Sugar binding properties of the melibiose permease in Escherichia coli membrane vesicles. Effects of Na+ and H+ concentrations.
The substrate binding reaction of the melibiose carrier was analyzed by studying [3H]p-nitrophenyl-alpha-D-galactopyranoside (Np alpha Gal) binding to de-energized membrane vesicles from Escherichia coli RA11 as a function of H+ and Na+ (or Li+) concentrations. The data indicate first that Na+ (or Li+) activates Np alpha Gal binding at all pH values tested between 5.5 and 7.5 and second that H+ inhibits the Na+ (or Li+)-dependent activating effect on Np alpha Gal binding. Similar conclusions were drawn for melibiose and methyl-1-thio-beta-D-galactoside binding activities. Unexpectedly, Np alpha Gal, melibiose, and methyl-1-thio-beta-D-galactoside binding activities are insensitive to a variety of SH reagents which completely block transport activity. Quantitative analysis of the effects of H+ and Na+ ions on the parameters of Np alpha Gal binding show that 1) the maximal number of binding sites is constant irrespective of the concentration of Na+ or Li+ in the range of pH between 6 and 7.5 and 2) the apparent dissociation constant for Np alpha Gal binding varies with both Na+ and H+ according to a relation described by a linear combination of the concentration of H+ and the reciprocal of Na+ concentration. These results can be accounted for by a model which assumes sequential binding of the cation and substrate in this order and competition between Na+ and H+ for a common cationic binding site on the porter. Predictions of the proposed binding model for a carrier mechanism catalyzing sugar transport according to a Na+ symport mode or a H+ symport mode are discussed.
D050260:Carbohydrate Metabolism; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008433:Mathematics; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D027981:Symporters
D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008094:Lithium; D012964:Sodium