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.[1]References
- Beta-D-galactoside transport in Escherichia coli. Solubilization in organic solvent and reconstitution of binding. König, B., Sandermann, H. Eur. J. Biochem. (1984) [Pubmed]
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