Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom.
The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (ADDKNPLEEAFREADYEVFLEIAKNGL) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% alpha-helix, 19% beta-sheet, 10% beta-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 microg/ml), edema- (MED 4.8 microg/ml) and human platelet aggregation-inducing (ED50 33 microg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 microg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.
Ali
S A
SA
International Center for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Pakistan. saali66@hotmail.com
Stoeva
S
S
Abbasi
A
A
Alam
J M
JM
Kayed
R
R
Faigle
M
M
Neumeister
B
B
Voelter
W
W
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D053938:DNA Fragmentation; D004487:Edema / Q000139:chemically induced; D006461:Hemolysis / Q000187:drug effects; D006470:Hemorrhage / Q000139:chemically induced; D006801:Humans; D050830:L-Amino Acid Oxidase; D051379:Mice; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014757:Viper Venoms / Q000201:enzymology*
D014757:Viper Venoms; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase