Na Translocation Dominates over H-Translocation in the Membrane Pyrophosphatase with Dual Transport Specificity.

Cation-pumping membrane pyrophosphatases (mPPases; EC 7.1.3.1) vary in their transport specificity from obligatory H transporters found in all kingdoms of life, to Na/H-co-transporters found in many prokaryotes. The available data suggest a unique "direct-coupling" mechanism of H transport, in which the transported proton is generated from nucleophilic water molecule. Na transport is best rationalized by assuming that the water-borne proton propels a prebound Na ion through the ion conductance channel ("billiard" mechanism). However, the "billiard" mechanism, in its simple form, is not applicable to the mPPases that simultaneously transport Na and H without evident competition between the cations (Na,H-PPases). In this study, we used a pyranine-based fluorescent assay to explore the relationship between the cation transport reactions catalyzed by recombinant Na,H-PPase in membrane vesicles. Under appropriately chosen conditions, including the addition of an H ionophore to convert Na influx into equivalent H efflux, the pyranine signal measures either H or Na translocation. Using a stopped-flow version of this assay, we demonstrate that H and Na are transported by Na,H-PPase in a ratio of approximately 1:8, which is independent of Na concentration. These findings were rationalized using an "extended billiard" model, whose most likely variant predicts the kinetic limitation of Na delivery to the pump-loading site.