Key result
A high-affinity Ca2+-ATPase in porcine vascular smooth muscle requires minute Mg2+ for activity, has broad substrate specificity, and is distinct from the plasma membrane Ca2+-pump ATPase.
A distinct high-affinity Ca2+-ATPase coexists with the Ca2+-pump ATPase in the plasma membrane of porcine vascular smooth muscles, characterized by unique Mg2+ dependence and substrate specificity.
Does not inform clinical vascular management; leaves open the functional role of this distinct ATPase in human smooth muscle.
A Ca2+-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 μM) 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 0.1 μM 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, 10 μg/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 μM, 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 (0.6–9 μM) of vanadate or by 1–100 μM 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 1–100 μM 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.
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Sun et al. (1990) studied this question. Ca2+-ATPase characterization vs. Plasma membrane Ca2+-pump ATPase was evaluated on Enzyme activity and characteristics. A high-affinity Ca2+-ATPase in porcine vascular smooth muscle requires minute Mg2+ for activity, has broad substrate specificity, and is distinct from the plasma membrane Ca2+-pump ATPase.
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