Tyrosine-Specific Protein Kinase Activity is Associated with the Purified Insulin Receptor

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 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major b...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1983-04, Vol.80 (8), p.2137-2141
Main Authors: Kasuga, Masato, Fujita-Yamaguchi, Yoko, Blithe, Diana L., Kahn, C. Ronald
Format: Article
Language:English
Subjects:
Antibodies
Autoradiography
Cell growth
Electrophoresis
Female
Gels
Humans
Insulin
man
Phosphoamino acids
Phosphoproteins
Phosphorylation
Phosphotyrosine
Placenta
Pregnancy
protein kinase
Protein Kinases - metabolism
Receptor, Insulin - metabolism
Receptors
Substrate Specificity
Tyrosine - analogs & derivatives
Tyrosine - biosynthesis
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Summary: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 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [γ -32P]ATP in the presence of MnCl2(2 mM) and analyzed in NaDodSO4/acrylamide gel electrophoresis, only the Mr95,000 band was labeled. Preincubation with several concentrations of insulin increased the32P incorporation into this peptide in dose-dependent fashion, whereas insulin-like growth factors were ≈ 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 Mr95,000 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. These data suggest that 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.80.8.2137