Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium.
The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 micrometer) and high (65 micrometer) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidylethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1-2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1-2 mumol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenylphosphatase partial reaction.
D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases; D002147:Calmodulin / Q000494:pharmacology*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010743:Phospholipids / Q000494:pharmacology*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D010743:Phospholipids; D015636:Magnesium Chloride; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium