MECHANISM OF NA+/H+ EXCHANGE BY ESCHERICHIA-COLI NHAA 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 Delta pH-driven sodium uptake into NH4 Cl-loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non-radioactive Nat and placed into the experimental buffer containing low amounts of Na-22(+) under experimental conditions when both components of protonmotive force generated by the antiporter. Delta psi and Delta pH, were dissipated by corresponding ionophores. The apparent K-m for sodium counterflow is 1.1 mM, and V-max is 80 mu mol/min/mg of protein. External Na+ accelerates the downhill efflux of Na-22(+) suggesting that the translocation of the Na+-loaded form of the carrier is faster than the rest of the catalytic cycle.