Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis

Abstract

Ca2+ was accumulated in inside-out membrane vesicles of Bacillus subtilis when NADH was used as an energy source. A .DELTA.pH (acid interior) could also drive Ca2+ accumulation in the membrane vesicles and the accumulation was inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin plus K+. These results indicate the presence of a Ca2+/H+ antiporter (exchanger) in this organism. The antiporter was isolated and purified to homogeneity from the membrane proteins by chromatography on hydroxyapatite, diethylaminoethyl(DEAE)-Toyopearl 650 M and butyl-Toyopearl 650 M. The purified antiporter has a molecular mass of about 45,000 daltons and an isoelectric point of 5.0. The fluorescence quenching of a cyanine dye (3,3''-dipropylthiodicarbocyanine iodide [diS-C3-(5)]) during Ca2+ accumulation in proteoliposomes by the purified antiporter showed the generation of a membrane potential (interior negative) suggesting a H+/Ca2+ stoichiometry above 2 in the transport. This was also supported by the result that the K+-diffusion potential, interior positive, stimulated the Ca2+ uptake in the presence of a .DELTA.pH. The apparent Km for Ca2+ of the antiporter was about 40 .mu.M and La3+ inhibited the transport. Amino acid analysis of the purified antiporter indicated the presence of large amounts of glutamic and aspartic acids and small amounts of histidine, lysine and arginine. This is consistent with the low isoelectric point (about 5.0) of the protein.