Zsindely A, Szabolcs M, Aradi J, Schablik M, Kiss A, Szabó G
Acta Biol Acad Sci Hung. 1977;28(3):281-90.
The inositol-dependent mutant of Neurospora crassa lacks inositol-1-phosphate synthetase activity. This defect can be revorted by the addition of high-molecular DNA isolated from the wild type. To elucidate the biochemical background of inositol dependence, inositol-1-phosphate synthetase was studied. A method has been developed fro the isolation of the enzyme from the wild type strain in 10 mg scale by salt fractionation, gel filtration and ion-exchange chromatography. The specific activity of the purified enzyme is 4750 U/mg protein and its purity has increased about 100-fold. Polyacrylamide gel electrophoresis indicated that, in addition to the main enzymatically active band, several accompanying proteins occur in very small amount. The molecular weight of the enzyme is 225,000 daltons. Probably it consists of four subunits, two with a molecular weight of 64,000 daltons and another two of 50,000 daltons. An enzymatically inactive protein has been isolated from the mutant with the same procedure as that of the enzyme; it migrated at gel electrophoreis similarly to the enzyme. It may be supposed that the isolated protein is the defective enzyme molecule.
粗糙脉孢菌的肌醇依赖型突变体缺乏肌醇-1-磷酸合成酶活性。通过添加从野生型中分离的高分子量DNA可恢复此缺陷。为阐明肌醇依赖性的生化背景，对肌醇-1-磷酸合成酶进行了研究。已开发出一种方法，可通过盐分级分离、凝胶过滤和离子交换色谱从野生型菌株中以10毫克规模分离该酶。纯化酶的比活性为4750 U/毫克蛋白，其纯度提高了约100倍。聚丙烯酰胺凝胶电泳表明，除了主要的酶活性条带外，还有几种伴随蛋白的含量非常少。该酶的分子量为225,000道尔顿。它可能由四个亚基组成，两个亚基的分子量为64,000道尔顿，另外两个亚基的分子量为50,000道尔顿。已用与分离该酶相同的方法从突变体中分离出一种无酶活性的蛋白；它在凝胶电泳中的迁移情况与该酶相似。可以推测分离出的蛋白是有缺陷的酶分子。
