Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom
2
조회
0
다운로드
2
인용
0
참고문헌
키워드
초록
Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen,gelatin, and bovine serum albumin were investigated. Results :1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially Aα-chain and slowly Bβ-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.
Abstract
Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen,gelatin, and bovine serum albumin were investigated. Results :1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially Aα-chain and slowly Bβ-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.
참고문헌 (21)
- Markland FS, "Snake venoms and the hemostatic system", Toxicon, 36, pp.1749~1800 (1998)
- Bjarnason JB, "Hemorrhagic metalloproteinases from snake venoms", Pharmacol. Ther, 62, pp.325~372 (1994)
- Marsh NA, "Snake venom affecting the haemostatic mechanism - a consideration of their mechanisms, practical applications and biological significance", Blood Coagul. Fibrinolysis, 5, pp.399~410 (1994)
- Kamiguti AS, "Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases", Toxicon, 34, pp.627~642 (1996)
- Suhr SM, "Identification of the snake venom substance that induces apoptosis", Biochem. Biophys. Res. Commun, 224, pp.134~139 (1996)
- Matsui T, "Snake venom proteases affecting hemostasis and thrombosis", Biochim. Biophys. Acta, 1477, pp.146~156 (2000)
- Swenson S, "Snake venom fibrin(ogen)olytic enzymes", Toxicon, 45, pp.1021~1039 (2005)
- Ramos OHP, "Snake venom metalloproteases - structure and function of catalytic and disintegrin domains", Comp. Biochem. Physiol. Part C, 142, pp.328~346 (2006)
- Marsh N, "Practical applications of snake venom toxins in haemostasis", Toxicon, 45, pp.1171~1181 (2005)
- McDiarmid RW, "Snake species of the world: A taxonomic and geographic reference, vol. 1" (1999), The Herpetologists’ league
- Mehrtens JM, "Living snakes of the world in color" (1987), Sterling Publishers
- Terada S, "Purification of a metalloprotease from Chinese Mamushi (Agkistrodon halys brevicaudus) venom", Fukuoka Univ. Sci. Rep, 21, pp.147~153 (1981)
- Fujimura S, "Purification and characterization of non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus ve-nom", Biochim. Biophys. Acta, 1243, pp.94~100 (1995)
- Terada S, "Purification and amino acid sequence of Brevilysin L6, a non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom", J. Biochem, 125, pp.64~69 (1999)
- Matsui T, "Purification and amino sequence of halystase from snake venom of Agkistrodon halys brevicaudus, a serine protease that cleaves specifically fibrinogen and kininogen", Eur. J. Biochem, 252, pp.69~575 (1998)
- Lee JW, "Purificiation and characterization of brevinase, a heterogenous two-chain fibrinolytic enzyme from the venom of Korean snake, Agkistroden blomhoffii brevicaudus", Biochem. Biophys. Res. Commun, 260, pp.665~670 (1999)
- Astrup T, "The fibrin plate method for estimating of fibrinolytic activity", Archs. Biochem. Biophys, 40, pp.346~351 (1952)
- Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T-4", Nature (London), 227, pp.680~685 (1970)
- Baramova EN, "Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases", Arch. Biochem. Biophys, 275, pp.63~71 (1989)
- Shannon JD, "Amino acid sequence of a Croatalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin", J. Biol. Chem, 264, pp.11575~11583 (1989)
- Johnson EK, "Isolation of hemorrhagic toxin from the venom of Agkistrodon contortrix laticinetus (broad-banded copperhead) and pathogenesis of the hemorrhage induced by the toxin in mice", Int. J. Biochem, 25, pp.267~278 (1993)