Further studies on the interaction of actin with heavy meromyosin and subfragment 1 in the presence of ATP.

It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.