Effects of phosphorylation, calcium ion, and tropomyosin on actin-activated adenosine 5'-triphosphatase activity of mammalian smooth muscle myosin.
Actomyosin isolated from bovine stomach muscle contains the endogenous light-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 light 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 light-chain kinase and phosphatase, but the myosin itself is free of the activities for both enzymes. The adenosine 5'-triphosphatase (ATPase) activity of myosin was activated by rabbit skeletal muscle actin only when the 20 000-dalton light chain was phosphorylated. The level of activation correlated with the amount of phosphate bound to the light chain. The maximum activation by pure actin was observed when the molar ratio of myosin to actin was 1:20. The activation was dependent on the amount of phosphate bound to the myosin light chain at all levels of actin concentrations. The actin-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 actin-activated ATPase activity was higher when the actin was complexed with tropomyosin. The highest level of activation was obtained when the myosin was fully phosphorylated and the actin was complexed with tropomyosin at a molar ratio of 1:6 (Tm/A). The potentiation of actin-activated ATP hydrolysis by tropomyosin is not dependent on Ca2+. These data indicate that tropomyosin plays a major role in the actin-activated ATP hydrolysis by smooth muscle myosin in the absence of other regulatory proteins.
D000199:Actins / Q000494:pharmacology; D000205:Actomyosin / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000302:isolation & purification; D010766:Phosphorylation; D014335:Tropomyosin / Q000494:pharmacology
D000199:Actins; D014335:Tropomyosin; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium