Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells.
Autophosphorylation of the insulin receptor was studied using a glycoprotein fraction solubilized and purified partially from the rat hepatoma cell line, Fao. Incubation of this receptor preparation with [gamma-32P] ATP, Mn2+, and insulin yielded a single insulin-stimulated phosphoprotein of Mr = 95,000 which corresponds to the beta-subunit of the insulin receptor. At 22 degrees C, incorporation of 32P was half-maximal at 30 s and about 90% complete after 2 min. At steady state, about 200 pmol of 32P were incorporated per mg of protein; this value corresponded to about 2 molecules of phosphate per insulin binding site estimated from Scatchard plots. Insulin increased the Vmax for autophosphorylation of the insulin receptor kinase nearly 20-fold with no effect on the Km for ATP. Mn2+ stimulated autophosphorylation by decreasing the Km of the kinase for ATP, whereas Mg2+ had no effect. Dilution of the insulin receptor over a 10-fold concentration range did not decrease the rate of autophosphorylation suggesting that it may occur by an intramolecular mechanism. When the phosphorylated beta-subunit of the insulin receptor was digested with trypsin, at least 5 phosphopeptides could be separated by high performance liquid chromatography on a mu Bondapak C18 reverse-phase column. Insulin stimulated the phosphorylation of all sites. These phosphate acceptor sites varied in their rate and degree of phosphorylation. Phosphopeptides pp4 and pp5 were phosphorylated very rapidly and reached steady state within 20 s, whereas phosphorylation of pp1 and pp2 required several minutes to reach steady state.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D010710:Phosphates; D008345:Manganese; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin