Shroyer L A, Varandani P T
Arch Biochem Biophys. 1985 Jan;236(1):205-19. doi: 10.1016/0003-9861(85)90620-4.
A thiol peptidase that catalyzes at near neutral pH the hydrolysis of insulin, the isolated A and B chains of insulin, and glucagon was purified from rat liver cytosol by fractionation on Sephadex G-200, Affi-Gel Blue, and Spherogel TSK-G 3000 SW. The purified enzyme showed a single component by chromatography on a Spherogel TSK column and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 180,000 and consists of two subunits having pI's of 5.9 and 6.3. Studies on its substrate specificity showed that the purified enzyme degrades glucagon, insulin, insulin B chain, and insulin A chain, but it does not degrade proinsulin, ACTH, or denatured hemoglobin. Kinetic analyses were performed on three substrates. The Km values were: 34 nM for insulin, 276 nM for insulin B chain, and 3.5 microM for glucagon. The kcat and Vm/Km values were glucagon greater than B chain greater than insulin. Thus, the enzyme has the highest affinity/lowest efficiency for insulin, an intermediate affinity/intermediate efficiency for B chain of insulin and the lowest affinity/highest efficiency for glucagon. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. The enzyme activity was markedly inhibited by N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and was partially inhibited by dithiothreitol, by the chelating agents EDTA and EGTA, and by phenylmethylsulfonyl fluoride (PMSF). Bacitracin inhibited the activity of the enzyme, but the protease inhibitors aprotinin, leupeptin, pepstatin, and phosphoramidon had little or no effect. Reduced glutathione, iodoacetate, and N alpha,p-tosyl-L-lysine chloromethyl ketone (TLCK) also had little or no effect on the enzyme activity.
一种硫醇肽酶在接近中性的pH条件下催化胰岛素、胰岛素分离出的A链和B链以及胰高血糖素的水解，通过在葡聚糖凝胶G - 200、Affi - Gel Blue和Spherogel TSK - G 3000 SW上分级分离，从大鼠肝细胞溶胶中纯化得到该酶。通过在Spherogel TSK柱上进行色谱分析以及在葡聚糖凝胶G - 200柱上进行凝胶过滤，纯化后的酶显示为单一成分。天然酶的分子量约为180,000，由两个亚基组成，其等电点分别为5.9和6.3。对其底物特异性的研究表明，纯化后的酶可降解胰高血糖素、胰岛素、胰岛素B链和胰岛素A链，但不能降解胰岛素原、促肾上腺皮质激素或变性血红蛋白。对三种底物进行了动力学分析。Km值分别为：胰岛素34 nM，胰岛素B链276 nM，胰高血糖素3.5 μM。kcat和Vm/Km值为胰高血糖素大于B链大于胰岛素。因此，该酶对胰岛素具有最高亲和力/最低效率，对胰岛素B链具有中等亲和力/中等效率，对胰高血糖素具有最低亲和力/最高效率。研究了几种潜在的激活剂和抑制剂对该酶活性的影响。该酶活性受到N - 乙基马来酰亚胺、对氯汞苯甲酸、碘乙酰胺和N - 对甲苯磺酰 - L - 苯丙氨酸氯甲基酮（TPCK）的显著抑制，并受到二硫苏糖醇、螯合剂乙二胺四乙酸（EDTA）和乙二醇双四乙酸（EGTA）以及苯甲基磺酰氟（PMSF）的部分抑制。杆菌肽抑制该酶的活性，但蛋白酶抑制剂抑肽酶、亮抑肽酶、胃蛋白酶抑制剂和磷酰胺素几乎没有影响或没有影响。还原型谷胱甘肽、碘乙酸和Nα，对甲苯磺酰 - L - 赖氨酸氯甲基酮（TLCK）对该酶活性也几乎没有影响或没有影响。
