L-amino Acid Oxidase Activity Research Articles

Advances in detection methods of L-amino acid oxidase activity.

L-amino acid oxidase (LAAO) is widely distributed in many different organisms and found to play important biological roles, thus attracting a great deal of attention for characterization of its activity. Diverse detection methods with their own properties have been established. This review advanced different LAAO activity assays based on substrate consumption, cofactor amount, and product accumulation. The description of benefits and drawbacks of each method is expected to help researchers find appropriate detection method of LAAO activity for their own purpose.

Snake venomics ofBothrops punctatus, a semiarboreal pitviper species from Antioquia, Colombia

Bothrops punctatus is an endangered, semi-arboreal pitviper species distributed in Panamá, Colombia, and Ecuador, whose venom is poorly characterized. In the present work, the protein composition of this venom was profiled using the ‘snake venomics’ analytical strategy. Decomplexation of the crude venom by RP-HPLC and SDS-PAGE, followed by tandem mass spectrometry of tryptic digests, showed that it consists of proteins assigned to at least nine snake toxin families. Metalloproteinases are predominant in this secretion (41.5% of the total proteins), followed by C-type lectin/lectin-like proteins (16.7%), bradykinin-potentiating peptides (10.7%), phospholipases A2 (93%), serine proteinases (5.4%), disintegrins (38%), L-amino acid oxidases (3.1%), vascular endothelial growth factors (17%), and cysteine-rich secretory proteins (1.2%). Altogether, 6.6% of the proteins were not identified. In vitro, the venom exhibited proteolytic, phospholipase A2, and L-amino acid oxidase activities, as well as angiotensin-converting enzyme (ACE)-inhibitory activity, in agreement with the obtained proteomic profile. Cytotoxic activity on murine C2C12 myoblasts was negative, suggesting that the majority of venom phospholipases A2 likely belong to the acidic type, which often lack major toxic effects. The protein composition of B. punctatus venom shows a good correlation with toxic activities here and previously reported, and adds further data in support of the wide diversity of strategies that have evolved in snake venoms to subdue prey, as increasingly being revealed by proteomic analyses.

A Highly Sensitive Method for Quantitative Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Ferric-Xylenol Orange Formation

L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (FeIIIXO) formation was established. This method showed that due to the conversion of FeII to FeIII by H2O2 and subsequent formation of FeIIIXO complex halo in agar medium, the logarithm of H2O2 concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red FeIIIXO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This FeIIIXO agar assay is highly sensitive to detect H2O2 down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel FeIIIXO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.

Genome analysis of Pseudoalteromonas flavipulchra JG1 reveals various survival advantages in marine environment.

BackgroundCompetition between bacteria for habitat and resources is very common in the natural environment and is considered to be a selective force for survival. Many strains of the genus Pseudoalteromonas were confirmed to produce bioactive compounds that provide those advantages over their competitors. In our previous study, P. flavipulchra JG1 was found to synthesize a Pseudoalteromonas flavipulchra antibacterial Protein (PfaP) with L-amino acid oxidase activity and five small chemical compounds, which were the main competitive agents of the strain. In addition, the genome of this bacterium has been previously sequenced as Whole Genome Shotgun project (PMID: 22740664). In this study, more extensive genomic analysis was performed to identify specific genes or gene clusters which related to its competitive feature, and further experiments were carried out to confirm the physiological roles of these genes when competing with other microorganisms in marine environment.ResultsThe antibacterial protein PfaP may also participate in the biosynthesis of 6-bromoindolyl-3-acetic acid, indicating a synergistic effect between the antibacterial macromolecule and small molecules. Chitinases and quorum quenching enzymes present in P. flavipulchra, which coincide with great chitinase and acyl homoserine lactones degrading activities of strain JG1, suggest other potential mechanisms contribute to antibacterial/antifungal activities. Moreover, movability and rapid response mechanisms to phosphorus starvation and other stresses, such as antibiotic, oxidative and heavy metal stress, enable JG1 to adapt to deleterious, fluctuating and oligotrophic marine environments.ConclusionsThe genome of P. flavipulchra JG1 exhibits significant genetic advantages against other microorganisms, encoding antimicrobial agents as well as abilities to adapt to various adverse environments. Genes involved in synthesis of various antimicrobial substances enriches the antagonistic mechanisms of P. flavipulchra JG1 and affords several admissible biocontrol procedures in aquaculture. Furthermore, JG1 also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. The analysis of the genome of P. flavipulchra JG1 provides a better understanding of its competitive properties and also an extensive application prospect.

