尖吻蝮蛇酸性磷脂酶A_2的纯化和初步晶体学  

Purification, Crystal Growth and Preliminary X-ray Analysis of a Phospholipase A_2 from Venom of Agkistrondon acutus

作　　者：张海龙[1] 林政炯[1] 杜晓燕[2] 周元聪[2] 

机构地区：[1]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101 [2]中国科学院上海生物化学研究所,上海200031

出　　处：《生物化学与生物物理学报》2000年第4期337-341,共5页

基　　金：中国科学院重大项目资助 !No .KJ95 1 A1 60 1&&

摘　　要：为进一步揭示影响血小板聚集活性的结构基础 ,纯化了江西尖吻蝮蛇 (Agkistrodonacutus)酸性磷脂酶A2 。江西尖吻蝮蛇蛇毒经CM Sepharose离子交换柱层析 ,两步DEAE Sepharose离子交换柱层析以及Mono QFPLC离子交换柱层析 ,得到了SDS 聚丙烯酰胺凝胶电泳和等电聚焦电泳均一的磷脂酶A2 (PLA2 ) ,其分子量约为 16 .5kD ,等电点为 4.3,经测定 ,该酶具有抑制ADP诱导的人血小板聚集活性。采用悬滴气相扩散法 ,利用CrystalScreenII(98种配方 )搜索出两种晶体生长条件 ,精化并培养得到可供X射线衍射分析的单晶。对这两种晶体进行了初步X射线晶体学研究 ,结果表明它们均属于单斜晶系和P2 1空间群 ,晶型I的晶胞参数为a =43.48 (1 =0 .1nm) ,b =71.49 ,c =43.85 ,β=116 .32°,每个不对称单位含 2个分子 ,晶型II的晶胞参数为a =49.2 5 ,b =38.33 ,c =70 .2 5 ,β =99.2 0° ,每个不对称单位也含 2个分子。并分别收集了中等分辨率的X射线衍射数据。An acidic phospholipase A 2 from Agkistrondon acutus venom has been purified to homogeneity via four steps using CM Sepharose, two times DEAE Sepharose, and Mono Q FPLC. The molecular weight of the protein was about 16.5 kD and the isoelectric point was 4.3. The purified enzyme showed a potent inhibitory effect on platelet aggregation induced by ADP in human platelet enriched plasma. The enzyme was then crystallized by hanging drop diffusion method using 2 methyl 2,4 pentanediol as a precipitant. Two kinds of single crystals suitable for X ray crystallographic studies were obtained. X ray crystallographic analysis showed that both crystal forms belong to monoclinic system and space group P 2 1. The cell dimensions of form I crystals were a = 43.48 ?, b = 71.49 ?, c = 43.85 ? and β = 116.32°; those of form II crystals were a = 49.25 ?, b = 38.33 ?, c = 70.25 ? and β = 99.20°. Complete diffraction data sets have been collected to medium resolution for the two crystal forms.

关 键 词：磷脂酶A2 尖吻蝮蛇 晶体结构 血小板 聚集活性 

分 类 号：Q51[生物学—生物化学]