Physicochemical studies on denaturation of myosin-adenosinetriphosphatase

Abstract

Denaturation of myosin A- and myosin B-ATPase was investigated in 0.5–0.6 M KC1 and at 30 °C. and pH 7.0. The denaturation of myosin A proceeded in accordance with the first-order law until ATPase disappeared, while that of myosin B proceeded according to the first-order law to a constant value of ATPase. From the results on denaturation of the three parts of myosin B separated by ultracentrifugation and of synthetic actomyosin, it was shown that the rapid denaturation of the first-order myosin B was due to myosin A contaminant in the preparation and/or released from the “heavy” components of myosin B. Under present conditions, the denaturation of myosin A was not affected by the addition of cysteine and by passing nitrogen or oxygen through the solution. By salting-out analysis, it was found that the main peak of myosin A moved from 38.5 to 32% saturation of ammonium sulfate with denaturation, and that a subunit, occupying about 10% of the total, was revealed in the precipitation range of 50–60%. This subunit was water soluble and considerably heat stable and traveled in the ultra-centrifuge as one peak, its s 20 w being 2.1–2.8. The reduced viscosity of myosin A and the weight-average molecular weight of myosin A increased with decrease of ATPase. When PP was added and ATPase was assayed using Ca ++ as an activator of ATPase, a lag phase appeared in the denaturation of ATPase. After the lag phase, the denaturation proceeded with a smaller velocity than in the absence of PP, while, when Mg ++ was used as a modifier of ATPase, the effect of PP could not be observed. From these results a reaction scheme was proposed which satisfactorily explained the course of the denaturation under various conditions.