Differential labeling of the erythrocyte hexose carrier by N-ethylmaleimide: correlation of transport inhibition with reactive carrier sulfhydryl groups

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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-[ 3 H] ethylmaleimide , and to localize it to the M r 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 M r 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.