title: Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus.
abstract: 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.
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotogecho, Hirakata, Osaka 573-0101, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Komori
authors.forename: Motomi
authors.initials: M
authors.lastname: Konishi
authors.forename: Yuji
authors.initials: Y
authors.lastname: Maruta
authors.forename: Michihisa
authors.initials: M
authors.lastname: Toriba
authors.forename: Atsushi
authors.initials: A
authors.lastname: Sakai
authors.forename: Akira
authors.initials: A
authors.lastname: Matsuda
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Hori
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Nakatani
authors.forename: Naoto
authors.initials: N
authors.lastname: Minamino
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Akizawa
mesh_terms: D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017812:Colubridae; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005088:Exocrine Glands / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020782:Matrix Metalloproteinases / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D008970:Molecular Weight; D010446:Peptide Fragments; D012910:Snake Venoms / Q000201:enzymology*; D013379:Substrate Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2
chemical_list: D010446:Peptide Fragments; D012910:Snake Venoms; D019716:Tissue Inhibitor of Metalloproteinase-2; D020782:Matrix Metalloproteinases