β‐d‐Galactoside Transport in Escherichia coli

Abstract

At relatively low concentrations (< 3 M) the aprotic solvents dimethylsulfoxide, N-methylpyrrolidone, tetramethylurea and hexamethylphosphoric triamide inhibited .beta.-D-galactoside transport by whole cells and the derived membrane vesicles of E. coli. Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure. At high concentrations of aprotic solvent (9 parts hexamethylphosphoric triamide or N-methylpyrrolidone/1 part buffer, 100 mM LiCl), 50-80% of the total protein of transport-positive membrane vesicles from E. coli ML 308-225 were solubilized. When these extracts were added, with constant sonication, to intact transport-negative vesicles derived from E. coli ML 35, lactose uptake was reconstituted according to the following criteria. Uptake (12 nmol/mg protein) required an energy source, provided by oxidation of D-lactate or ascorbate/phenazine methosulfate or by a K+-diffusion potential established with the aid of valinomycin. Uptake was completely abolished in the presence of uncoupler (carbonyl cyanide m-chlorophenylhydrazone, 1 .mu.M) or of high-affinity substrate (thio-.beta.-D-digalactoside, 5 mM).