Specific labeling of the lac carrier protein in membrane vesicles of Escherichia coli by a photoaffinity reagent.
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.
D000345:Affinity Labels; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D007763:Lac Operon; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010782:Photolysis; D027981:Symporters
D000345:Affinity Labels; D029968:Escherichia coli Proteins; D005697:Galactosides; D006027:Glycosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose