Interaction of actin with N-ethylmaleimide modified heavy meromyosin in the presence and absence of adenosine triphosphate

N-Ethylmaleimide modified heavy meromyosin in only 3-fold activated by actin rather than 200-fold as is normal heavy meromyosin (Silverman, R., Eisenberg, E., and Kielley, W. W. (1972), Nature (London) 240, 207). Ultracentrifuge studies demonstrated that in the absence of ATP the N-ethylmaleimide modified heavy meromyosin binds to actin at a ratio of 2 actins to 1 N-ethylmaleimide modified heavy meromyosin. However, it was found that most of the N-ethylmaleimide modified heavy meromyosin was not bound to actin during ATP hydrolysis. Ultracentrifuge studies demonstrated that in the presence of 25 or 50 mM KCl under conditions where the ATPase is maximally activated by actin, less than 5% of the N-ethylmaleimide modified heavy meromyosin was bound to actin. In the absence of KCl there was limited binding but even this binding did not appear to correlate with the N-ethylmaleimide modified heavy meromyosin ATPase rate. Turbidity and viscosity studies also indicated that in the presence of ATP under conditions of maximal actin activation the N-ethylmaleimide modified heavy meromyosin and actin are almost completely dissociated, whereas there is a marked increase in turbidity and viscosity after all of the ATP is hydrolyzed. These results suggest that in the presence of ATP and actin N-ethylmaleimide modified heavy meromyosin exists most of the time in a refractory state unable to bind to actin and only a small part of the time in a nonrefractory state which can interact with actin. It follows that the major rate-limiting step during actin activation is the transition from the refractory to the nonrefractory state. Since the actin activation of N-ethylmaleimide modified heavy meromyosin is lower than that of normal heavy meromyosin this transition may be slower for N-ethylmaleimide modified heavy meromyosin than for normal heavy meromyosin.