Reconstitution of calcium transporters from Azotobacter vinelandii membranes

Plasma membranes from Azotobacter vinelandii contain two Ca2+ transport activities: an electrophoretic uniporter and an electroneutral Ca2+/2H+ exchanger (P. Zimniak and E. M. Barnes, Jr. J. Biol. Chem. 255, 10,140 (1980)). Both activities were reconstituted by the freeze-thaw technique of M. Kasahara and P. C. Hinkle (J. Biol. Chem. 252, 7384 (1977)) using phosphatidylcholine/phosphatidylethanolamine (1:1) at a lipid-to-protein ratio of 40. Reconstitution was evidenced both by expansion of the intravesicular volume accessible to Ca2+ and by transfer of the transport activities to vesicles with a buoyant density less than that of native membranes. The Ca2+ transporters, reconstituted into K+-filled proteoliposomes, retained their dependence on the membrane potential or delta pH induced by the addition of valinomycin or nigericin, respectively. The kinetic parameters of the reconstituted activities were similar to those in native membranes, as was their sensitivity to inhibitors. The sensitivities of the electrophoretic Ca2+ transporter to ruthenium red, morpholinoethanesulfonate, and external K+ and of the Ca2+/2H+ antiporter to Sr2+ and heat treatment were also retained by the reconstituted system.