Biomaterial Database

Specific labeling of the lac carrier protein in membrane vesicles of Escherichia coli by a photoaffinity reagent. 1980

G J Kaczorowski, and G LeBlanc, and H R Kaback

4-Nitrophenyl-alpha-D-galactopyranoside (NPG) was used as a photoaffinity reagent to specifically inactivate the beta-galactoside transport system in Escherichia coli ML 308-225 membrane vesicles. Photolysis of NPG produced time-dependent, irreversible loss of transport activity with corresponding incorporation of [3H]NPG into the membrane. Both processes were blocked by beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside, a high-affinity substrate of the lac carrier protein and inactivation of lactose transport was specific because NPG photolysis did not affect proline uptake or the ability of the vesicles to generate an electrochemical proton gradient. Arylation of the lac carrier protein was stoichiometric and resulted in the formation of 0.25 nmol of NPG adduct per mg of membrane protein. All attempts to regenerate transport activity by reillumination in the presence of externally added nucleophiles failed, indicating that arylation is functionally irreversible. When vesicles labeled with [3H]NPG under defined experimental conditions were solubilized and analyzed by gel electrophoresis, only one radioactive peak with an apparent molecular weight of 30,000 was observed, confirming that the reaction is highly specific. The results demonstrate that NPG is an active-site-directed photoaffinity label for the lac carrier protein.

UI	MeSH Term	Description	Entries
D007763	Lac Operon	The genetic unit consisting of three structural genes, an operator and a regulatory gene. The regulatory gene controls the synthesis of the three structural genes: BETA-GALACTOSIDASE and beta-galactoside permease (involved with the metabolism of lactose), and beta-thiogalactoside acetyltransferase.	Lac Gene,LacZ Genes,Lactose Operon,Gene, Lac,Gene, LacZ,Genes, Lac,Genes, LacZ,Lac Genes,Lac Operons,LacZ Gene,Lactose Operons,Operon, Lac,Operon, Lactose,Operons, Lac,Operons, Lactose
D007785	Lactose	A disaccharide of GLUCOSE and GALACTOSE in human and cow milk. It is used in pharmacy for tablets, in medicine as a nutrient, and in industry.	Anhydrous Lactose,Lactose, Anhydrous
D009004	Monosaccharide Transport Proteins	A large group of membrane transport proteins that shuttle MONOSACCHARIDES across CELL MEMBRANES.	Hexose Transport Proteins,Band 4.5 Preactin,Erythrocyte Band 4.5 Protein,Glucose Transport-Inducing Protein,Hexose Transporter,4.5 Preactin, Band,Glucose Transport Inducing Protein,Preactin, Band 4.5,Proteins, Monosaccharide Transport,Transport Proteins, Hexose,Transport Proteins, Monosaccharide,Transport-Inducing Protein, Glucose
D010782	Photolysis	Chemical bond cleavage reactions resulting from absorption of radiant energy.	Photodegradation
D002462	Cell Membrane	The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.	Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D002474	Cell-Free System	A fractionated cell extract that maintains a biological function. A subcellular fraction isolated by ultracentrifugation or other separation techniques must first be isolated so that a process can be studied free from all of the complex side reactions that occur in a cell. The cell-free system is therefore widely used in cell biology. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p166)	Cellfree System,Cell Free System,Cell-Free Systems,Cellfree Systems,System, Cell-Free,System, Cellfree,Systems, Cell-Free,Systems, Cellfree
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005697	Galactosides	Glycosides formed by the reaction of the hydroxyl group on the anomeric carbon atom of galactose with an alcohol to form an acetal. They include both alpha- and beta-galactosides.
D006027	Glycosides	Any compound that contains a constituent sugar, in which the hydroxyl group attached to the first carbon is substituted by an alcoholic, phenolic, or other group. They are named specifically for the sugar contained, such as glucoside (glucose), pentoside (pentose), fructoside (fructose), etc. Upon hydrolysis, a sugar and nonsugar component (aglycone) are formed. (From Dorland, 28th ed; From Miall's Dictionary of Chemistry, 5th ed)	Glycoside
D000345	Affinity Labels	Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids.	Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling