Werries E, Franz A, Hippe H, Acil Y
Fachbereich Biologie/Chemie, Universität Osnabrück, Abteilung Biochemie, Germany.
J Protozool. 1991 Jul-Aug;38(4):378-83. doi: 10.1111/j.1550-7408.1991.tb01374.x.
The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.
蓝氏贾第鞭毛虫滋养体匀浆中存在的、对偶氮酪蛋白和尿素变性血红蛋白有活性的蛋白酶活性，通过一系列纯化程序被分离成两种不同的酶。这些程序包括在Fractogel TSK HW - 55(F)上进行凝胶过滤、有机汞琼脂糖亲和色谱以及在DEAE - 纤维素上进行离子交换色谱。通过在Sephadex G - 100上进行色谱分析，两种纯化的酶的相对分子量分别为Mr = 95,000和35,000 +/- 10%。基于硫醇试剂的抑制作用以及二硫苏糖醇和半胱氨酸对这种作用的消除，它们被鉴定为半胱氨酸蛋白酶。蛋白酶I(Mr = 95,000)和蛋白酶II(Mr = 35,000)对释放特征性片段的胰岛素β链有活性。然而，通过使用代表胰岛素β链1 - 6、8 - 18和20 - 30序列的合成肽，可以观察到这两种酶在底物特异性上的差异。此外，合成四肽Arg - Gly - Phe - Phe、Arg - Gly - Leu - Hyp和Arg - Arg - Phe - Phe被这两种蛋白酶水解，分别释放出Phe - Phe和Leu - Hyp。与Arg - Gly - Phe - Phe相比，在1 mM底物浓度下，Arg - Gly - Leu - Hyp和Arg - Arg - Phe - Phe的水解速率分别为蛋白酶I的91%和63%以及蛋白酶II的80%和57%。
