蛋氨酸合酶活性筛选体系的建立  

Purification and activity evaluation of methionine synthase

作　　者：郭莹[1] 李超[1] 张志丽[1] 田超[1] 王孝伟[1] 刘俊义[1] 

机构地区：[1]北京大学药学院化学生物学系,北京大学天然药物及仿生药物国家重点实验室,北京100083

出　　处：《药学学报》2012年第11期1463-1469,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(20972011,21042009,21172014);国家科技部基金资助项目(2009ZX09301-010)

摘　　要：钴胺素依赖的蛋氨酸合酶催化N5-甲基四氢叶酸转移甲基至同型半胱氨酸生成蛋氨酸和四氢叶酸,直接参与蛋氨酸循环、叶酸循环及含硫氨基酸代谢,与DNA、蛋白质合成及生物甲基化有密切关系。本研究采用蛋白层析技术,将大鼠肝匀浆经超声破碎和高速离心处理后,依次经过DE-52批处理、Q Sepharose Fast Flow离子交换层析和CHT陶瓷羟基磷灰石吸附柱层析进行纯化,并对纯化产物进行了SDS-PAGE和Western blotting鉴定。采用分光光度法测定蛋氨酸合酶的活性,对纯化酶的酶促反应动力学进行了研究,确定了最佳反应条件,动力学结果显示蛋氨酸合酶的双底物酶促反应的机制为乒乓机制。研究表明,采用层析技术纯化得到的蛋氨酸合酶适用于以其为靶点的化合物高通量筛选。Methionine synthase （MS, EC2.1.1.13）, a key enzyme in the folate metabolism area catalyzing methyl transfer from NLmethyltetrahydrofolate to homocysteine to give tetrahydrofolate and methionine, takes a core position in folate cycle, one-carbon-unit transfer and sculpture amino acid pathways. Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme was purified 609-fold to near homogeneity by batch chromatography on DE-52, anion-exchange chromatography on Q Sepharose Fast Flow and CHT-I hydroxyapatite column and was identified by SDS-PAGE and Western blotting. The enzyme activity was determined by spectrophotometric assay. In addition, the influencing factor and optimal reaction condition were performed. The steady state kinetic of rat liver methionine synthase was similar to that of other mammalian cobalamin-dependent methionine synthase which employed a Ping-Pong mechanism. The result indicated that cobalamin-dependent methionine synthase purified from rat liver is suitable for screening and studying methionine synthase specific inhibitors.

关 键 词：蛋氨酸合酶 蛋白层析 分离纯化 酶促反应动力学 叶酸拮抗剂 

分 类 号：R963[医药卫生—微生物与生化药学]