Ca2+/H+ countertransport and electrogenicity in proteoliposomes containing erythrocyte plasma membrane Ca-ATPase and exogenous lipids.
A reconstituted proteoliposomal system was obtained with Ca-ATPase purified from human erythrocyte membrane (plasma membrane, PM ATPase), and liposomes prepared by reverse-phase evaporation. The reconstituted PM ATPase behaved as an electrogenic Ca2+/H+ exchanger and, under optimal conditions, utilization of 1 mol of ATP was accompanied by uptake of one Ca2+ by the vesicles, and ejection of one H+ from the lumen of the vesicles. Ca2+ uptake was greatly (5-fold) stimulated by the addition of calmodulin, and by collapsing the H+ gradient with the ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of calmodulin and p-trifluoromethoxyphenylhydrazone, the reconstituted system sustained transport rates of 1.00 +/- 0.12 mumol of Ca2+/mg of protein min-1 (30 degrees C), reaching asymptotic levels of 8.05 +/- 0.41 mumol of Ca2+/mg of protein (i.e. 20 mM lumenal Ca2+). The corresponding net charge transfer produced a maximal electrical gradient of 40.5 +/- 1.8 mV at steady state. Demonstration of the electrogenic behavior of the PM ATPase, obtained for the first time with these experiments, was critically dependent on the detergent used in the reconstitution procedure. The lumenal pH rise had a much greater rate-limiting effect on the pump, than the electrical potential developed by the pump.
Hao
L
L
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Rigaud
J L
JL
Inesi
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D011510:Proteolipids / Q000378:metabolism*
D011510:Proteolipids; C020485:proteoliposomes; D006859:Hydrogen; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium