Reoxidation of the class I disulfides of the rat adipocyte insulin receptor is dependent upon the presence of insulin: the class I disulfide of the insulin receptor is extracellular.
Elements of the quaternary structure of the native and dithiothreitol- (DTT) reduced rat adipocyte insulin receptor have been elucidated by vectorial probing and subunit cross-linking. The charged reducing agents glutathione and beta-mercaptoethylamine were used to reduce the class I disulfides of the receptor in intact adipocytes, demonstrating the extracellular location of the disulfide directly. This interpretation was confirmed by use of DTT as a reducing agent and the nonpermeant sulfhydryl blocking reagent Thiolyte MQ to prevent the reoxidation of the class I sulfhydryl groups which occurred when they were not blocked. It was found that the above reoxidation of the receptor is dependent on the concentration of insulin in the nanomolar range, not occurring measurably at 4 degrees C in its absence. Cross-linking studies with ethylene glycol bis(succinimidyl succinate) demonstrated that the alpha subunits could not be cross-linked to each other after reduction of the class I disulfides, suggesting that the interaction between the receptor heterodimers may be due primarily to the disulfide bonds.
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D010084:Oxidation-Reduction; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004220:Disulfides; D007328:Insulin; D046911:Macromolecular Substances; D013388:Succinimides; C028722:ethylene glycolylbis(succinimidyl succinate); D011972:Receptor, Insulin; D005978:Glutathione; D004229:Dithiothreitol