ABSTRACT
Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.
Subject(s)
Disaccharides/metabolism , Escherichia coli/metabolism , Melibiose/metabolism , Membrane Transport Proteins/metabolism , Symporters , Biological Transport, Active , Carbohydrates , Liposomes , Membrane Transport Proteins/isolation & purification , Substrate SpecificityABSTRACT
Family studies show that the Huntington's disease gene is linked to a polymorphic DNA marker that maps to human chromosome 4. The chromosomal localization of the Huntington's disease gene is the first step in using recombinant DNA technology to identify the primary genetic defect in this disorder.
Subject(s)
Chromosomes, Human, 4-5 , Huntington Disease/genetics , Chromosome Mapping , Cloning, Molecular , DNA Restriction Enzymes , Female , Genetic Linkage , Humans , Huntington Disease/diagnosis , Male , Pedigree , Polymorphism, GeneticABSTRACT
A strain of EScherichia coli was constructed containing a plasmid from the Clarke-Carbon collection that showed high levels of melibiose transport activity. Membranes from the plasmid-containing strain were extracted with octyl-beta-D-glucopyranoside and melibiose transport was reconstituted in liposomes. The proteoliposomes exhibited counterflow activity, as well as membrane potential and sodium gradient-driven melibiose accumulation.
Subject(s)
Escherichia coli/genetics , Membrane Transport Proteins/genetics , Plasmids , Symporters , Escherichia coli/enzymology , Kinetics , Melibiose/metabolism , Membrane Transport Proteins/isolation & purification , Membrane Transport Proteins/metabolism , Solubility , Species SpecificityABSTRACT
p-Nitrophenyl-alpha-galactoside (alpha-pNPG) was found to be a substrate for the melibiose transport system of Escherichia coli. This sugar enters induced cells via the carrier and is split by alpha-galactosidase to galactose and p-nitrophenol. In mutant cells lacking the alpha-galactosidase [3H]-alpha-pNPG accumulated to concentrations 15 times higher than the external medium. The transport of alpha-pNPG is inhibited by both Na+ and Li+. Na+ (10 mM) reduced the Km for alpha-pNPG from 0.45 to 0.18 mM and reduced the Vmax from 6.7 nmoles/min/mg dry wt to a value of 3.0.
