In vivo association of [125I]-insulin with a cytosolic insulin-degrading enzyme: detection by covalent cross-linking and immunoprecipitation with a monoclonal antibody.
A cytosolic insulin-degrading enzyme (Mr = 110,000) was found to be cross-linked to [125I]-insulin in intact human hepatoma cells, HepG2, incubated with the hormone and treated with the bifunctional cross-linker, disuccinimidyl suberate. The labeling of this protein was greatly increased by concurrent treatment of the cells with N-ethylmaleimide, to the extent that the amount of [125I]-insulin cross-linked to the enzyme in these cells was approximately 20 to 50% that cross-linked to the insulin receptor. The labeling of the insulin-degrading enzyme required the prior interaction of [125I]-insulin with its receptor as well as a temperature- and energy-dependent processing of the hormone. The present work therefore supports a role for this protease in the cellular processing of insulin.
D000911:Antibodies, Monoclonal; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D002460:Cell Line; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D007339:Insulysin / Q000378:metabolism*; D008113:Liver Neoplasms / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D013388:Succinimides / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007328:Insulin; D013388:Succinimides; C008147:insulin, iodo-; D010447:Peptide Hydrolases; D007339:Insulysin; C019358:disuccinimidyl suberate