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  • Title: Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability.
    Author: Lee EH, Park JE, Park JW, Lee JS.
    Journal: Int J Mol Med; 2014 Oct; 34(4):1180-90. PubMed ID: 25069477.
    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.
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