In vitro comparison of enzymatic effects among Brazilian Bothrops spp. venoms

In various types of snake venom, the major toxic components are proteinases and members of the phospholipase A2 family, although other enzymes also contribute to the toxicity. In this study, we evaluated the proteolytic, phospholipase, and l-Amino acid oxidase activities in the venom of five Bothrops species—Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, and Bothrops alternatus—all of which are used in the production of commercial antivenom, prepared in horses. The enzymatic activities of each species' venom were classified as high, moderate, or low. B. moojeni venom demonstrated the highest enzymatic activity profile, followed by the venom of B. neuwiedi, B. jararacussu, B. jararaca, and B. alternatus. To our knowledge, this is the first study to compare all of these enzymes from multiple species, which is significant in view of the activity of l-amino acid oxidase across Bothrops species.

In-Gel Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Prussian Blue-Forming Reaction

L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples.

A thermostable L-aspartate oxidase: a new tool for biotechnological applications

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80°C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

Antimicrobial Profile of Selected Snake Venoms and Their Associated Enzymatic Activities

Aims: To investigate the antiviral and antibacterial profile of several crude snake venoms and to assess some of their enzymatic activities. Methodology: The antiviral activities of Naja haje, Bitis arietans, Naja nigricollis and Echis carinatus snake venoms were investigated against Herpes simplex virus type1, Rift valley fever virus and Vesicular stomatitis virus using the end point of cytopathic effect method. Antibacterial activities of Bitis arietans, Cerastes cerastes, Echis carinatus, Vipera lebetina, Naja naja, Pseudechis australis, Naja nigricollis and Naja haje venoms were examined against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa using disc diffusion method. Microdilution method was used to determine the venom's minimum inhibitory concentration. L-amino acid oxidase and phospholipase A2 activities of crude venoms were evaluated using enzymatic assays. Results: Naja nigricollis, Bitis arietans and Echis carinatus snake venoms exhibited significant antiviral activities against all test viruses, except for N. haje treated cells. The mean depletion of viral infectivity titer of venom pretreated cells was higher than its depletion post viral infection for all three venoms showing antiviral activities. Naja nigricollis exhibited the highest antiviral activity against test viruses and recorded a mean depletion of viral infectivity titer in venom pretreated cells of 3.8 log (10) / ml , 3.2 log (10) / ml and 2.5 Research Article British Microbiology Research Journal, 2(4): 251-263, 2012 252 log (10) / ml for HSV-1, RVFV and VSV, respectively. Pseudechis australis, followed by Naja naja and Naja nigricollis venoms, showed the highest inhibitory activity against test bacteria with inhibition zones ranging from 11-17 mm, 8-14 mm and 8-13 mm, respectively. Minimum inhibitory concentrations of test venoms against different bacterial strains ranged from 156 μg / ml to 1.25 mg / ml. Maximum Lamino oxidase activity was detected in Naja naja, Cerastes cerastes and Pseudechis australis. The highest Phospholipase A2 activity was identified in Bitis arietans, Pseudechis australis, Naja naja and Naja nigricollis. Conclusion: It can be concluded that snake venoms or their bioactive derivatives can be promising therapeutic agents against some microbial infections. Further investigations will be carried out for purification and more characterization of the biologically active components in snake venoms.

Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI=5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0–12.0), after heating to 55°C for 30min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H2O2 and other reactive components since it was not counteracted by H2O2 scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.

