Separation and characterization of two Mg(2+)-ATPase activities from the human erythrocyte membrane.
Two distinct Mg(2+)-ATPase activities were isolated from Triton X-100-solubilized human erythrocyte membranes using a combination of calmodulin-agarose to remove Ca(2+)-ATPase and ion exchange chromatography to separate the Mg(2+)-ATPase activities. The activity designated Mg(2+)-ATPase A was inhibited by low concentrations of vanadate (IC50 approximately 2 microM) and by calcium (IC50 approximately 1.5 mM), La3+ (80% inhibition at 0.5 mM), and F- (IC50 approximately 1 mM). Inhibition by F- increased in the presence of 10 microM 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 stimulated 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, Mg(2+)-ATPase B was slightly stimulated by vanadate and by calcium but was unaffected by LaCl3, and by F- +/- AlCl3. The activity was further stimulated 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 maximum 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 stimulated 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.
Department of Pharmacy, University of Sydney, NSW, Australia.
M E
ME
Auland
M B
MB
Morris
B D
BD
Roufogalis
D000255:Adenosine Triphosphate / Q000378:metabolism*; D017607:Aluminum Compounds / Q000494:pharmacology; D001692:Biological Transport; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002124:Calcium Fluoride / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D017607:Aluminum Compounds; D010743:Phospholipids; D015636:Magnesium Chloride; D014638:Vanadates; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D002124:Calcium Fluoride; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride