Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom.
A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.
Toyama
Marcos H
MH
UNESP, Campus do Litoral Paulista, São Vicente, São Paulo, São Paulo, Brazil. mhtjpn@yahoo.com
Toyama
Daniela de O
Dde O
Passero
Luiz F D
LF
Laurenti
Marcia D
MD
Corbett
Carlos E
CE
Tomokane
Thaise Y
TY
Fonseca
Fabiana V
FV
Antunes
Edson
E
Joazeiro
Paulo P
PP
Beriam
Luis O S
LO
Martins
Maria Alice C
MA
Monteiro
Helena S A
HS
Fonteles
Marasses C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects*; D003435:Crotalid Venoms / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050830:L-Amino Acid Oxidase / Q000302:isolation & purification / Q000494:pharmacology*; D007896:Leishmaniasis / Q000188:drug therapy; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D013997:Time Factors
D000900:Anti-Bacterial Agents; D003435:Crotalid Venoms; D010975:Platelet Aggregation Inhibitors; D006861:Hydrogen Peroxide; D050830:L-Amino Acid Oxidase