title: Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom.
abstract: BACKGROUND
  Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.
  
  
  METHODS
  The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.
  
  
  RESULTS
  Bl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H₂O₂. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC(50) of 0.07μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H₂O₂ in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.
  
  
  CONCLUSIONS
  Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H₂O₂ which kill the cells.
  
  
  CONCLUSIONS
  These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.
authors.affiliation: Research and Development Center, Ezequiel Dias Foundation, 30510-0103, Belo Horizonte, Brazil.
authors.forename: Gustavo B
authors.initials: GB
authors.lastname: Naumann
authors.forename: Liliana F
authors.initials: LF
authors.lastname: Silva
authors.forename: Luciana
authors.initials: L
authors.lastname: Silva
authors.forename: Gilson
authors.initials: G
authors.lastname: Faria
authors.forename: Michael
authors.initials: M
authors.lastname: Richardson
authors.forename: Karla
authors.initials: K
authors.lastname: Evangelista
authors.forename: Markus
authors.initials: M
authors.lastname: Kohlhoff
authors.forename: Celia M F
authors.initials: CM
authors.lastname: Gontijo
authors.forename: Alexei
authors.initials: A
authors.lastname: Navdaev
authors.forename: Flavia F
authors.initials: FF
authors.lastname: de Rezende
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Eble
authors.forename: Eladio F
authors.initials: EF
authors.lastname: Sanchez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D017837:Bothrops / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004795:Enzyme Stability; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050830:L-Amino Acid Oxidase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007892:Leishmania braziliensis / Q000187:drug effects / Q000254:growth & development; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects*; D012910:Snake Venoms / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D012910:Snake Venoms; D006861:Hydrogen Peroxide; D050830:L-Amino Acid Oxidase