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A calcium activated magnesium dependent atpase with high affinities for both calcium and magnesium in vascular smooth muscle microsomes comparison with plasma membrane calcium pump atpase



A calcium activated magnesium dependent atpase with high affinities for both calcium and magnesium in vascular smooth muscle microsomes comparison with plasma membrane calcium pump atpase



Journal of Biochemistry (Tokyo) 108(5): 730-736



A Ca2+-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 .mu.M) 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 .mu.M 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 nucleotide 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 .mu.g/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 .mu.M, 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 (0.5-9 .mu.M) of vanadate or by 1-100 .mu.M 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 .mu.M 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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Accession: 006931600

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