Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane.
Solubilized and purified high-affinity (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the human erythrocyte membrane (Wolf, H.U., Dieckvoss, G. and Lichtner, R. (1977) Acta Biol. Ger. 36, 847) has been phosphorylated and dephosphorylated under various conditions with respect to Ca2+ and Mg2+ concentrations. In the range, 0.001--100 mM, the rate of phosphorylation was dependent on Ca2+ concentration, showing a maximum at 10 mM. The phosphorylation rate was nearly independent of the Mg2+ concentration within the range 0.01-1 mM. This enzyme has at least three Ca2+ binding sites with different affinities and regulatory functions: (1) binding to the high-affinity site yields phosphorylation of the enzyme; (2) binding to a low-affinity site (Ca2+ concentrations higher than 40 microM) inhibits dephosphorylation or the conformational change which is necessary for dephosphorylation; (3) by binding to an additional low-affinity site, Ca2+ at concentrations higher than 1 mM abolishes negative cooperative behaviour (shown below 1 mM Ca2+) and causes weak positive cooperativity between at least two catalytic subunits in the phosphorylation reaction. The phosphoprotein obtained at Ca2+ concentrations above 1 mM dephosphorylates spontaneously after removal of the divalent metal ions. Addition of Mg2+ accelerates the dephosphorylation rate. Affinities of the inhibitory Ca2+ binding sites are reduced by the binding of substrate or K+.
D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000097:blood / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding
D004533:Egtazic Acid; D004492:Edetic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D011188:Potassium; D002118:Calcium