Garcia J V, Stoppelli M P, Decker S J, Rosner M R
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
J Cell Biol. 1989 Jan;108(1):177-82. doi: 10.1083/jcb.108.1.177.
We have recently described the purification and characterization of an insulin-degrading enzyme (IDE) from Drosophila melanogaster that can cleave porcine insulin, is highly conserved through evolution and is developmentally regulated. We now report that the IDE is, in fact, an insulin EGF-binding protein (dp100) that we had isolated previously from Drosophila using an antihuman EGF receptor antiserum. This conclusion is based upon the following evidence. (a) dp100, identified by its ability to cross-link to labeled insulin, EGF, and transforming growth factor-alpha (TGF-alpha), and to be immunoprecipitated by anti-EGF receptor antisera, copurifies with the IDE activity. Thus, the purified IDE can be affinity labeled with either 125I-insulin, 125I-EGF, or 125I-TGF-alpha, and this labeling is specifically inhibited with unlabeled insulin, EGF, and the insulin B chain. (b) The antiserum to the human EGF receptor, which recognizes dp100, is able to specifically immunoprecipitate the insulin-degrading activity. (c) The purified IDE preparation contains a single protein of 110 kD which is recognized by both the anti-EGF receptor antiserum and anti-Drosophila IDE antiserum. (d) Polyclonal antiserum to the purified IDE, which specifically recognized only the 110-kD band in Drosophila Kc cells, immunoprecipitates dp100 cross-linked to 125I-TGF-alpha and dp100 cross-linked to 125I-insulin from the purified IDE preparation. (e) EGF, which competes with insulin for binding to dp100, also inhibits the degradation of insulin by the purified IDE. These results raise the possibility that a functional interaction between the insulin and EGF growth factor families can occur which is mediated by the insulin-degrading enzyme.
我们最近描述了从黑腹果蝇中纯化和鉴定出的一种胰岛素降解酶（IDE），它能够切割猪胰岛素，在进化过程中高度保守且受发育调控。我们现在报告，事实上，该IDE是一种胰岛素表皮生长因子结合蛋白（dp100），我们之前使用抗人表皮生长因子受体抗血清从果蝇中分离得到了它。这一结论基于以下证据。（a）dp100可通过与标记的胰岛素、表皮生长因子（EGF）和转化生长因子-α（TGF-α）交联，并被抗表皮生长因子受体抗血清免疫沉淀来鉴定，它与IDE活性共同纯化。因此，纯化的IDE可用125I-胰岛素、125I-EGF或125I-TGF-α进行亲和标记，且这种标记可被未标记的胰岛素、EGF和胰岛素B链特异性抑制。（b）识别dp100的抗人表皮生长因子受体抗血清能够特异性免疫沉淀胰岛素降解活性。（c）纯化的IDE制剂含有一种110kD的单一蛋白质，它可被抗表皮生长因子受体抗血清和抗果蝇IDE抗血清识别。（d）针对纯化的IDE的多克隆抗血清，它在果蝇Kc细胞中仅特异性识别110-kD条带，可从纯化的IDE制剂中免疫沉淀与125I-TGF-α交联的dp100和与125I-胰岛素交联的dp100。（e）与胰岛素竞争结合dp100的EGF，也能抑制纯化的IDE对胰岛素的降解。这些结果增加了胰岛素和表皮生长因子家族之间可能通过胰岛素降解酶介导发生功能相互作用的可能性。