L-Amino acid oxidase from filamentous fungi: screening and optimization

Twenty-seven fungal isolates recovered on medium containing L-lysine were found to have the potentiality for producing extracellular L-amino acid oxidase (L-AAO). Aspergillus oryzae displayed the highest yield of enzyme (2.6 U/mg protein) and antioxidants (2.3 mg/ml) followed by Aspergillus flavipes and Trichoderma viride. Upon optimization of the fermentation medium, the maximum enzyme yield (4.6 U/mg protein) was obtained on a medium containing L-lysine (80 mM), glucose (0.6%), KH2PO4 (0.1%), KCl (0.05%) and MgSO4⋅7H2O (0.05%), pH 7.0, under submerged conditions. Supplementing the medium with K+, Ni2+ and Cu2+ ions increased enzyme biosynthesis by about twofold compared to the control. The activity of extracellular L-AAO from the submerged cultures of A. oryzae was twofold higher than that of the intracellular L-AAO. Eleven agro-industrial byproducts were tested as substrates under solid state fermentation conditions; of these, rice bran (3.3 U/mg) was the optimum solid substrate for the induction of L-AAO and antioxidants, followed by wheat bran (2.8 U/mg) and cotton seed cake (2.5 U/mg), possibly due to their higher crude protein content than ash and fiber. Glucose (1%) and L-phenylalanine were the best co-metabolic and co-inductive carbon and nitrogen source, respectively, for maximum L-AAO production by A. oryzae under solid state fermentation, at an initial pH of 7.0. The productivity of L-AAO by A. oryzae under solid state fermentation was higher than that of the submerged cultures by about 1.4-fold under optimum conditions, justifying the former fermentation processes for overproduction of this enzyme in terms of both economics and practicality.

Novel l-amino acid oxidase with algicidal activity against toxic cyanobacterium Microcystis aeruginosa synthesized by a bacterium Aquimarina sp.

A brownish yellow pigmented bacterial strain, designated antisso-27, was recently isolated from a water area of saltpan in Southern Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain antisso-27 belongs the genus Aquimarina in the family Flavobacteriacea and its only closest neighbor is Aquimarina spongiae (96.6%). Based on screening for algicidal activity, strain antisso-27 exhibits potent activity against the toxic cyanobacterium Microcystis aeruginosa. Both the strain antisso-27 bacterial culture and its culture filtrate show algicidal activity against the toxic cyanobacterium, indicating that an algicidal substance is released from strain antisso-27. The algicidal activity of strain antisso-27 occurs during the late stationary phase of bacterial growth. Strain antisso-27 can synthesize an algicidal protein with a molecular mass of 190 kDa, and its isoelectric point is approximately 9.4. This study explores the nature of this algicidal protein such as l-amino acid oxidase with broad substrate specificity. The enzyme is most active with l-leucine, l-isoleucine, l-methionine and l-valine and the hydrogen peroxide generated by its catalysis mediates algicidal activity. This is the first report on an Aquimarina strain algicidal to the toxic M. aeruginosa and the algicidal activity is generated through its enzymatic activity of l-amino acid oxidase.

Within-clutch variation in venoms from hatchlings of Deinagkistrodon acutus (Viperidae)

We used 17 hatchling five-paced pit-vipers snakes (Deinagkistrodon acutus) to study within-clutch variation in snake venoms. We measured venom yield and total protein content, and examined the correlations between venom yield and hatchling size [snout-vent length (SVL) and body mass]. We also analyzed the electrophoretic profiles and enzymatic activities of venoms from hatchlings. Lyophilized venom mass was not correlated with SVL, nor with body mass. Liquid venom mass and total protein content were not correlated with body mass, but were positively correlated with SVL. Venom composition, as shown in SDS-PAGE chromatograms did vary among individuals but there were biochemical differences in activity which had to be due to subtle venom composition differences between the sexes. Female hatchlings showed higher esterolytic and fibrinogenolytic activities but lower proteolytic, collagenolytic, phosphomonoesterase and fibrinolytic activities than male hatchlings. We did not find sexual differences in 5′ nucleotidase, phospholipase A2 and hyaluronidase activities, and l-amino acid oxidase activities in either female or male hatchlings. Within-clutch variation in venoms from D. acutus hatchlings should be attributed to the individual-based differences in presence or absence, and the relative amount of the protein components, and might have a genetic basis.

The structure of a nativel-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 Å resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers. The binding of four Zn(2+) ions stabilizes the enzymatically active quaternary structure and is considered important for the biological activity of LAAO and other flavoproteins. Each monomer consists of three domains with a cofactor bound between the FAD and substrate binding domains, and a solvent exposed glycosylation site which is considered crucial for the toxicity. Comparison of LAAO structures in the absence and presence of a substrate indicates conformational changes in the dynamic active site. The active site H-bond network involving the triad Lys326-Water-N5 of FAD is formed only upon substrate binding, and results in the increased mobility of the isoalloxazine system. Details of the catalytic transformation of amino acid substrates are discussed.

