Tyrosine-specific protein kinase activity is associated with the purified insulin receptor.

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Abstract
Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed 3 major bands of MW 135,000, 95,000 and 52,000 in NaDodSO4[sodium dodecylsulfate]/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All 3 bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [.gamma.-32P]ATP in the presence of MnCl2 (2 mM) and analyzed in NaDodSO4/polyacrylamide gel electrophoresis, only the MW 95,000 band was labeled. Preincubation with several concentrations of insulin increased the 32P incorporation into this peptide in dose-dependent fashion, while insulin-like growth factors were .apprxeq. 2% as potent and epidermal growth factor had little or no effect, consistent with their known affinities for the insulin receptor. Insulin stimulation of phosphorylation of the MW 95,000 subunit of the receptor was observed also in immunoprecipitates of this highly purified insulin receptor by anti-insulin-receptor antibodies. Phosphoamino acid determination revealed only phosphotyrosine in both the basal and insulin-stimulated states. Apparently a tyrosine-specific protein kinase activity is closely associated with insulin receptor, and this may be important in the signal transmission required for insulin action.