Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom.
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.
Park
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D
Signal Transduction Lab., Mogam Biotechnology Research Institute, Kyunggido, South Korea.
Kim
H
H
Chung
K
K
Kim
D S
DS
Yun
Y
Y
D017836:Agkistrodon / Q000502:physiology*; D000595:Amino Acid Sequence; D000721:Ancrod / Q000032:analysis*; D000818:Animals; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism / Q000494:pharmacology*; D010960:Plasminogen Activators / Q000032:analysis*; D011994:Recombinant Proteins; D012910:Snake Venoms / Q000737:chemistry*
D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D012910:Snake Venoms; D010958:Plasminogen; D000721:Ancrod; D010960:Plasminogen Activators