Characterization of the insulin receptor kinase purified from human placental membranes.
The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.
D000818:Animals; D001717:Birds; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification*; D011505:Protein-Tyrosine Kinases; D011817:Rabbits; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D001357:Sarcoma, Avian / Q000378:metabolism
D002364:Caseins; D006657:Histones; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin