Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport.
Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant "uncoupled" for beta-galactoside/H+ symport [Wong, P. T. S., Kashket, E. R., & Wilson, T. H. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 63], are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H+ electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H+ influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide [Viitanen, P., Garcia, M. L., Foster, D. L., Kaczorowski, G. J., & Kaback, H. R. (1983) Biochemistry 22, 2531]. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent Km. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
D000911:Antibodies, Monoclonal; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008756:Methylgalactosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010101:Oxygen Consumption; D011392:Proline / Q000378:metabolism; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002250:Carbon Radioisotopes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; C021499:lactose permease; D011392:Proline