Substructure of the myosin molecule: IV. Interactions of myosin and its subfragments with adenosine triphosphate and F-actin

Abstract

The enzymic activity of several single-headed subfragments of myosin (HMM S-1 and single-headed HMM) has been compared to the double-headed derivative of myosin (HMM) both in the presence and absence of aetin. Under the assay conditions of our experiments, we find that HMM hydrolyses ATP at approximately twice the rate of any single-headed species. These results suggest a relatively independent functional role for each of the two heads of the myosin molecule. An attempt has been made to determine the stoichiometry of association between subfragments and actin, either in the absence of nucleotide or during the hydrolysis of ATP. It was originally thought that a comparison of the maximum turnover rate of HMM at infinite concentrations of actin with the maximum rate at infinite concentrations of enzyme (but with a fixed amount of actin) would yield the combining ratio of actin to HMM. However, the considerable variation of ATP turnover rates with the conditions of the experiment has made it impossible to reach any firm conclusions regarding stoichiometry. A more direct approach to the question of stoichiometry is possible in the absence of ATP. By reacting varying amounts of F-actin with a given concentration of subfragment and centrifuging the resulting complex, it is possible to determine the unbound concentration of subfragment in the supernatant. These data provide sufficient information to construct a Scatchard plot and show that twice as many moles of actin are bound by HMM as by HMM S-1. Furthermore, the association constant of actin for HMM is several orders of magnitude higher than that for the single-headed species. In connection with the question of why myosin has two “heads”, we have examined the ability of single-headed molecules to undergo the phenomenon of “superprecipitation”. We find that single-headed myosin (the preparation of which was discussed in the preceding paper) is able to superprecipitate in much the same manner as native myosin. We conclude from these studies that each head of the myosin molecule is able to function in a relatively independent fashion. These studies do not, of course, exclude the possibility of more subtle interactions between the heads of myosin which our techniques are not able to detect.