Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport.
The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.
May
J M
JM
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
D019325:3-O-Methylglucose; D000345:Affinity Labels; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D003545:Cysteine / Q000494:pharmacology; D003571:Cytochalasin B / Q000097:blood / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004357:Drug Synergism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008301:Maleimides / Q000097:blood* / Q000494:pharmacology; D008320:Maltose / Q000494:pharmacology; D008757:Methylglucosides / Q000097:blood; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D010693:Phloretin / Q000494:pharmacology; D010777:Photochemistry; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000097:blood*
D000345:Affinity Labels; D008301:Maleimides; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; C044107:glutathione maleimide; D019325:3-O-Methylglucose; C043592:maleimide; D003571:Cytochalasin B; D008320:Maltose; D003545:Cysteine; D010693:Phloretin; D004229:Dithiothreitol