Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids.
The Ca2+-pumping ATPase from human erythrocyte membranes, purified nearly to homogeneity (Niggli, V., Penniston, J. T., and Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958), can be reconstituted into phospholipid vesicles. The purified and the reconstituted forms of the enzyme displayed the properties expected of the intact Ca2+ pump; they had an appropriate (Ca2+-Mg2+)-ATPase activity which displayed a relatively low affinity for Ca2+. Added calmodulin increased both the maximum rate and the affinity for Ca2+ of the enzyme. Mg2+ alone caused no significant ATP hydrolysis in the purified enzyme, indicating that the Mg2+-ATPase is a separate enzyme. Vesicles of the reconstituted enzyme accumulated Ca2+ with a ratio of Ca2+ accumulated to ATP hydrolyzed of approximately 1. Ca2+ accumulation and ATPase of the reconstituted enzyme were inhibited concurrently by vanadate ion, with a K 1/2 for inhibition which was indistinguishable from that observed for the (Ca2+-Mg2+)-ATPase in whole erythrocyte ghosts. While the above properties were all consistent with those observed for the (Ca2+-Mg2+)-ATPase in whole erythrocyte ghosts, the purified enzyme displayed an unexpected response to acidic phospholipids. Enzyme reconstituted with or prepared in phosphatidylserine acted as if calmodulin were already present, and added calmodulin caused no effect beyond that due to phosphatidylserine. This mimicry of the calmodulin effect by acidic phospholipids is similar to that reported for cyclic nucleotide phosphodiesterase (Wolff, D. J., and Brostrom, C. O. (1976) Arch. Biochem. Biophys., 173, 720-723).
D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D002147:Calmodulin / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010743:Phospholipids / Q000494:pharmacology*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D008081:Liposomes; D010743:Phospholipids; D000001:Calcimycin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium