Characterization of affinity-purified insulin receptor/kinase. Effects of dithiothreitol on receptor/kinase function.
The insulin receptor/kinase was purified to near homogeneity from human placenta. The purified kinase exhibited a specific activity of 300 nmol/min/mg of protein at 30 degrees C using the synthetic peptide, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, as substrate in the presence of insulin. Treatment of the receptor/kinase with dithiothreitol (DTT) reduced insulin binding by 40-50% and also inhibited tyrosine kinase activity. Phosphorylation and activation of the receptor/kinase did not prevent the DTT-induced loss of binding but completely protected it from the deleterious effects of reducing agent on enzymic activity. Analyses of the structure of the receptor/kinase following phosphorylation and treatment with DTT indicated that the class I disulfide bonds were reduced under the conditions employed, but the tetrameric structure of the receptor/kinase was essentially unaltered. These findings indicate that intact class I disulfides are required for insulin binding but are not necessary for maintenance of the preactivated kinase. DTT was also found to enhance the autoactivation of the insulin receptor/kinase and to promote the reversal of the autophosphorylation reaction. Thus disulfide bonds appear to have multiple roles in the function of the insulin receptor/kinase.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000302:isolation & purification; D011972:Receptor, Insulin / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D008274:Magnesium; D004229:Dithiothreitol