Elementary steps of the actin-activated ATPase reaction of cardiac muscle myosin subfragment-1

The rates of the elementary steps of the actomyosin ATPase reaction were measured using the myosin subfragment-1 of porcine left ventricular muscle. The results could be explained only by the two-route mechanism for actomyosin ATPase (Inoue, Shigekawa, & Tonomura (1973) J. Biochem. 74, 923-934), in which ATP is hydrolyzed via routes with or without accompanying dissociation of actomyosin. The dependence on the F-actin concentration of the rate of the acto-S-1 ATPase reaction in the steady state was measured in 5 mM KCl at 20 degrees C. The maximal rate, Vmax, and the dissociation constant for F-actin of the ATPase, Kd, were 3.0 s-1 and 2.2 mg/ml, respectively. The Kd value was almost the same as that determined from the extent of binding of S-1 with F-actin during the ATPase reaction. The rate of recombination of the S-1-phosphate-ADP complex, S-1ADPP, with F-actin, vr, was lower than that of the ATPase reaction in the steady state. Thus, ATP is mainly hydrolyzed without accompanying dissociation of acto-S-1 into S-1ADPP and F-actin. In the cardiac acto-S-1 ATPase reaction, the rate of the ATPase reaction in the steady state and that of recombination of S-1ADPP with F-actin were about 1/5 those of the skeletal acto-S-1 ATPase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)