Properties of (Mg2+ + Ca2+)‐ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP, and protein activator

Abstract

Erythrocyte membranes prepared by three different procedures showed (Mg²⁺ + Ca²⁺)-ATPase activities differing in specific activity and in affinity for Ca²⁺. The (Mg²⁺ + Ca²⁺)-ATPase activity of the three preparations was stimulated to different extents by a Ca²⁺-dependent protein activator isolated from hemolystes. The Ca²⁺ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg²⁺ + Ca²⁺)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca²⁺ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca²⁺ affinity with changes in ATP and MgCl₂ concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg²⁺ + Ca²⁺)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.