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 80869-V/asset/equation/feb2001457939380869v-math-si1.gif?v=1&s=4fbbeeb0223e8194b6156baaf69d8925940d87c8)
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.
