Effects of Ca2+, Mg2+ and calmodulin on the formation and decomposition of the phosphorylated intermediate of the erythrocyte Ca2+-stimulated ATPase.
Formation of the phosphorylated intermediate (ECaP) of the human erythrocyte Ca2+-stimulated ATPase (Ca2+-ATPase) was more rapid and reached steady state sooner at 400 microM-Ca2+ than at 1 microM-Ca2+. Calmodulin increased the apparent rate of ECaP formation at 1 microM-Ca2+, whereas at 400 microM-Ca2+, calmodulin decreased the steady-state level of the ECaP without affecting its apparent rate of formation. Removal of endogenous Mg2+ with trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid, which decreased both the velocity and Ca2+-sensitivity of the Ca2+-ATPase, did not alter the Ca2+-sensitivity or the apparent rate of formation of ECaP. ECaP formation at high Ca2+ concentrations was not affected by Mg2+ concentrations as high as 1 mM, and the ECaP could be dephosphorylated by ADP and ATP along either the forward or reverse pathways. The results suggest that high Ca2+ concentrations inhibit Ca2+-ATPase activity by preventing dephosphorylation of the E2P complex, rather than by inhibition of the transformation from E1CaP ('high-Ca2+-affinity' ECaP) to E2CaP ('lower-energy' ECaP).
Allen
B G
BG
Division of Pharmacology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Katz
S
S
Roufogalis
B D
BD
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000097:blood*; D002147:Calmodulin / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation
D002147:Calmodulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium