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.