鸵鸟脑μ-钙蛋白酶和钙蛋白酶抑制蛋白的纯化与特性分析

The purification and characterization of mu-calpain and calpastatin from ostrich brain.

作者信息

Mkwetshana Noxolo, Naudé Ryno J, Muramoto Koji

机构信息

Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, 6000, Port Elizabeth, South Africa.

出版信息

Int J Biochem Cell Biol. 2002 Jun;34(6):613-24. doi: 10.1016/s1357-2725(02)00004-3.

DOI:10.1016/s1357-2725(02)00004-3

PMID:11943592

Abstract

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.

摘要

钙激活中性蛋白酶（CANPs）及其内源性特异性抑制剂钙蛋白酶抑制蛋白存在于多种脊椎动物和无脊椎动物组织中。CANPs是半胱氨酸蛋白酶，其活性绝对需要Ca(2+)。μ-钙蛋白酶和钙蛋白酶抑制蛋白通过在Toyopearl-Super Q 650S和Pharmacia Mono Q HR 5/5柱上连续色谱步骤进行纯化。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），该酶的分子量为84KDa，通过氨基酸分析其最小分子量为79KDa，等电点为5.2。使用变性梯度PAGE，钙蛋白酶抑制蛋白的分子量为323KDa，等电点为4.7。μ-钙蛋白酶的氨基酸组成显示有689个残基，其最适pH和温度分别为7.5和37℃。μ-钙蛋白酶发生了Ca(2+)依赖性自催化水解，产生了一个82KDa的片段。μ-钙蛋白酶的N端序列与人及牛的μ-钙蛋白酶显示出24%和18%的序列同一性。