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

Abstract

During the hydrolysis of ATP, both normal and SH1-[fast reacting thio group located on heavy chain of subfragment 1] blocked heavy meromyosin [HMM] undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. [Rabbit skeletal muscle was studied.] The fraction of HMM or subfragment 1 [S-1] which remained unbound to actin when the ATPase equaled Vmax should have the same properties as the original protein. The unbound protein had normal ATPase activity. It was unbound to actin for a kinetic reason rather than because it was a permanently altered form of the myosin. If the HMM heads acted independently half as much S-1 as HMM should bind to actin. Experiments in the ultracentrifuge demonstrated that about half as much S-1 as HMM sediments with the actin at Vmax. The ATP turnover rate per actin monomer at infinite HMM concentration was much higher than the ATP turnover rate per HMM head at infinite actin concentration. This was true for SH1-blocked HMM since, even at very high concentrations of SH1-blocked HMM in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked HMM concentration. All of these results confirmed the refractory state model for both SH1-blocked HMM and unmodified HMM and S-1. However, the nature of the small amount of HMM which did bind to actin in the presence of ATP at high actin concentration remains unclear.