Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: Δψ-Dependent 22Na+ efflux from whole cells

Abstract

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (Δψ). In the absence of a Δψ, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a Δψ, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the Δψ. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the Δψ caused a pronounced increase in the V(max) for 22Na+ efflux. The K(m) values for Na+ were unaffected by the Δψ. Upon imposition of a Δψ at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the Δψ. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low Δψ generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibitied by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a Δψ at either pH 7.0 or 9.0.