Beta-D-galactoside transport in Escherichia coli. Solubilization in organic solvent and reconstitution of binding.
The beta-D-galactoside transport protein (y-gene product) of Escherichia coli, strain T 206, was solubilized in 85-95% yield using the organic solvents hexamethylphosphoric triamide at pH 7.5 or butan-1-ol at pH 4.2. The transport protein obtained with the former solvent could be incorporated into a defined lipid/protein aggregate of density 1.12 g/ml, but no beta-D-galactoside binding was restored. Diacylglycerol kinase regained activity in the same lipid/protein aggregates. In control experiments, liposomes formed from hexamethylphosphoric triamide were found to be active in the valinomycin-mediated uptake of Rb+ ions. beta-D-Galactoside binding (3.6-5.7 nmol/mg protein) as well as diacylglycerol kinase activity [7 nmol min-1 (mg protein)-1] was reconstituted into proteoliposomes from butan-1-ol solution by adaptation of a published procedure [Wright, J. K. et al. (1982) Eur. J. Biochem. 124, 545-552]. A microparticulate nature of the butan-1-ol-solubilized transport protein could be excluded by gel permeation chromatography on a newly synthesized matrix, hydroxypropyl-Sephacryl S-300.
D000440:Butanols; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006492:Hempa; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D012995:Solubility; D012997:Solvents; D027981:Symporters
D000440:Butanols; D029968:Escherichia coli Proteins; D005697:Galactosides; D006027:Glycosides; C050221:LacY protein, E coli; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D012997:Solvents; D027981:Symporters; C007816:beta-galactoside; C021499:lactose permease; D006492:Hempa