Pike L J, Eakes A T, Krebs E G
J Biol Chem. 1986 Mar 15;261(8):3782-9.
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.
胰岛素受体/激酶从人胎盘中纯化至接近均一状态。纯化后的激酶在30℃下，以合成肽Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly为底物、胰岛素存在的情况下，表现出300 nmol/分钟/毫克蛋白质的比活性。用二硫苏糖醇（DTT）处理受体/激酶可使胰岛素结合减少40%-50%，并抑制酪氨酸激酶活性。受体/激酶的磷酸化和激活并不能阻止DTT诱导的结合丧失，但能完全保护其免受还原剂对酶活性的有害影响。对磷酸化及DTT处理后的受体/激酶结构分析表明，在所采用的条件下I类二硫键被还原，但受体/激酶的四聚体结构基本未改变。这些发现表明完整的I类二硫键对于胰岛素结合是必需的，但对于维持预激活的激酶并非必要。还发现DTT可增强胰岛素受体/激酶的自身激活，并促进自身磷酸化反应的逆转。因此，二硫键在胰岛素受体/激酶的功能中似乎具有多种作用。
