江浙蝮蛇毒L-氨基酸氧化酶的分离纯化及其性质鉴定  

Purification and Characterization of L-amino Acid Oxidase from Agkistrodon halys Pallas Venom

作　　者：刘杰武[1] 柴敏强[1] 杜晓燕 宋建国[1] 周元聪[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2002年第3期305-310,共6页

摘　　要：从江浙蝮蛇粗毒中经SephadexG 15 0凝胶过滤、DEAE SepharoseCL 6B以及FPLCSuperose 12分离出一种蛋白质。该蛋白质经SDS PAGE测定在非还原和还原条件下分子量均为 5 7kD左右 ,等电聚焦测得其等电点约为 4.9。生物活性测定表明该蛋白质具有L 氨基酸氧化酶活性 ,能够抑制由ADP和胶原所引起的血小板聚集 ,具有抑制革兰氏阴性菌的作用 ;并且具有细胞毒性 。L-amino acid oxidase (LAO, EC 1.4.3.2) is widely found in snake venoms and is thought to contribute to the toxicity in envenoming. By using of Sephadex G-150, DEAE-Sepharose CL-6B and FPLC Superose 12 chromatography, a protein with L-amino acid oxidase activity was purified and characterized from Agkistrodon haly Pallas venom. Its molecular mass was 57 kD as determined by SDS-PAGE analysis under both reducing and non-reducing conditions, and its pI was about 4.9. The protein catalysed the stereospecific oxidative deamination of L-amino acid substrate. It inhibited the platelet aggregation induced by ADP and collagen dose-dependently, even at low concentrations of 0.2 μmol/L and 0.08 μmol/L, respectively. The LAO had antibacterial effect to E.coli K12D31, and the effective concentration was as low as 0.03 g/L. Furthermore, the LAO showed cytotoxicity in crystal violet assay and apoptosis-inducing acitvity in the A549 cells. After 24 h treatment with 5 mg/L LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by using of agrose gel electrophoresis.

关 键 词：江浙蝮蛇毒 L-氨基酸氧化酶 分离纯化 性质 鉴定 血小板聚集 抑菌作用 细胞凋亡 

分 类 号：Q55[生物学—生物化学]