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 hemolysates. 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 microM 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 strength, 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.

Divalent cation dependent ATPase activities of red blood cell membranes: Influence of the oxidation of membrane thiol groups close to each other

“…It is relevant to note that our membranes were prepared in EDTA buffers (see Materials and Methods), which, according to the reports of Katz et a1 [23] may lead to membrane preparations devoided of activator protein. On the other hand, Lynch and Cheung [l 11 have demonstrated that erythrocyte membranes can not be completely depleted of calmodulin, even by treatment with such a strong Ca2+ chelator as EGTA.…”

“…The gel pictures in Figure 1 A, B, and C, when compared with the reports by other authors [21, 221 who used preparation buffers without EDTA, seem to exclude any important qualitative loss of the main protein bands. However, it is important to note that SDS-gel electrophoresis cannot exclude the possibility of a selective extraction of the activator protein by EDTA buffers, as described by other authors [23].…”

Characterization of the Ca2+‐ and Mg2+‐Dependent ATPases in Electrophorus Electroplax Microsomes

“…To assay for the low-affinity ATPase activity, higher concentrations of ATP were used (3 mM). In red cells, Katz et al (1979) found that only the high-affinity enzyme was active at low ATP concentrations (2-20 p M ) , whereas higher concentrations of ATP were needed for the expression of the low-affinity ATPase (above 200 p M ) . When the ATP concentration was more than 1 mM, phosphate was determined by the Lowry-Lopez method (1946), with phosphate standards of 0 to 160 nmolilOO pl.…”

Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum

“…This is comparable to results obtained by a number of other researchers. Katz et al (1979) showed that maximal calmodulin concentrations would 2+ 2+ stimulate Mg -Ca -ATPase 111% in identical membranes, at 0.1 /(M free 2+ Ca . Niggli et al (1979) using EDTA treated membranes, which they 2+ 2+ refer to as hypotonic ghosts, obtained a 301% stimulation of Mg -Ca ATPase activity in the presence of 2/4g calmodulin/mg ghost protein and 2+ 50/vj M Ca .…”