Smithers G W, O'Sullivan W J
Biochem Med. 1984 Aug;32(1):106-21. doi: 10.1016/0006-2944(84)90013-9.
A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.
基于外源蛋白的热变性和GMP-琼脂糖亲和层析，已开发出一种简便快速的纯化方法，用于从人脑中纯化次黄嘌呤磷酸核糖转移酶。通过十二烷基硫酸钠和梯度聚丙烯酰胺凝胶电泳判断，获得了一种均一的酶制剂。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计该酶的亚基分子量为24,000。通过梯度凝胶电泳测定的天然分子量约为100,000。这些结果表明，人脑中的次黄嘌呤磷酸核糖转移酶是一种四聚体，这与最近报道的人红细胞酶的结果一致。通过非变性聚丙烯酰胺凝胶电泳、等电聚焦和层析聚焦，区分出了人脑酶的至少三种电荷变异形式。估计三种主要形式的酸性pI值约为5.7、5.5和5.0。
