Regulation of actin-activated ATP hydrolysis by arterial myosin.

Abstract

Myosin was isolated from the main pulmonary artery of swine and was phosphorylated or dephosphorylated by utilizing the endogenous kinase or phosphatase, respectively. The myosins, phosphorylated to various degrees, were purified free of kinase and phosphatase activities by gel filtration on Sepharose CL-4B agarose columns. The level of actin-activated ATPase activity was dependent upon the degree of myosin L chain phosphorylation. Fully phosphorylated myosin reconstituted with actin and tropomyosin (actin/tropomyosin = 6:1) had the highest ATPase activity (0.1 .mu.mol Pi/mg .cntdot. min). The actin-activated ATPase activity showed maximal (60-65%) Ca2+ sensitivity at 2 mol Ca2+ bound/mol myosin. The actin-activated ATPase activity, Ca2+ binding and Ca2+ sensitivity of arterial myosin were dependent upon Mg2+ concentration. The ATPase activity was maximal at 2-3 mM Mg2+ and at low (0.5 mM) Mg2+ concentrations, the activity was only 1/3 of the maximal activity. Increasing the Mg2+ above 3 mM was not associated with a further increase in ATPase activity, but the Ca2+ binding and Ca2+ sensitivity decreased with increasing Mg2+ concentration. The maximal Ca2+ sensitivity was observed at 2-3 mM Mg2+, a concentration at which the myosin bound 2 mol Ca2+/mol. Both the ATPase activity and the Ca2+ sensitivity were more remarkable when actin that contained tropomyosin was used to activate the ATPase activity. Ca regulates the actin-activated ATP hydrolysis not only by its effects on the phosphorylation system but also by direct binding to the myosin.