Influence of EGTA on the apparent Ca2+ affinity of Mg2+-dependent, Ca2+-stimulated ATPase in the human erythrocyte membrane.
The apparent Ca2+ affinity of Mg2+-dependent, Ca2+-stimulated ATPase (Mg2+ + Ca2+)-ATPase) in human erythrocyte membranes increased with increasing concentrations of EGTA used to buffer free Ca2+. The shift in apparent Ca2+ affinity was seen in membranes prepared by hypotonic hemolysis and in membranes depleted of endogenous activators by EDTA treatment. The effect of EGTA differed from that of calmodulin, as it increased Ca2+ affinity without increasing V. EGTA also increased the apparent Ca2+ affinity when calmodulin was present in the assay medium. ATP-stimulated calcium binding to membranes was greater at 1 mM EGTA than at 0.1 mM EGTA. Similarly to ATPase activation, whereas binding decreased as Ca2+ was raised above 35 microM at 1.0 mM EGTA, binding progressively increased up to 100 microM or more free Ca2+ at 0.1 mM EGTA. EGTA also increased the Ca2+ affinity of Triton X-100-solubilized (Mg2+ + Ca2+)-ATPase, indicating that its effect did not depend on an intact membrane. Analysis of the kinetic data by a computerized nonlinear curve fitting procedure showed that a low Ca2+ affinity state of the enzyme was converted to a high Ca2+ affinity state in the presence of EGTA. The species associated with the enzyme interconversion appeared to be [CaEGTA]2-.
D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005026:Ethylene Glycols / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D011092:Polyethylene Glycols / Q000494:pharmacology
D002147:Calmodulin; D005026:Ethylene Glycols; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium