Ca2+ activation of membrane-bound (Ca2++Mg2+)-dependent ATPase from human erythrocytes prepared in the presence or absence of Ca2+.
The kinetics of Ca2+ activation of membrane-bound (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase EC 3.6.1.3) from human erythrocytes was studied. The ATPase from membrane prepared in the presence of 0.7-500muM Ca2+ showed positively cooperative behaviour and a Km for Ca2+ of between 1 and 4 muM. If the membranes were prepared in the absence of Ca2+ the Km increased, and an enzyme model with at least four calcium-binding sites accounted for the kinetic change assuming that one calcium-binding site decreased its affinity. Mg2+ or Mg-ATP could not replace Ca2+. Continuous-flow centrifugation involving a shear stress on membranes was necessary to obtain the high affinity ATPase activity. Using ordinary centrifugation the Ca2+-prepared membranes behaved as membranes prepared in the absence of Ca2+. The Ca2+-stimulated ATPase from membranes prepared without Ca2+ showed reduced maximum activity, but dialyzed, membrane-free hemolysates, whether prepared with Ca2+ present or not, recovered the activity when the hemolysate was present during the ATPase assay. It is suggested that the different Ca2+-affinities of the Ca2+-stimulated ATPase correspond to two different states of the calcium-pump.
D000251:Adenosine Triphosphatases / Q000097:blood*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002021:Buffers; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D003956:Dialysis; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid; D004912:Erythrocytes / Q000201:enzymology*; D005615:Freezing; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood; D006461:Hemolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D011485:Protein Binding
D002021:Buffers; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium