J Toxicol Sci 2006 May 01;312:157-68. doi: 10.2131/jts.31.157.

Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus.

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During the characterization of hemorrhagic factor in venom of Rhabdophis tigrinus tigrinus, so-called Yamakagashi in Japan, one of the Colubridae family, a novel metalloproteinase with molecular weight of 38 kDa in the Duvernoy's gland of Yamakagashi was identified by gelatin zymography and by monitoring its proteolytic activity using a fluorescence peptide substrate, MOCAc-PLGLA2pr(Dnp)AR-NH2, which was developed for measuring the well-known matrix metalloproteinase (MMP) activity. After purification by gel filtration HPLC and/or column switch HPLC system consisting of an affinity column, which was immobilized with a synthetic BS-10 peptide (MQKPRCGVPD) originating from propeptide domain of MMP-7 and a reversed-phase column, the N-terminal amino acid sequence of the 38 kDa metalloproteinase was identified as FNTFPGDLK which shared a high homology to Xenopus MMP-9. The 38 kDa metalloproteinase required Zn2+ and Ca2+ ions for its proteolytic activity. In addition, the proteolytic activity was almost completely inhibited by BS-10, a MMP inhibitor, but not by the serine proteinase inhibitors, cysteine proteinase inhibitors and aspartic proteinase inhibitors. Together these results demonstrated that the 38 kDa proteinase is a novel snake verom metalloproteinase (SVMP) containing HExGHxxGxxH motif which possesses high affinity to the BS-10 peptide, into its molecule, and the enzymatic properties are closed to that of MMPs. Based on the results obtained in the present study, we concluded that the 38 kDa metalloproteinase is a novel metalloproteinase whose activity may be regulated by the cysteine switch mechanism, and could be classified as one of the matrix metalloproteinases rather than snake venom metalloproteinases.

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