Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20,000-dalton light chains

Abstract

The proteolysis of [turkey] gizzard myosin by S. aureus protease produces both heavy meromyosin and subfragment 1 in which the 20,000-dalton L chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin H chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly H meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at .apprx. 68,000 and 94,000 daltons from the N-terminus of the myosin H chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20,000-dalton L chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax .apprx. 5.6 s-1 and Ka (the apparent dissociation constant for actin) .apprx. 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax .apprx. 0.2 s-1 and Ka .apprx. 7 mg/ml. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/ml, respectively. The Mg2+- and Ca2+-ATPase activities of subfragment 1 are distinct from those of heavy meromyosin, and the Mg2+-ATPase activity of subfragment 1 is not affected by phosphorylation. The phosphorylation of the 20,000-dalton L chain of subfragment 1 apparently is not required for actin-activated ATPase activity. Regulation via phosphorylation involves the interaction of the myosin heads with other parts of the molecule, possibly in the subfragment 2 region.