Discovery of serum L-amino acid oxidase in the rockfish Sebastes schlegeli: Isolation and biochemical characterization

Fish have a complex innate defense mechanism against microbial invasion. In particular, epidermal mucus and serum in fish play important roles in innate immunity and contain a variety of bioactive substances such as complements, lectins and lysozymes, involved in host defense. Recently, L-amino acid oxidases (LAOs) with antibacterial activity were isolated from the skin and/or gill mucous secretions of rockfish, great sculpin and flounder, and were identified to be a novel type of antibacterial protein in the integument of fish. In the present study, we found LAO activity in the serum of rockfish Sebastes schlegeli. The LAO was isolated from the serum by sequential column chromatography of Con-A lectin affinity chromatography, anion exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC, and characterized. The LAO (a molecular mass of 160 kDa) comprised subunits with a molecular mass of 53 kDa and showed strict substrate specificity, catalyzing only L-lysine with Km 0.37 mM and kcat 57.1s(-1). The serum LAO exhibited a broad antibacterial activity against Gram positive and negative bacteria, most potently against Aeromonas hydrophila and Aeromonas salmonicida with a minimum inhibitory concentration of 0.078 μg/mL. This is the first report of LAO in the serum of fish and its involvement in innate immunity in the rockfish body.

L-Amino acid oxidase activity present in fractions of Bothrops jararaca venom is responsible for the induction of programmed cell death in Trypanosoma cruzi

Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti- T. cruzi activity was associated with fractions that present l-amino acid oxidase ( l-AAO) activity. l-AAO produces H 2O 2, which is highly toxic. The addition of catalase to the medium, a H 2O 2 scavenger, reverted the killing capacity of venom fractions. The anti- T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for l-AAO activity. These results were confirmed with a commercial purified l-AAO. Treatment for 24 h with fractions that present l-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H 2O 2. These results indicate that H 2O 2, the product of l-AAO reaction, induces programmed cell death explaining the anti- T. cruzi activity of B. jararaca venom.

Involvement of an l-amino acid oxidase in the activity of the marine bacterium Pseudoalteromonas flavipulchra against methicillin-resistant Staphylococcus aureus

In this study, a yellow pigmented bacterial strain, designated C2, was recently isolated from the encrusting pore coral Montipora aequituberculata, which was collected from the Kenting coast of Southern Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain C2 belongs to the genus Pseudoalteromonas and that its only closest neighbor is Pseudoalteromonas flavipulchra NCIMB 2033 T strain. Based on the antibiogram assay, strain C2 shows potent antibacterial activity against several methicillin-resistant Staphylococcus aureus (MRSA) strains. Strain C2 can synthesize an antibacterial protein with a molecular mass of 60 kDa, and its isoelectric point is approximately 9.4. Liquid chromatography–tandem mass spectrometry analyses reveal close similarity of this antibacterial protein with the antibacterial protein AlpP from the marine bacterium Pseudoalteromonas tunicata D2. This study explores the nature of this antibacterial protein such as l-amino acid oxidase with broad substrate specificity. The enzyme is most active with l-Lys, l-Met, l-Glu, l-Leu, l-Gln, l-Tyr and l-Phe, and the hydrogen peroxide generated by its catalysis mediates antibacterial activity. This is the first report on a marine bacterium P. flavipulchra producing antibacterial activity by generating hydrogen peroxide through its enzymatic activity of l-amino acid oxidase against MRSA.

Distribution of free D‐aspartic acid and D‐aspartate oxidase in frog Rana esculenta tissues

In this paper, we examined the distribution pattern of D-aspartic acid (D-Asp), as well as D-aspartate oxidase (D-AspO), D-amino acid oxidase (D-AAO), and L-amino acid oxidase (L-AAO) activities in different tissues of frog, Rana esculenta. High concentrations of free D-Asp were found in the testes (0.21+/-0.02 micromol/g b.w), in the liver (0.20+/-0.03 micromol/g b.w), and in the Harderian gland (HG) (0.19+/-0.03 micromol/g b.w). A higher activity of both D-AspO and D-AAO with respect to L-AAO was endogenously present in all examined frog tissues, particularly within the kidney, liver, and brain. Our in vivo experiments, consisting of i.p. injections of 2.0 micromol/g b.w. D-Asp in frogs, revealed that all examined tissues can take up and accumulate D-Asp and that this amino acid specifically triggers D-AspO activity. Indeed, no increase in both D-AAO and L-AAO was found in all frog tissues after D-Asp treatment. The optimum pH for D-AspO activity was around 8.2 and the optimum temperature was about 37 degrees C. Furthermore, its activity linearly increased with increasing D-Asp incubation times. In vitro experiments assaying the substrate specificity of D-AspO indicated that the enzyme had greater affinity for N-methyl-D-aspartate than for D-Asp and D-glutamate. This study provides evidence of the presence of free D-Asp in frog R. esculenta tissues, along with its role in triggering D-AspO activity. These findings suggest that D-AspO could play an essential role in decreasing excessive amounts of D-Asp in frog tissues, a phenomenon that, if left unchecked, could have detrimental physiological effects on the animal.

