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  • Title: Lactose carrier protein of Escherichia coli. Reconstitution of galactoside binding and countertransport.
    Author: Wright JK, Schwarz H, Straub E, Overath P, Bieseler B, Beyreuther K.
    Journal: Eur J Biochem; 1982 Jun; 124(3):545-52. PubMed ID: 7049697.
    Abstract:
    A procedure for the reconstitution of the lactose carrier protein, a galactoside:proton symporter in Escherichia coli, is described. Starting from cytoplasmic membranes derived from carrier-overproducing strains, essentially all proteins including 89% of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl O-beta-D-maltoside. In the micellar state the carrier binds substrates with reduced affinity. Addition of E. coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50-60-nm diameter). In these vesicles, about 70% of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding. After fusion of the small vesicles into larger vesicles (1-5 micrometers), rapid countertransport of galactosides is demonstrated. Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons.
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