title: Insulin-stimulated tyrosine protein kinase. Characterization and relation to the insulin receptor.
abstract: Utilizing histone phosphorylation as the basis for a quantitative assay, the insulin-stimulated protein kinase in human placenta has been characterized. The kinase copurifies through wheat germ agglutinin-Sepharose and DEAE-cellulose in constant ratio to the insulin binding function. Both activities are bound to the same extent on insulin-Sepharose, and the immobilized kinase, after extensive washing, exhibits activity versus histone, which closely approaches that of the insulin-stimulated, solubilized kinase. In addition, the bound kinase retains the ability to phosphorylate the Mr = 95,000 subunit of the bead-bound receptor. Elution of the beads with sodium dodecyl sulfate yields on electrophoresis two major peptides of Mr = 130,000 and 95,000. Thus, insulin binding and insulin-stimulated histone kinase copurify in a constant stoichiometric ratio in close physical relation and are likely functional expressions of the same molecule. After the DEAE step, the insulin-stimulated kinase phosphorylates histone subfraction 2b exclusively on tyrosine residues. Insulin increases the Vmax for H2b by 3-5-fold and increases the rate of the histone phosphorylation in direct correspondence to the steady state level of specifically bound insulin. ATP is the preferred phosphate donor. The reaction is supported by either Mn2+ or Mg2+. At [ATP] less than 0.5 mM, insulin-stimulated kinase is substantially higher with Mn2+ as the sole divalent cation, as compared to Mg2+. At [ATP] greater than or equal to 0.5 mM, the rates observed with Mn2+ have plateaued, whereas the rates in the presence of Mg2+ show a continued increase such that maximal activity is seen with Mg2+ and 2-3 mM ATP. Under these conditions, the estimated turnover number of the kinase ranges between 30 and 100 pmol of 32P transferred per min/pmol of insulin bound. Thus, the tyrosine kinase activity of the insulin receptor is quantitatively comparable to that estimated for several serine protein kinases and is unlikely to reflect the side reaction of another enzymatic function.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D006656:Protamine Kinase / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D007328:Insulin; D008345:Manganese; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D006656:Protamine Kinase; D008274:Magnesium