牛胰脏组织蛋白酶L的纯化和酶学性质

Purification and Characterization of Cathepsin L from Bovine Pancreas

新鲜牛胰脏匀浆物经酸化粗提后,再通过盐析、柱层析等步骤纯化,制备了0.58 mg纯酶,纯化倍数达530.54。经凝胶电泳分析,该酶有2个亚基,分子质量为29.1 k D和18.9 k D。牛胰脏组织蛋白酶L的最适反应温度为50℃,最适反应pH值为6.5。巯基还原剂二硫苏糖醇、L-半胱氨酸均明显激活了该酶活性,10μmol/L的N-(反式-环氧丁二酰基)-L-亮氨酸-4-胍基丁基酰胺(E-64)可完全抑制其活性。1 mmol/L的Zn2+对酶活性有明显抑制作用。该纯化酶可水解苄氧羰基-苯丙氨酰-精氨酰-甲基香豆素(Z-Phe-Arg-MCA),其Km值为3.52μmol/L。

In this study, 0.58 mg of purified enzyme was prepared from acidification and subsequent extraction of fresh bovine pancreas homogenate followed by purification through salting out and column chromatography. The purification fold was 530.54. The purified enzyme had two subunits with molecular weights of 18.9 and 29.1 kD, respectively on SDS- polyacrylamide gel electrophoresis （SDS-PAGE）. The optimum reaction temperature for bovine pancreas cathepsin L was 50 ℃, and the optimum pH was 6.5. Its enzyme activity was efficiently activated by dithiothreitol （DTT） and L-cysteine （L-Cys）, while it could be completely inhibited by 10μmol/L E-64 and evidently suppressed by 1 mmol/L Zn2＋. The purified enzyme could hydrolyze Z-Phe-Arg-MCA with a Km value of 3.52 μmol/L.