Na +/solute symport in membrane vesicles from Bacillus alcalophilus

Abstract

The characteristics of α-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited α-aminoisobutyric acid uptake upon energization with ascorbate and N, N, N′, N′-tetramethyl- p-phenylenediamine. The presence of Na + caused a pronounced reduction in the Km for α-aminoisobutyric acid in wild-type but not KM23 vesicles; the maximum velocity ( V) was unaffected in vesicles from both strains. Passive efflux and exchange of α-aminoisobutyric acid from wild-type vesicles were Na +-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of α-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na +-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in l-methionine transport indicated that the porters, i.e., the solute-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na +-independent, H +-coupled porters upon mutation of B. alcalophilus to non-alkalophily.