Krug E L, Kent C
Arch Biochem Biophys. 1984 Jun;231(2):400-10. doi: 10.1016/0003-9861(84)90403-x.
A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.
已设计出一种从产气荚膜梭菌中纯化磷脂酶C的新方法，该方法可得到基本纯净的酶。该方法包括硫酸铵分级分离、在QAE-葡聚糖凝胶上进行离子交换色谱以及在与琼脂糖相连的磷脂酰胆碱上进行亲和色谱。通过十二烷基硫酸钠-凝胶电泳、氨基酸分析和凝胶过滤测定，该酶的分子量为43,000；等电点为pH 5.4。该酶在脱氧胆酸钠中分散的磷脂酰胆碱存在下活性最佳，不过在乙醇中分散的磷脂酰胆碱或鞘磷脂存在下也观察到了可观的活性。测定中对金属离子的需求可由多种不同离子满足。发现纯酶每摩尔酶含有2摩尔锌，因此表明它是一种锌金属酶。
