Separation and Characterization of Two Mg2+-ATPase Activities from the Human Erythrocyte Membrane

Two distinct Mg2+-ATPase activities were isolated from Triton X-100-solubilized human erythrocyte membranes using a combination of calmodulin-agarose to remove Ca2+-ATPase and ion exchange chromatography to separate the Mg2+-ATPase activities. The activity designated Mg2+-ATPase A was inhibited by low concentrations of vanadate (IC50 ≍ 2 μM) and by calcium (IC50 ≍ 1.5 mM), La3+ (80% inhibition at 0.5 mM), and F− (IC50 ≍ 1 mM). Inhibition by F− increased in the presence of 10 μM AlCl3. The activity appeared to be dependent on the concentration of MgATP. Two millimolar deoxyATP supported nearly full activity and 2 mM GTP supported 40% of the activity compared to that seen with 2 mM ATP. The activity was stimicrolated twofold by the addition of 1% (w/v) phosphatidylserine. These properties are consistent with a role of this enzyme in the control of red blood cell shape, possibly through association with the ATP-dependent translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the bilayer. In contrast, Mg2+-ATPase B was slightly stimicrolated by vanadate and by calcium but was unaffected by LaCl3, and by F− ± AlCl3. The activity was further stimicrolated by added magnesium in excess of fixed concentrations of ATP. In addition, at a fixed concentration of 10 mM MgCl2, the activity with increasing ATP was biphasic, with the maximicrom at 1 mM. GTP 2 mM failed to support activity while 2 mM deoxyATP supported only 30% of activity compared to that seen with 2 mM ATP. The activity was not stimicrolated by oxidized glutathione or the glutathione conjugate lithocholic acid, suggesting it is not involved in the ATP-dependent transport of such compounds out of the erythrocyte.