title: Primary structure and characterization of a non hemorrhagic metalloproteinase with fibrinolytic activity, from the snake venom of Protobothrops tokarensis (Tokara-habu).
abstract: A low molecular weight metalloproteinase, named PT-H₂ protease, with fibrinolytic activity, was purified from the venom of Protobothrops tokarensis (Tokara-habu) by gel-filtration using Sephadex G-100, and ion-exchange chromatographies using CM-Sepharose Fast Flow and Mono S HR 5/5. By this procedure, about 85 mg of purified protein were obtained from 1.0 g of P. tokarensis venom. The purified protein showed a single protein band with a molecular weight of about 22.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed a single band of 6.8. This proteinase showed a strong fibrinolytic activity. Further, this proteinase showed fibrinogenase activity and proteolytic activity against synthetic substrates for matrix metalloproteinase, ADAM-17, and TACE (tumor necrosis factor converting enzyme). These proteolytic activities were inhibited by metalloproteinase inhibitors such as EDTA. PT-H₂ protease consisted of 201 amino acid residues and had a calculated molecular weight of 22,994.7 Da. This protein showed conservation of the Zn²⁺-binding HEXXHXXGXXHD sequence. PT-H₂ protease showed high homology from 51.7 to 99.5% with amino acid sequences of other snake venom metalloproteinases.
authors.affiliation: Department of Hygienic Chemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, Japan. oyama@my-pharm.ac.jp
authors.forename: Etsuko
authors.initials: E
authors.lastname: Oyama
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kitagawa
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Takahashi
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003911:Dextrans; D004492:Edetic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012910:Snake Venoms / Q000737:chemistry*; D013329:Structure-Activity Relationship; D017841:Trimeresurus
chemical_list: D003911:Dextrans; D004791:Enzyme Inhibitors; D005343:Fibrinolytic Agents; D012910:Snake Venoms; C025614:sephadex; D004492:Edetic Acid; D051722:ADAM Proteins; D008666:Metalloendopeptidases; C069317:hemorrhagic metalloproteinase; D000072198:ADAM17 Protein