Mechanism of glutamate transport in Escherichia coli B. 2. Kinetics of glutamate transport driven by artificially imposed proton and sodium ion gradients across the cytoplasmic membrane

Abstract

Simultaneous imposition of a pH gradient (delta pH, interior alkaline) and a sodium gradient (delta pNa, [Na+]out greater than [Na+]in) across cytoplasmic membrane vesicles from Escherichia coli B led to a several hundred fold accumulation of glutamate. Although less effective, delta pH (interior alkaline)( alone caused accumulation of glutamate in the presence of Na+. In addition, delta pNa ([Na+]out greater than [Na+]in) alone also drove the transport system, where the maximum level of glutamate accumulation was affected by the pH of the medium. A membrane potential imposed by valinomycin-induced K+ diffusion (interior negative) enhanced the accumulation, indicating that the system operation in an electrogenic manner. The Michaelis constant of glutamate transport was greatly affected by changes in the concentrations of both Na+ and H+ and could be expressed by a linear combination of the reciprocals of the Na+ and H+ concentrations in the medium. On the contrary, a membrane potential (interior negative) exerted its effect by increasing the maximum velocity. When membrane vesicles were loaded with glutamate and Na+, but not with glutamate alone, rapid efflux of glutamate with Na+ as the cocation down the concentration gradients took place upon dilution. These results indicate that both Na+ and H+ are syn-coupled ions of glutamate transport in E. coli B and that the carrier/Na+/H+/Glu- complex observed in the binding reaction is an intermediate in the transport.