A Ca²⁺-Activated, Mg²⁺-Dependent ATPase with High Affinities for Both Ca²⁺ and Mg²⁺ in Vascular Smooth Muscle Microsomes: Comparison with Plasma Membrane Ca²⁺-Pump ATPase

Abstract

A Ca²⁺-ATPase with a high affinity for free Ca²⁺ (apparent K_m of 0.13μM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2⁺-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca²⁺-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg²⁺, but required a minute amount of Mg²⁺ for its activity as evidenced by the findings that it was fully active in the presence of 0.1μM free Mg²⁺ but lost the activity in a reaction mixture containing trans-cyclohexane-1, 2-diamine-N, N, N', N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca²⁺ and Mg²⁺. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10μg/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240μM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca²⁺-ATPase activity was not modified either by low concentrations (0.5-9μM) of vanadate or by 1-100μM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity. In contrast, the plasma membrane Ca²⁺-pump ATPase purified from porcine aorta required millimolar Mg²⁺ for activity and could utilize only ATP as a substrate. Furthermore, the Ca²⁺-pump ATPase activity was sensitive to stimulation by calmodulin and inhibition by low concentrations of vanadate. In the presence of 1-100μM p-chloromercuribenzoic acid, the purified Ca²⁺-pump ATPase lost most of its activity. Therefore, it is concluded that the high-affinity Ca²⁺-ATPase is a distinct entity from the Ca²⁺-pump ATPase and coexists with the Ca²⁺-pump ATPase in the plasma membrane of porcine vascular smooth muscles.