Interaction of a permeant maleimide derivative of cysteine with the erythrocyte glucose carrier. Differential labelling of an exofacial carrier thiol group and its role in the transport mechanism.
S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5'-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.
May
J M
JM
Diabetes Research and Training Center, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2230.
D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability; D003545:Cysteine / Q000031:analogs & derivatives; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008301:Maleimides; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000378:metabolism*; D010449:Peptide Mapping; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin
D008301:Maleimides; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C062020:S-(bismaleimidoethyl ether)cysteine; D014357:Trypsin; D003545:Cysteine