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Purification and properties of the human placental insulin receptor.

1981; Elsevier BV; Volume: 256; Issue: 17 Linguagem: Inglês

10.1016/s0021-9258(19)52540-6

1083-351X

Todd W. Siegel, S Ganguly, Steven Jacobs, O M Rosen, C S Rubin,

Pregnancy and preeclampsia studies

Human placental insulin receptorwas purified 11,000-fold to near homogeneity using DEAE-cellulose chromatography and affinity chromatography on insulin-Sepharose.Approximately 200 to 300 pg of purified receptor were obtained from a single placenta.In solution, the native receptor is a complex (Mr = 440,000) of an acidic, multi-subunit protein with a M, of 350,000 and bound detergent accounting for the remainder of the mass.The receptor protein is asymmetric ( f / f o = 1.4) and consists of a single Coomassie blue staining polypeptide of M, = 135,000.In addition to the 135,000dalton polypeptide, two smaller polypeptides of M, = 45,000 and 90,000 were observed upon autoradiography of 12'I-labeled receptor subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.These two smaller polypeptides did not stain with Coomassie blue but migrated with native receptor activity on isoelectric focusing gels and were coimmunoprecipitated with the 135,000-dalton polypeptide by anti-insulin receptor antibody.The 135,000-dalton subunit was specifically labeled by '"1-insulin using the bifunctional cross-linking reagent dissucinimidyl suberate (P.F. Pilch, and M. P. Czech, (1979) J. Biol.Chem.254,3375-3381), suggesting that this component contains the insulin binding domain.The initial step in insulin action is the specific binding of the hormone by a high affinity receptor at the target cell surface.Plasma membrane receptors for insulin were recognized more than a decade ago, but progress toward a detailed description of the structural components of the receptor and the elucidation of their roles in sequestering insulin and transducing hormone binding into physiological effects has been sporadic and limited because of the low concentrations of receptors in target tissues and difficulties encountered in purifying highly active forms of this integral membrane protein.Cuatrecasas (1,2) fist demonstrated that the receptor could be quantitatively solubilized from rat liver and fat cell membranes by the nonionic detergent Triton X-100 without alteration in its affinity, specificity, and kinetics of insulin binding and susceptibility to proteases and denaturants.Subsequently, Cuatrecasas (3) and Jacobs et al. (4) isolated a highly purified preparation of rat liver insulin receptor using a combination of conventional procedures and affinity chromatography on insulin-agarose.Analysis of the insulin receptor on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions revealed a single Coomassie blue-stained poly-