Reconstitution of calcium transporters from Azotobacter vinelandii membranes

Abstract

Plasma membranes from Azotobacter vinelandii contain two Ca 2+ transport activities: an electrophoretic uniporter and an electroneutral Ca 2+ 2 H + 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 Ca 2+ and by transfer of the transport activities to vesicles with a buoyant density less than that of native membranes. The Ca 2+ transporters, reconstituted into K +-filled proteoliposomes, retained their dependence on the membrane potential or ΔpH 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 Ca 2+ transporter to ruthenium red, morpholinoethanesulfonate, and external K + and of the Ca 2+ 2 H + antiporter to Sr 2+ and heat treatment were also retained by the reconstituted system.