Mechanism of Na⁺/H⁺exchange byEscherichia coliNhaA in reconstituted proteoliposomes

Purified NhaA, a Na⁺/H⁺ antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein. Homologous Na⁺/Na⁺ exchange as well as Na⁺/Li⁺ exchange via NhaA were detected by monitoring the effects of external Li⁺ and Na⁺ ions on the ΔpH‐driven sodium uptake into NH₄ Cl‐loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non‐radioactive Na⁺ and placed into the experimental buffer containing low amounts of ²²Na⁺ under experimental conditions when both components of protonmotive force generated by the antiporter. ΔΨ and ΔpH, were dissipated by corresponding ionophores. The apparent K _m for sodium counterflow is 1.1 mM, and V _max is 80 of protein. External Na⁺ accelerates the downhill efflux of ²²Na⁺ suggesting that the translocation of the Na⁺loaded form of the carrier is faster than the rest of the catalytic cycle.