Cytotoxic proteins of Amanita virosa Secr. mushroom: Purification, characteristics and action towards mammalian cells

A new hemolytic lectin was purified from the fruit bodies of Amanita virosa Secr. mushroom by the affinity chromatography on the cross-linked ovomucin. This lectin destroyed erythrocytes of human and animals of various species, and its hemolytic activity decreased in the row: rabbit > rat > human > dog. The erythrocytes of sheep, cow and carp were resistant to such hemolytic action of the lectin (1 mg/mL). The lectin-mediated hemolysis was blocked by the polyethylene glycol with molecular mass over 1350. A. virosa lectin, unlike Amanita phalloides lectin, did not interact with tested monosaccharides. However, the 4-nitrophenyl derivates of the monosaccharides inhibited the action of A. virosa lectin which did not prefer targeting O-type glycoproteins over the N-type glycoproteins. Murine leukemia cells of L1210 line and human leukemia T-cells of CEM T4 and Jurkat lines were shown to be sensitive to toxic effect of the lectin and another protein toxovirin isolated from A. virosa fruit bodies It was found that toxovirin possessed an enzymatic activity of l-amino acid oxidase. Since both toxic proteins – the lectin and toxovirin – are sensitive to an elevated temperature, it is suggested that they play a significant role in human poisoning only when the unbaked mushroom is eaten.

Isolation and cDNA cloning of an antibacterial L-amino acid oxidase from the skin mucus of the great sculpin Myoxocephalus polyacanthocephalus

The skin mucus of the great sculpin Myoxocephalus polyacanthocephalus showed both antibacterial and L-amino acid oxidase (LAO) activities. Antibacterial LAOs were purified from the skin mucus of the M. polyacanthocephalus by column chromatography and named MPLAO1, MPLAO2, and MPLAO3, based on the order of elution by ion-exchange high performance liquid chromatography. cDNA cloning of MPLAO3 revealed that the full-length cDNAwas 2659 bp and encoded the signal peptide (Met1-Ala26) and the mature protein (Val28-Phe520). A homology search using the BLAST program revealed that MPLAO3 shared sequence identity with LAO family proteins, and had 74% identity with the antibacterial LAO from the skin mucus of the rockfish Sebastes schlegeli. MPLAO3 catalyzed the oxidation of only L-lysine with a Km of 0.16 mM. MPLAO3 exhibited potent antibacterial activity against both Gram-positive and Gramnegative bacteria, and was most active against Aeromonas salmonicida JCM7874 with a minimum inhibitory concentration of 0.02 microg/mL. The antibacterial activity was attributable to H2O2, because the activity was completely lost in the presence of catalase. The antibacterial LAOs may be involved in the innate immunity of the great sculpin M. polyacanthocephalus skin.

Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) — Relation to pharmacological activities

The venom proteome of Daboia russelli siamensis, a snake of medical importance in several Asian countries, was analysed by 2-D electrophoresis, subsequent MS/MS and enzymatic assays. The proteome comprises toxins from six protein families: serine proteinases, metalloproteinases, phospholipases A(2), L-amino acid oxidases, vascular endothelial growth factors and C-type lectin-like proteins. The venom toxin composition correlates with the clinical manifestation of the Russell's viper bite and explains pathological effects of the venom such as coagulopathy, oedema, hypotensive, necrotic and tissue damaging effects. The vast majority of toxins are potentially involved in coagulopathy and neurotoxic effects. The predominant venom components are proteinases capable of activating blood coagulation factors and promoting a rapid clotting of the blood, and neurotoxic phospholipase A(2)s. The analysis of the venom protein composition provides a catalogue of secreted toxins. The proteome of D. r. siamensis exhibits a lower level of toxin diversity than the proteomes of other viperid snakes. In comparison to the venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, the venom from D. r. siamensis showed quantitative differences in the proteolytic, phospholipase A(2), L-amino acid oxidase and alkaline phosphatase activities.