title: Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability.
abstract: In the present study, a novel fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom was purified and characterized in terms of enzyme kinetics and substrate specificity. The purified enzyme [termed snake venom metalloprotease-Macrovipera mauritanica (SVMP‑MM)] was composed of a single polypeptide with an apparent molecular weight of 27 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of the enzyme was composed of NH(2)-QRFAPRYIEL-COOH, as determined by N-terminal sequencing. The Aα- and the Bβ-chains of fibrinogen were completely cleaved by SVMP-MM within 20 and 480 min, respectively. However, the γ-chain was much more resistant to digestion by the enzyme. The enzyme also exhibited proteolytic activity, cleaving the α-α polymer of cross-linked fibrin, but did not effectively digest the γ-γ polymer. To determine the kinetic parameters for SVMP-MM, a fluorescence-quenching peptide (termed o-aminobenzoic acid-HTEKLVTS-2,4-dinitrophenyl‑NH(2)) containing a K-L sequence for SVMP-MM cleavage was designed and synthesized. The optimal pH and temperature for the enzyme activity were found to be 5.5 and 37˚C, respectively, when the fluorogenic substrate was synthesized and used as a substrate. Among the various divalent cations tested, Ni(2+) and Cu(2+) showed strong inhibitory effects on enzyme activity, with an average of 69.6% inhibition. The enzyme activity was also inhibited by treatment with 1,10-phenanthroline, ethylenediaminetetraacetic acid and glycol-bis-(2‑aminoethylether)-N,N,N',N'-tetra-acetic acid, but not with aprotinin, tosyl-lysine chloromethyl ketone and tosyl-phenylalanyl chloromethyl ketone, suggesting that SVMP-MM is a metalloprotease and not a serine protease. The enzymatic parameters, including the K(M), k(cat), and k(cat)/K(M) values were estimated to be 0.015 mM, 0.031 sec(-1), and 20.67 mM(-1)sec(-1), respectively. SVMP-MM induced vascular permeability by digesting type IV collagen. The results obtained in our study demonstrate that SVMP-MM is a fibrin(ogen)olytic P-I class metalloprotease, which can induce a hemorrhagic reaction in vivo.
authors: Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea.
authors: Eun Hee
authors: EH
authors: Lee
authors: Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea.
authors: Jung Eun
authors: JE
authors: Park
authors: Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea.
authors: Jong Woo
authors: JW
authors: Park
authors: Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea.
authors: Jung Sup
authors: JS
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D024141:Collagen Type IV / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D005453:Fluorescence; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D024141:Collagen Type IV; D010455:Peptides; D011480:Protease Inhibitors; D014757:Viper Venoms; D005340:Fibrinogen; D045726:Metalloproteases