Differential labeling of the erythrocyte hexose carrier by N-ethylmaleimide: correlation of transport inhibition with reactive carrier sulfhydryl groups.
Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10-13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[3H]ethylmaleimide, and to localize it to the Mr 19,000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23,000-40,000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.
May
J M
JM
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232.
D019325:3-O-Methylglucose; D000478:Alkylation; D001692:Biological Transport / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D005033:Ethylmaleimide / Q000097:blood / Q000494:pharmacology*; D008757:Methylglucosides / Q000097:blood; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood; D010693:Phloretin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000097:blood*; D014357:Trypsin
D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D004228:Dithionitrobenzoic Acid; D014357:Trypsin; D005033:Ethylmaleimide; D010693:Phloretin