EXPRESSION AND CHARACTERIZATION OF A NOVEL PLASMINOGEN-ACTIVATOR FROMAGKISTRODON HALYS VENOM

A venom gland cDNA library of Agkistrodon halys was constructed and sc reened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open readin g frames of two selected positives which were found to encode novel se rine 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 sh owed the highest similarity of 82% to the previously characterized pla sminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem., 270, 10 246-10255). Haly 2 displayed a 78% similarity to beta-fibrinogenase (H ung et al. 1994, B. B. R. C., 205, 1707-1715). Haly-PA was successfull y expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake veno m, anti-Haly-PA antibody detected the same size of band indicating tha t this enzyme is a component of snake venom. Recombinant Haly-PA was p urified 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 ac tive plasmin. These results indicate that Haly-PA is a plasminogen act ivator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin. (C) 1998 Elsevier Science Ltd. All rights rese rved.