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Kinetic and functional studies of (Mg+Ca)-ATPase activities in human erythrocyte membranes Quist, E. E.


The presence of both high and low affinity (Mg+Ca)-ATPase activities was shown kinetically in a number of membrane preparations derived from human erythrocytes. The (Mg+Ca)-ATPase activity in membranes prepared in both the presence and absence of EDTA had similar affinity for Ca²⁺refuting the hypothesis that low affinity (Mg+Ca)-ATPase activity is an artifact resulting from denaturation of high affinity (Mg+Ca)-ATPase to a low affinity form. It was found that high affinity (Mg+Ca)-ATPase activity was lost in a pH dependent manner from ghost membranes incubated in low ionic strength media, (less than 2 mosm). The loss of high affinity (Mg+Ca)-ATPase activity corresponded to a loss of approximately 25% of the membrane proteins. The soluble protein fraction itself had no (Mg+Ca)-ATPase activity . However, if the protein fraction was concentrated and recombined with the residual membranes, high affinity (Mg+Ca)-ATPase activity was restored. The soluble component required for restoration of high affinity (Mg+Ca)-ATPase activity was found to be protein consisting of two peptide chains of molecular weight greater than 200,000 daltons when analyzed by SDS acrylamide gel electrophoresis. A number of observations suggested that low affinity (Mg+Ca)-ATPase activity was phospholipid dependent in contrast to high affinity (Mg+Ca)-ATPase activity. Treatment of intact erythrocytes or resealed ghosts with trypsin on the outer surface of cells resulted in a reduction of high and low affinity (Mg+Ca)-ATPase activities in ghost membranes prepared from these cells. This result was an indication that polypeptides required for both activities spanned the membrane. Ruthenium red and LaCl₃ were found to be non-selective inhibitors of high and low (Mg+Ca)-ATPase activities. Both (Mg+Ca)-ATPase activities were inhibited with similar potencies by these agents. A stoichiometry of two was determined for calcium transport in resealed erythrocytes using lanthanum as a selective inhibitor. Addition of 0.1 mM lanthanum in the external medium of ghosts resealed with 3 mM CaCl₂ inhibited calcium transport 100% and total Mg²⁺ and Ca²⁺ dependent ATPase activities by 50%, indicating that all (Mg+Ca)-ATPase activity in resealed ghosts was not associated with calcium transport. Schatzmann (1973) previously estimated the stoichiometry to be one. A stoichiometry of two was found here in resealed ghosts prepared in the presence or absence of 1.0 mM EDTA during the hemolysis precedure. In resealed vesicles containing only low affinity (Mg+Ca)-ATPase activity, ATP dependent calcium uptake into inside-out vesicles and low affinity (Mg+Ca)-ATPase activity were stimulated by external Ca²⁺ With similar affinities.. This finding suggests that calcium transport is associated with low affinity (Mg+Ca)-ATPase activity in contrast to the suggestion by Schatzmann (1973) that calcium transport is associated with high affinity (Mg+Ca)-ATPase activity in resealed ghosts. Restoration of high affinity (Mg+Ca)-ATPase activity by the addition of soluble protein fraction containing spectrin increased (Mg+Ca)-ATPase activity over two fold without affecting the velocity of calcium uptake. Therefore, high affinity (Mg+Ca)-ATPase activity may be associated with some function other than calcium transport. Kinetic plots revealed that the Ca²⁺ activation of low affinity (Mg+Ca)-ATPase activity showed negative cooperativity. A negative cooperative mechanism for calcium transport would be advantageous for erythrocyte survival, since calcium could be transported efficiently over a wide range of intracellular calcium concentrations (0.3 to 300 μm Ca²⁺).

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