Park D, Kim H, Chung K, Kim D S, Yun Y
Signal Transduction Lab., Mogam Biotechnology Research Institute, Kyunggido, South Korea.
Toxicon. 1998 Dec;36(12):1807-19. doi: 10.1016/s0041-0101(98)00090-7.
A venom gland cDNA library of Agkistrodon halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively. Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem. 270, 10246- 10255). Haly 2 displayed a 78% similarity to beta-fibrinogenase (Hung et al. 1994, B. B. R. C., 205, 1707 1715). Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom. Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column. In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasimin. These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.
构建了蝮蛇毒腺cDNA文库，并用基于毒液丝氨酸蛋白酶共有序列的探针进行筛选。接下来，我们测定了两个选定阳性克隆的完整开放阅读框序列，发现它们分别编码长度为234和233个氨基酸的新型丝氨酸蛋白酶，分别命名为Haly-PA和Haly 2。在蛋白质数据库搜索中，Haly-PA与先前鉴定的纤溶酶原激活剂TSV-PA显示出82%的最高相似度（Zhang等人，1995年，《生物化学杂志》，270卷，10246 - 10255页）。Haly 2与β-纤维蛋白原酶显示出78%的相似度（Hung等人，1994年，《生物化学与生物物理研究通讯》，205卷，1707 - 1715页）。使用杆状病毒系统成功表达了Haly-PA，并作为一种32 kDa的糖蛋白分泌到培养基中。在蛇毒的western分析中，抗Haly-PA抗体检测到相同大小的条带，表明这种酶是蛇毒的一个成分。使用阴离子交换和凝胶过滤柱相结合的方法将重组Haly-PA纯化至同质。在纤维蛋白（原）溶解试验中，重组Haly-PA显示出依赖纤溶酶原存在的间接纤维蛋白（原）溶解活性，并将纤溶酶原切割生成活性纤溶酶。这些结果表明，Haly-PA是一种纤溶酶原激活剂，通过将纤溶酶原转化为纤溶酶而显示出纤维蛋白（原）溶解活性。
