Dynamic interaction between actin and myosin subfragment 1 in the presence of ADP

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Abstract
The equilibrium and dynamics of the interaction between actin, myosin subfragment 1 (S1), and ADP have been investigated by using actin which has been covalently labeled at Cys-374 with a pyrene group. The results are consistent with actin binding to S1.cntdot.ADP (M.cntdot.D) in a two-step reaction, A + M.cntdot.D .dblarw. A-M.cntdot.D .dblarw. A.cntdot.M.cntdot.D, in which the pyrene fluorescence only monitors the second step. In this model, K1 = 2.3 .times. 104 M-1 (k+1 = 4.6 .times. 104 M-1 s-1) and K2 = 10 (k+2 .ltoreq. 4 s-1); i.e., both steps are relatively slow compared to the maximum turnover of the ATPase reaction. ADP dissociates from both M.cntdot.D and A-M.cntdot.D at 2 s-1 and from A.cntdot.M.cntdot.D at .gtoreq. 500 s-1; therefore, actin only accelerates the release of product from the A.cntdot.M.cntdot.D state. This model is consistent with the actomyosin ATPase model proposed by Geeves et al. [(1984) J. Muscle Res. Cell Motil. 5,351]. The results suggest that A-M.cntdot.D cannot break down at a rate > 4 s-1 by dissociation of ADP, by dissociation of actin, or by isomerizing to A.cntdot.M.cntdot.D. It is therefore unlikely to be significantly occupied in a rapidly contracting muscle, but it may have a role in a muscle contracting against a load where the ATPase rate is markedly inhibited. Under these conditions, this complex may have a role in maintaining tension with a low ATP turnover rate.