The Pre-steady State of the Myosin-Adenosine Triphosphate System<subtitle>X. The Reaction Mechanism of the Myosin-ATP System and a Molecular Mechanism of Muscle Contraction<xref ref-type="fn" rid="fn1"><sup>*</sup></xref></subtitle>

Abstract

1. With ITP as substrate, the initial burst of P1-liberation was about 1 mole/4×105g of myosin at high concentrations of MgCl2 (1–10 mm), but it decreased with decrease in the concentration of MgCl2 below 1 mm. 2. The time-course of TCA-labile 32P1-liberation from the myosin-γ-32P-ATP system was measured after adding the stoichiometric amount of γ-32P-ATP to myosin, together with various amounts of ITP, deoxy-ATP or ADP. The amount of initial burst of 32P1-liberation decreased with increase in the concentration of ITP, deoxy-ATP or ADP, but its initial rate was not affected by adding ATP analogues. 3. The initial rapid liberation of H+ after mixing the stoichiometric amount of ATP with myosin was measured using a stopped-flow method. The rate of the initial rapid H+-absorption was too fast to be observed by this method. 4. In addition to the rapid stoichiometric H+-liberation, a slow stoichiometric liberation was observed with a pH-stat after adding the stoichiometric amount of ATP to myosin. The rate constants of the slow liberation were 0.7 and 0.4 min−1, respectively, in 0.5 and 2.8 m KC1. The rate in 0.5 m KC1 was much lower than that after adding a large amount of ATP. The rate in 2.8 m KC1 was almost equal to the initial rate after adding a large amount of ATP, but the latter decreased gradually to that in the steady state. 5. The time-course of change in optical density at 293 mμ after mixing the stoichiometric amount of ATP with myosin was equal to that of the initial rapid H+-liberation under the same conditions. 6. When the stoichiometric amount of ATP was mixed with myosin, the ultraviolet spectrum of myosin changed rapidly, as mentioned above, and then the change decayed gradually. The time for half maximum decay, t½, was about 50 sec. This value was in good agreement with the t½ value for ADP-liberation from the myosin-phosphate-ADP complex, E2⋱P⋰ADP1 7. The amount of change in the spectrum of myosin induced by ATP remained constant between pH6.0 and 9.5. The value of t½ of the decay was maximal at pH 7.5 and decreased on both acidic and alkaline sides. The value of t½ increased with increase in the KC1 concentration. 8. When myosin was incubated with NTP*** for 2hr in the presence of 2mm ATP, 0.6 m KC1 and 10 μm MgCl2, 2moles of NTP were bound to 4×103g of myosin and the extra-burst was completely inhibited. However, the stoichiometric initial burst of P1-liberation and the ATPase activity in the steady state were unaffected, and the ability of myosin to bind to F-actin was preserved. 9. ATP did not induce superprecipitation of actomyosin reconstituted from F-actin and NTP(2)-myosin produced by the method described above. The ATPase activity of actomyosin was slightly inhibited in 0.05 m KC1, 10 μm MgCl2 and 0.1 mm ATP, by treatment of myosin with NTP. 10. On the basis of the reaction mechanism of the myosin-ATP system described in this series of papers, a molecular mechanism of muscle contraction was proposed: sliding of F-actin filaments past myosin filaments is induced by a conformational change of myosin coupled with the cyclic phosphorylation and dephosphorylation of myosin, and by the dissociation of actomyosin caused by formation of the myosin-phosphate ADP complex, E2⋱P⋰ADP1⁠. Which of these two reactions occurs depends on the conformation of myosin. *This investigation was supported by United States Public Health Service Research Grant AM-08303, and by grants from the Muscular Dystrophy Associations of America, Inc., Toyo Rayon Science Foundation and the Ministry of Education of Japan.