title: The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes.
abstract: 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.
mesh_terms: D019325:3-O-Methylglucose; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008301:Maleimides / Q000494:pharmacology*; D008757:Methylglucosides / Q000378:metabolism*; D008759:Methylglycosides / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents
chemical_list: D002352:Carrier Proteins; D007328:Insulin; D008301:Maleimides; D008757:Methylglucosides; D008759:Methylglycosides; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; C044107:glutathione maleimide; D019325:3-O-Methylglucose; D005033:Ethylmaleimide