Calcium-sensitive binding of heavy meromyosin to regulated actin in the presence of ATP

Abstract

Regulated actin, F-actin plus troponin-tropomyosin, activates the myosin ATPase in the presence but not in the absence of calcium. Ultracentrifugation has been used to examine the interaction of regulated actin with two proteolytic fragments of myosin, heavy meromyosin (HMM), a two-headed species, and subfragment 1 (S-1), a single head. In the presence of ATP, the association constant (Ka) for the binding of S-1 to regulated actin is approximately 1.5 X 10(4) M-1, whether or not calcium is present. This is true for S-1 with and without the Mr = 19,000 phosphorylatable light chain. These results confirm the observations of Chalovich et al. (Chalovich, J., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256, 575-578) and support their suggestion that the inhibition of the regulated actin-activated ATPase of S-1 by removal of calcium does not result from preventing S-1 binding. The binding of HMM to regulated actin in the presence of ATP, however, is calcium sensitive. Removal of calcium results in approximately a 4-fold decrease in Ka, from 1.1 X 10(4) M-1 to 2.5 X 10(3) M-1. The results with HMM are more easily reconciled with the low stiffness of relaxed muscle and suggest a functional difference between S-1 and HMM.