KINETIC-PROPERTIES OF ELECTROGENIC NA+/H+ ANTIPORT IN MEMBRANE-VESICLES FROM AN ALKALOPHILIC BACILLUS SP

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of DELTA-psi (membrane potential, interior negative) and/or DELTA-pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed DELTA-psi increased the Na+ efflux rate (V) linearly, and the slope of V versus DELTA-psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the DELTA-psi caused a pronounced increase in the V(max) for Na+ efflux, whereas the K(m) values for Na+ were unaffected by the DELTA-psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent K(m) of the Na+ efflux reaction. These results have also been observed in DELTA-pH-driven Na+ efflux experiments. When Na+-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of DELTA-psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.