Gutensohn W, Huber M, Jahn H
Hoppe Seylers Z Physiol Chem. 1976 Dec;357(10):1379-85. doi: 10.1515/bchm2.1976.357.2.1379.
Three major approaches to the complete purification of hypoxanthine phosphoribosyltransferase from human erythrocytes and rat brain are described. Preparative isoelectric focusing which has been used for the isolation of the human enzyme was not fully successful in the case of rat brain. Preparative polyacrylamide-gel electrophoresis in gel blocks yields enzyme samples of high purity as judged by analytical gel electrophoresis, but with a comparatively low specific enzyme activity. The most rapid and convenient method, a modification of the affinity chromatography on GMP agarose first described by Hughes[5] gives hypoxanthine phosphoribosyltransferase which is superior to the other preparations in its homogeneity and its specific activity. All three methods produce an identical enzyme protein detected by polyacrylamide electrophoresis on nondenaturing and sodium dodecylsulfate gels. Molecular data of hypoxanthine phosphoribosyltransferase derived from these studies are: Isoelectric points of 5.60; 5.85 and 5.90 for three isozyme peaks of the rat brain enzyme; and a molecular weight of 72000 for the native rat brain enzyme and of 25000-27000 for the subunit of human and rat enzyme. Guanylate kinase does not interfere with the purification of hypoxanthine phosphoribosyltransferase on GMP agarose and moreover is itself partially purified by this chromatography.
本文描述了从人红细胞和大鼠脑中完全纯化次黄嘌呤磷酸核糖基转移酶的三种主要方法。用于分离人酶的制备性等电聚焦法在大鼠脑中并不完全成功。凝胶块中的制备性聚丙烯酰胺凝胶电泳产生的酶样品经分析凝胶电泳判断具有高纯度，但比酶活性相对较低。最快速便捷的方法是对休斯[5]首次描述的GMP琼脂糖亲和色谱法进行改进，得到的次黄嘌呤磷酸核糖基转移酶在纯度和比活性方面优于其他制备方法。所有三种方法在非变性和十二烷基硫酸钠凝胶上通过聚丙烯酰胺电泳检测都产生相同的酶蛋白。从这些研究中获得的次黄嘌呤磷酸核糖基转移酶的分子数据如下：大鼠脑酶的三个同工酶峰的等电点分别为5.60、5.85和5.90；大鼠脑天然酶的分子量为72000，人和大鼠酶亚基的分子量为25000 - 27000。鸟苷酸激酶不干扰GMP琼脂糖上的次黄嘌呤磷酸核糖基转移酶的纯化，而且其本身也通过这种色谱法得到部分纯化。
