The effects of pH on proton sugar symport activity of the lactose permease purified from Escherichia coli.
The lactose permease, which catalyzes galactoside-proton symport into Escherichia coli, has been purified and reconstituted in active form into artificial lipid vesicles. The roles of many detergents and phospholipids in solubilization and stabilization of the activity of the permease have been examined with a view to its eventual crystallization. Initial rates of uptake into reconstituted proteoliposomes determined by rapid mixing techniques proved that the activity of the permease can be comparable to that observed in the intact cell, while the best values for uptake rates obtained with conventional techniques were comparable to those reported for vesicles. The activity of the purified protein has been monitored over time periods of hours to weeks. It is shown that, under the best current conditions, the permease retains full activity for 1 to 2 weeks. Although this is still marginal for its crystallization, future improvements can now be assayed by rather stringent criteria. The mechanism of galactoside transport into reconstituted proteoliposome has been investigated by examining the effects of pH on influx into the vesicles. It is shown that the observed effects are entirely consistent with the predictions of a simple model of proton symport. The apparent increase in rate of uptake that is observed in the presence of a pH gradient is not so much due to an acceleration by a component of the protonmotive force as to the relaxation of inhibition by a product (internal protons) of the symport reaction.
D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008081:Liposomes; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids / Q000378:metabolism; D011510:Proteolipids; D013379:Substrate Specificity; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D008553:Melibiose; D007785:Lactose