Influence of EGTA on the apparent Ca 2+ affinity of Mg 2+-dependent, Ca 2+-stimulated ATPase in the human erythrocyte membrane

Abstract

The apparent Ca 2+ affinity of Mg 2+-dependent, Ca 2+-stimulated ATPase ((Mg 2+ + Ca 2+)-ATPase) in human erythrocyte membranes increased with increasing concentrations of EGTA used to buffer free Ca 2+. The shift in apparent Ca 2+ 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 Ca 2+ affinity without increasing V. EGTA also increased the apparent Ca 2+ 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 Ca 2+ was raised above 35 μM at 1.0 mM EGTA, binding progressively increased up to 100 μM or more free Ca 2+ at 0.1 mM EGTA. EGTA also increased the Ca 2+ affinity of Triton X-100-solubilized (Mg 2+ + Ca 2+)-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 Ca 2+ affinity state of the enzyme was converted to a high Ca 2+ affinity state in the presence of EGTA. The species associated with the enzyme interconversion appeared to be [CaEGTA] 2−.