β‐ d ‐Galactoside Transport in Escherichia coli

At relatively low concentrations (< 3 M) the aprotic solvents: dimethylsulfoxide, N‐methyl‐pyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited β‐D‐galactoside transport by whole cells, and the derived membrane vesicles of Escherichia 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‐methyl‐pyrrolidone/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. a) 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. b) Uptake was completely abolished in the presence of uncoupler (carbonyl cyanide m‐chlorophenylhydrazone, 1 μM) or of high‐affinity substrate (thio‐β‐d‐digalactoside, 5 mM).