title: Insulin receptor is an insulin-dependent tyrosine protein kinase: copurification of insulin-binding activity and protein kinase activity to homogeneity from human placenta.
abstract: The insulin-binding activity and insulin-dependent tyrosine protein kinase activity of the insulin receptor have been purified 2000-fold to homogeneity from human placental membranes. The purified receptor has one high-affinity binding site for insulin per mol of receptor. Its Vmax for phosphorylating angiotensin is 80 nmol of phosphate per min per mg of protein at 23 degrees C. The procedure used to purify the receptor includes chromatography on wheat germ agglutinin-agarose and on insulin-Sepharose. The purified receptor was eluted from insulin-Sepharose with 0.5 M NaCl and 1 mM dithiothreitol at pH 5.5. The addition of dithiothreitol was essential for recovery of the protein kinase. A silver-stained gel of the reduced purified receptor showed two major bands, Mr 95,000 (beta subunit) and Mr 135,000 (alpha subunit). The component of Mr 95,000 comigrated with the autophosphorylated beta subunit of the receptor. The latter was phosphorylated exclusively on tyrosine residues by an intramolecular process. In the presence of insulin, approximately 2 mol of phosphate was incorporated per mol of beta subunit. Two major beta subunit tryptic phosphopeptides were resolved by high-pressure liquid chromatography after autophosphorylation of the purified receptor in the presence or absence of insulin. It is concluded that the insulin binding and the insulin-dependent protein kinase are intrinsic components of the same oligomer since (i) they copurify to homogeneity, (ii) the purified receptor protein kinase is immunoprecipitated by polyclonal and monoclonal antibodies to the human insulin receptor, and (iii) phosphorylation of the beta subunit of the receptor occurs by an intramolecular reaction.
mesh_terms: D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D019000:Phosphotyrosine; D010920:Placenta / Q000502:physiology*; D011247:Pregnancy; D011485:Protein Binding; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin