The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.