Effects of phosphorylation, calcium ion, and tropomyosin on actin-activated adenosine 5'-triphosphatase activity of mammalian smooth muscle myosin

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Abstract
Actomyosin isolated from bovine stomach muscle contains the endogenous L chain kinase and phosphatase. Myosin can be separated from other proteins by gel filtration on a Sepharose 4B-agarose column. The amount of phosphate covalently bound to the 20,000-dalton L chains of purified myosin can be controlled by phosphorylation or dephosphorylation using the endogenous enzymes prior to column purification. The purified myosin can serve as a substrate for exogenously added L chain kinase and phosphatase, but the myosin itself is free of the activities for both enzymes. The ATPase activity of myosin was activated by rabbit skeletal muscle actin [A] only when the 20,000-dalton L chain was phosphorylated. The level of activation correlated with the amount of phosphate bound to the L chain. The maximum activation by pure A was observed when the molar ratio of myosin to A was 1:20. The activation was dependent on the amount of phosphate bound to the myosin L chain at all levels of A concentrations. The A-activated ATPase activity of stomach muscle myosin is not dependent on Ca2+ concentration once the myosin is phosphorylated and is free of kinase and phosphatase activity. The A-activated ATPase activity was higher when the A was complexed with tropomyosin [Tm]. The highest level of activation was obtained when the myosin was fully phosphorylated and the A was complexed with Tm at a molar ratio of 1:6 (Tm/A). The potentiation of A-activated ATP hydrolysis by Tm is not dependent on Ca2+. Tm apparently plays a major role in the A-activated ATP hydrolysis by smooth muscle myosin in the absence of other regulatory proteins.