利用硫胺素焦磷酸亲和柱制备大肠杆菌丙酮酸氧化酶。

Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column.

作者信息

O'Brien T A, Schrock H L, Russell P, Blake R, Gennis R B

出版信息

Biochim Biophys Acta. 1976 Nov 8;452(1):13-29. doi: 10.1016/0005-2744(76)90054-1.

Abstract

An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogenous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.

摘要

本文报道了一种改进的纯化大肠杆菌丙酮酸氧化酶（丙酮酸：铁细胞色素b1氧化还原酶，EC 1.2.2.2）的方法，该酶是一种外周膜黄素酶。与原始纯化方案相比，该方法的重复性更高，所需时间大大缩短。本方案的关键要素是一种新型的基于琼脂糖的亲和树脂，设计用于分离需要硫胺素焦磷酸的酶。文中描述了两种此类亲和树脂的合成、部分特性及应用。丙酮酸氧化酶从亲和树脂上洗脱下来时是一种纯的、同质的蛋白质。该酶是一种四聚体，亚基分子量约为60000。各亚基似乎是相同的。丙酮酸氧化酶的等电点为5.6。