Impermeant maleimides. Identification of an exofacial component of the human erythrocyte hexose transport mechanism.
The facilitated diffusion of D-glucose across human erythrocyte membranes requires an exofacial (outer surface) sulfhydryl group which can be alkylated by the impermeant reagents glutathione-maleimide and dextran-maleimide. The irreversible inhibition produced by these reagents is asymmetric; inhibition of glucose efflux considerably exceeds that of influx when transport is assayed in the absence of glucose on the opposite side of the membrane. Both D-glucose and cytochalasin B protect the exofacial transport site from alkylation by the impermeant maleimides. This masking effect provides the basis for a two-step procedure for differential labeling of the outer transport site with radioactive glutathione-maleimide. The method labels clearly and consistently a component of the membrane proteins which migrates in sodium dodecyl sulfate-polyacrylamide gels between Coomassie brilliant blue-stained Bands 4.2 and 5, corresponding to an apparent molecular weight of 65,000 to 70,000. Transport studies after inhibition with N-ethylmaleimide suggest that the hexose mechanism also requires a second sulfhydryl group which is not accessible at the cell surface.
D001665:Binding Sites; D001692:Biological Transport; D001786:Blood Glucose / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005978:Glutathione; D006801:Humans; D008301:Maleimides / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001786:Blood Glucose; D008301:Maleimides; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D003571:Cytochalasin B; D005978:Glutathione