Kinetic Properties of Dissociation of the H-Meromyosin-ADP Complex*

Abstract

The rate constants (k_d) of dissociation of ADP from the H-meromyosin (HMM)-ADP complex were measured at 5, 10, and 20° by the following four methods. (1) The dissociation constant of binding of ADP to HMM was determined by measuring the increase in fluorescence of HMM induced by ADP, and the rate of formation of the HMM-ADP complex, $k→$, after adding ADP to HMM was measured in terms of the change in UV absorption of HMM. Then the rate constant, k_d, of the dissociation step was calculated as the product of the dissociation constant and the binding rate constant. (2) The value of k_d was obtained as the value of $k→$ extrapolated to [ADP]=0. (3) The value of k_d was determined from the rate of change in the UV absorption after adding a sufficient amount of PP₁ to the HMM-ADP complex. (4) It was determined using ATP, instead of PP₁ as the displacing agent. The value of k_d could be estimated more accurately by methods (3) and (4) than by methods (1) and (2). At all temperatures used, the values of k_d estimated by these methods were more than 10 times that of the maximum rate of the myosin-ATPase [EC 3.6.1.3] reaction in the steady state under the same conditions.