Transport by reconstituted lactose carrier from parental and mutant strains of Escherichia coli.
The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lower Km but similar Vmax compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010447:Peptide Hydrolases; D027981:Symporters
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D010447:Peptide Hydrolases; D007785:Lactose