Sarcolemmal (Ca²⁺+Mg²⁺)-ATPase of Vascular Smooth Muscle and the Effects of Protein Kinases Thereupon

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To elucidate the regulation mechanisms for sarcolemmal Ca²⁺-pumping ATPase of vascular smooth muscle, the preparation of the membrane fraction of porcine aorta with which the enzyme activity could be analyzed was attempted. A Ca²⁺-activated, Mg²⁺-dependent ATPase ((Ca²⁺+Mg²⁺)-ATPase) activity with high affinity for Ca²⁺(K_m =79±18nM) was found in a sarcolemma-enriched fraction obtained from digitonin-treated microsomes that possessed the essential properties of plasma membrane (PM) Ca²⁺-pumping ATPases, as determined for the erythrocyte and cardiac muscle enzymes. The activity was stimulatd by calmodulin and inhibited by low concentrations of vanadate. Saponin had a stimulatory effect on it. The existence of the PM enzyme in the membrane fraction was substantiated by the Ca²⁺-dependent, hydroxylamine sensitive phosphorylation of a 130K protein, which could be selectively enhanced by LaCl₃. The enzyme activity was potentiated by either cGMP or a purified G-kinase. Purified protein kinase C potentiated the enzyme activity. However, none of these agents stimulated the activity of the enzyme purified from microsomes by calmodulin affinity chromatography. The results suggest that the sarcolem-mal Ca²⁺-pumping ATPase of vascular smooth muscle is regulated by these protein kinases not through phosphorylation of the enzyme itself but through phosphorylation of mem-brane components(s) other than the enzyme. Phosphatidylinositol phosphate was found to stimulate the enzyme, suggesting its role in mediation of the stimulatory effects of the protein kinases.