Chandler M L, Varandani P T
Biochim Biophys Acta. 1975 Aug 26;397(2):307-17. doi: 10.1016/0005-2744(75)90120-5.
Insulin degradation by glutathione-insulin transhydrogenase has been studied using three different assay procedures: the measurement of the change in insulin immunoreactivity; the formation of 5% trichloroacetic acid-soluble radioactivity from 125 I-labeled insulin and the formation of GSSG via coupling to the oxidation of NADPH with the use of glutathione reductase. The extent of reaction as measured by each assay was different, and the ratios between the assays were not constant with time. Kinetic experiments with the NADPH-coupled assay and the trichloroacetic acid assay yielded similar results: Line-weaver-Burke plots with insulin as variable and GSH as fixed substrate gave a set of straight, intersecting lines, and such plots with GSH as variable and insulin as fixed substrate were parabolic. Apparent Km values for insulin at 1 mM GSH were found to be quite similar by three assay techniques; however, the V values per unit of enzyme protein varied considerably with different procedures. The results are interpreted as indicating that immunoreactivity is lost after reduction of only one of the disulfide bonds of insulin whereas the two interchain disulfide linkages must be broken to produce the trichloroacetic acid-soluble A chain. The results of the NADPH-coupled assay suggest that all three disulfide bonds of insulin are possible substrates for the enzyme. The trichloroacetic acid precipitation assay seems to be the most practicable technique for general use because of the greater ease in performing large number of samples, precision and sensitivity.
利用三种不同的测定方法研究了谷胱甘肽 - 胰岛素转氢酶对胰岛素的降解作用：测量胰岛素免疫反应性的变化；由¹²⁵I标记的胰岛素形成5%三氯乙酸可溶性放射性物质，以及通过与谷胱甘肽还原酶氧化NADPH偶联形成GSSG。每种测定方法所测得的反应程度不同，且各测定方法之间的比率随时间并不恒定。用NADPH偶联测定法和三氯乙酸测定法进行的动力学实验得出了相似的结果：以胰岛素为变量、GSH为固定底物的Line - weaver - Burke图给出了一组直线且相交，而以GSH为变量、胰岛素为固定底物的此类图呈抛物线状。通过三种测定技术发现，在1 mM GSH时胰岛素的表观Km值相当相似；然而，每单位酶蛋白的V值因不同方法而有很大差异。结果表明，胰岛素只有一个二硫键还原后免疫反应性丧失，而两条链间二硫键必须断裂才能产生三氯乙酸可溶性A链。NADPH偶联测定法的结果表明，胰岛素的所有三个二硫键都可能是该酶的底物。三氯乙酸沉淀测定法似乎是最适合普遍使用的技术，因为它更易于处理大量样品，具有精度和灵敏度。
