Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp

Abstract

The effects of imposed proton motive force on the kinetic properties of the alkalophilic BaciUus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of A4 (membrane potential, interior negative) and/or ApH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed A* increased the Na+ efflux rate (F) linearly, and the slope of V versus A* was higher at pH 9 than at pH 8. Kinetic experiments indicated that the A* caused a pronounced increase in the VM.X for Na+ efflux, whereas the Km values for Na+ were unaffected by the A*. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in ApH-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 A* 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.Except in the case of some bacteria (6, 13), it has been generally accepted that the Na+/H+ antiporter plays a role in pH homeostasis of bacterial cells in an alkaline environment (11,15,27). In our previous paper (12), we reported that in alkalophilic Bacillus sp. strain N-6, Na+-or Li'-loaded right-side-out vesicles acidified the interior after the addition of respiratory substrates, ascorbate plus tetramethyl-p-phenylenediamine. Na+ was also extruded from Na+-loaded vesicles in the presence of electron donors. Furthermore, Na+ was extruded upon imposition of an outwardly directed K+ diffusion potential (interior negative). These results suggested that the Na+/H+ antiporter system in this strain operated electrogenically and played a role in pH homeostasis at alkaline pH range. The Na+/H+ antiporter has been assayed in bacterial membrane vesicles by several approaches (14), including assaying (i) the effect of Na+ on H+ efflux from everted membrane vesicles in the presence of electron donors (5, 22, 25), (ii) the effect of Na+ on H+ influx into right-side-out vesicles (24), and (iii) Na+ efflux from Na+-loaded right-side-out vesicles by imposed ApH (interior alkaline) and/or imposed A* (interior negative) (3, 4).As mentioned above, 22Na+ was extruded from 22Na+-loaded right-side-out vesicles in Bacillus sp. strain N-6 upon imposition of Ai (interior negative). If this Na+ efflux is coupled with H+ influx into vesicles through an Na+/H+ antiporter system, the acidification of the interiors of the vesicles could be observed with the imposition of...

Potassium-induced inhibition of photosynthesis and associated electron transport chain of Microcystis: Implication for controlling cyanobacterial blooms

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Enigmatic Microorganisms and Life in Extreme Environments

“…The challenge of alkaliphiles to the chemiosmotic theory is that reversed pH difference, ego 2.5 unit in a growth at pHIl, generates a reverse force of around 150mV (Grant, 1989). show a sodium dependent movement of proton (Kitada and Horikoshi, 1992). A sodium/proton antiporter is strongly implicated in the pH homeostasis in alkaliphiles.…”

Properties of two different Na+/H+ antiport systems in alkaliphilic Bacillus sp. strain C-125

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