摘要：

Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were affinity crosslinked with125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a Mrof 130,000, corresponding to the size of the hormone-binding subunit of insulin receptors on the surface of target cells; the second polypeptide exhibited a Mrof 200,000 and appears to be a component of the latent pool because it was unaffected when 3T3-L1 adipocytes were exposed to trypsin under conditions that result in a 95% reduction in cell surface insulin-binding activity and the loss of the Mr130,000 polypeptide in crosslinking experiments. Unexpectedly, the population of Mr200,000 molecules in intact cells was accessible for limited cleavage by chymotrypsin, yielding a Mr195,000 insulin-binding polypeptide. When 3T3-L1 adipocytes received a 15-min pulse of [35S]methionine, the predominant immunoprecipitated polypeptide had a Mrof 180,000. During a 1.5-hr chase, radioactivity in the Mr180,000 species rapidly declined while the latent Mr200,000 polypeptide became intensely labeled. After a 5-hr chase period, broad protein bands with MrSof 130,000 and 90,000 were visualized as the major immunoprecipitated radioactive polypeptides. Thus, the Mr180,000 species may be a very early biosynthetic precursor that may be subsequently processed to a Mr200,000 form and one or both of the smaller receptor subunits at the cell surface.

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