A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin

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Abstract
Treatment of extensively washed erythrocyte membranes with 0.1 mM-EDTA decreased their Mg2+-dependent, Ca2+-stimulated ATPase [(Mg2+ + Ca2+)-ATPase] activity. An activator released by this treatment restored the (Mg2+ + Ca2+)-ATPase to its original value in a Ca2+-dependent manner. This activator was different from calmodulin, as determined by a number of criteria. It was retained on an Amicon XM-100 ultrafiltration membrane (MW cut-off 100,000); it appeared in the void volume of Sephadex G-100 and G-75 columns; it was not retained on a DEAE-cellulose ion-exchange column at ionic strengths similar to those used to retain calmodulin; and it maximally activated (Mg2+ + Ca2+)-ATPase activity less than calmodulin and at a higher Ca2+ concentration. Like calmodulin, the activator is heat-stable. The activator fraction isolated on a 2.5-15% sucrose gradient in 0.16 mM-KCl showed a single band of MW 63,000 and no calmodulin on 10%-polyacrylamide/sodium dodecyl sulfate gels. A trace amount of calmodulin was detected in the activator fraction by radioimmunoassay (.apprx. 10 pg/ml of ghosts), but this amount was insufficient to account for (Mg2+ + Ca2+)-ATPase activation. Calmodulin-binding protein failed to inhibit (Mg2+ + Ca2+)-ATPase activity by more than 10-20% in the membrane preparations from which the activator was extracted. Erythrocyte membranes contain a (Mg2+ + Ca2+)-ATPase activator that may attenuate the activation of the Ca2+-transport ATPase by calmodulin.

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