myosin.

*This investigation was supported by grants from the Ministry of Education of Japan, and the Muscular Dystrophy Associations of America, Inc.


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