Structure of covalent insulin-receptor complexes (I-S-S-R) in isolated rat adipocytes and human placental membranes.
The structure of naturally-formed covalent disulphide-linked complexes between insulin and its receptor was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. To prevent destabilization of disulphide bonds at alkaline pH the standard discontinuous electrophoresis conditions were changed to a continuous buffer system at pH 7.0. 125I-insulin was first bound to either rat adipocytes or human placental membranes for 10 min at 37 degrees C. After washing, non-dissociable radioactivity was extracted from cells or membranes in Triton X-100 and immunoprecipitated with an antiserum (B-2) to the insulin receptor. Electrophoresis of the immune precipitate revealed the two smaller of the three reported species of native insulin receptor (Mr values approx. 350 000, 290 000 and 260 000); in addition, a species of Mr 200 000 was also frequently observed in adipocytes. When non-dissociable 125I-insulin was chemically crosslinked to adipocytes or placental membranes, prior to solubilization and immunoprecipitation, all three species of the native receptor were labelled; after reduction, only a single species of Mr 130000 was observed. These findings indicate that disulphide exchange of insulin occurs with the Mr 130000 (alpha) binding subunit within partially reduced species of the native, oligomeric receptor. The degree of disulphide binding of insulin could therefore depend on the relative abundance of partially reduced receptor species and on the redox state of the cell membrane.
D000273:Adipose Tissue / Q000032:analysis* / Q000166:cytology; D000818:Animals; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D008566:Membranes / Q000032:analysis; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin; D013388:Succinimides
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D013388:Succinimides; D011972:Receptor, Insulin; C019358:disuccinimidyl suberate