title: Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli.
abstract: NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, purified, and reconstituted in a functional state, demonstrating that a single polypeptide, the product of the nhaB gene, can catalyze full activity. NhaB is a minor protein that accounts for less than 0.1% of the total membrane protein. The use of proteoliposomes made possible the determination of important kinetic and pharmacological properties in the absence of passive and mediated leaks. The activity of NhaB was found to have some pH dependence; the apparent Km for Na+ changes by 10-fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the Vmax remains constant. It was demonstrated that NhaB is electrogenic and translocates more H+ than Na+ per cycle; the rate of sodium efflux from proteoliposomes was accelerated by a membrane potential, negative inside, and NhaB activity generated a membrane potential as monitored by two techniques. The stoichiometry of NhaB was estimated by a thermodynamic method in which the magnitude of delta psi generated by NhaB was measured at various Na+ gradients. A kinetic method, in which the electrophoretic movement of 86Rb+ (in the presence of valinomycin) was monitored in parallel with measurements of NhaB-mediated 22Na+ uptake, allowed us to determine a stoichiometry of 3H+/2Na+. The significance of the existence of two antiporters with different stoichiometries, NhaA and NhaB, active in the same cell, is discussed.
authors.affiliation: Division of Microbial and Molecular Ecology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
authors.forename: E
authors.initials: E
authors.lastname: Pinner
authors.forename: E
authors.initials: E
authors.lastname: Padan
authors.forename: S
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D013816:Thermodynamics
chemical_list: D017923:Sodium-Hydrogen Exchangers; D012964:Sodium