A Chemically Modified Subfragment-1 of Myosin from Skeletal Muscle as a Novel Tool for Identifying the Function of Actomyosin in Non-Muscle Cells1

Two kinds of subfragment-1 of myosin, S-1(T) and S-1(CT), were prepared by two-step tryptic EC 3.4.21.4 digestion of myosin that had been modified with about 1 mol of p-chloromercuribenzoate (CMB) per mol of myosin, and one-step chymotryptic EC 3.4.21.1 digestion of the myosin, respectively. The amount of bound CMB was about 0.82–0.90 mol per 2 mol of S-1. Both kinds of S-1 modified with CMB equally inhibited superprecipitation of mlyosin B from rabbit skeletal muscle. About 2 mol of CMB-S-1 (1 mol of CMB-S-1A) inhibited the function of 1 mol of actin monomer on the superprecipitation of actomyosin reconstituted from myosin and fibrous actin(FA) with relaxing protein (RP). CMB-S-1 also effectively inhibited superprecipitation of myosin B from the plasmodia of the slime mold Physarum polycephalum. The ATPase EC 3.6.1.3 activity of CMB-S-1(T) was similar to that of CMIB-S-1(CT) in the absence of FA, but was not enhanced as effectively by FA as the latter. In the presence of 0.3 mg/ml of FA with RP, the activity of CMB-S-1(T) was only one-fifth of that of CMB-S-1 (CT). CMB-S-1(T) did not affect the activities of ATPase from animal cells outside actomyosin systems, such as the Ca2+-dependent ATPase EC 3.6.1.3 of the SR prepared from rabbit skeletal muscle and the Na+, K+-dependent ATPase EC 3.6.1.3 from porcine kidney. It also scarcely affected Ca2+-uptake by the SR at concentrations lower than 0.2 mg/ml. However, CMB-S-1(T) strongly inhibited the polymerization and depolymerization of tubulin prepared from bovine brain. At 0.15 mol per mol of tubulin heterodimer, CMB-S-1(T) inhibited by 50% the extent of polymerization of 0.80 mg/ml tubulin (7.3 µM tubulin heterodimer). S-1(T) also inhibited tubulin polymerization as effectively as CMB-S-1(T). CMB-S-1(CMB-S-1A) also weakly bound itself to polymerized tubulin. It was concluded that CMB-S-1(T) can be used as a specific inhibitor of actin functions in non-muscle cells if the possible involvement of tubulin is excluded by other means.