牛组织蛋白酶B不同同工型的纯化及特性

Purification and properties of different isoforms of bovine cathepsin B.

作者信息

Deval C, Bechet D, Obled A, Ferrara M

机构信息

Unité de recherche sur les protéases, Institut national de la recherche agronomique SRV, Ceyrat, France.

出版信息

Biochem Cell Biol. 1990 Apr;68(4):822-6. doi: 10.1139/o90-121.

DOI:10.1139/o90-121

PMID:2223009

Abstract

A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30,000) and cleaved (Mr 25,000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 microM and 52 s-1, and 125 microM and 75 s-1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.

摘要

本文描述了一种从牛肝中快速纯化组织蛋白酶B的方法。制备粗溶酶体提取物后，该方法仅涉及DEAE Zeta-Prep-Disk色谱、凝胶过滤和Mono-S柱快速蛋白质液相色谱。区分出了组织蛋白酶B的两个活性峰（P1和P2）。两者均呈现未切割的（相对分子质量（Mr）30,000）和切割的（Mr 25,000 + Mr 5,000）链，但等电聚焦显示为不同的同工型。然而，这两种不同群体的组织蛋白酶B同工型表现出相似的酶学性质。P1和P2对Z-Arg-Arg-NMec水解的米氏常数（Km）和催化常数（kcat）分别为114 μM和52 s-1，以及125 μM和75 s-1。两者均被低浓度的E-64或亮抑酶肽快速抑制，但不受组织蛋白酶L特异性抑制剂Z-Phe-Phe-CHN2的影响。