KINETIC-PROPERTIES OF NHAB, A NA+ H+ ANTIPORTER FROM ESCHERICHIA-COLI/

NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, pur ified, and reconstituted in a functional state, demonstrating that a s ingle polypeptide, the product of the nhaB gene, can catalyze full act ivity. 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 t he absence of passive and mediated leaks. The activity of NhaB was fou nd to have some pH dependence; the apparent K-m for Na+ changes by 10- fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the V-max rem ains constant. It was demonstrated that NhaB is electrogenic and trans locates more H+ than Na+ per cycle; the rate of sodium efflux from pro teoliposomes 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 meas ured at various Na+ gradients. A kinetic method, in which the electrop horetic movement of Rb-86(+) (in the presence of valinomycin) was moni tored in parallel with measurements of NhaB-mediated Na-22(+) uptake, allowed us to determine a stoichiometry of 3H(+)/ 2Na(+). The signific ance of the existence of two antiporters with different stoichiometrie s, NhaA and NhaB, active in the same cell, is discussed.