L-aminoácido oxidase

Izidoro LF, Ribeiro MC, Souza GR, Sant'ana CD, Hamaguchi A, Homsi-Brandeburgo MI, Goulart LR, Beleboni RO, Nomizo A, Sampaio SV, Soares AM, Rodrigues VM.

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.

Samel M, Vija H, Ronnholm G, Siigur J, Kalkkinen N, Siigur E.

An L-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom L-amino acid oxidases. The purified protein catalyzed oxidative desamination of L-amino acids, the most specific substrate is L-Phe. The best substrates among the studied 20 amino acids were: L-Met, L-Leu, L-Phe, L-Ile, L-Arg and L-His. Five amino acids, L-Ser, L-Pro, Gly, L-Thr and L-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 microM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.

Sant`Ana, CD^(1,3); Curtarelli, MB⁽¹⁾; Pereira, ALA⁽¹⁾; Ticli, FK⁽³⁾; Mazzi, MV⁽³⁾; Marcussi, S^(1,2); Magalhães, MR⁽⁴⁾; Giglio, JR⁽²⁾; Sampaio, SV⁽³⁾; Pereira, PS⁽¹⁾; Marins, MA⁽¹⁾; Soares, AM⁽³⁾

Inibição da atividade nucleotídica de venenos de serpentes por extratos de plantas

Venenos de serpentes são compostos principalmente por enzimas das classes PLA2s, proteases, LAAOs, entre outras. Essas proteínas têm sido intensamente estudadas e utilizadas como ferramentas em laboratórios para fins diagnósticos, sendo até mesmo empregadas para o entendimento de patologias e com aplicação na clínica médica. Neste trabalho foi avaliada a inibição da atividade nucleotídica de venenos de serpentes dos gêneros Bothrops e Crotalus, por extratos aquosos de diferentes plantas (Pentacletra macroloba, Mikania glomerata, Casearia sylvestris e Mandevilla velutina). As partes das plantas utilizadas para a elaboração dos extratos foram escolhidas baseando-se em conhecimentos populares de seu uso anti-ofídico, sendo utilizados o caule de P. macroloba, as raízes de M. glomerata, folhas de C. sylvestris e xilopódio de M. velutina. As amostras de venenos e extratos foram previamente incubadas em volume final de 5µL durante 60min a 37ºC (n=3) e posteriormente avaliadas através da técnica de enzimodifusão, sendo utilizado um gel composto por agarose 2%, tampão Tris-HCl 0,05M com MgSO4 0,01M pH 7.4 acrescido de DNA de esperma de salmão como substrato e brometo de etídio para possibilitar a visualização da atividade em UV. Os extratos de P. macroloba e M. glomerata foram eficientes em inibir 100% da atividade nucleotídica induzida pelos venenos de Bothrops jararacussu (50µg), B. neuwiedi (50 e 100µg), C. d. terríficus (50 e 100µg) e C. d. collineatus (50µg), nas proporções de 1:15 e 1:30 (m/m). Os extratos de C. sylvestris e M. velutina inibiram 100% da atividade nucleotídica do veneno de B. jararacussu apenas nas primeiras 6 horas, mostrando o mesmo efeito para C. d. collineatus em 24 horas de experimento, na proporção de 1:12 (m/m). Os resultados apresentados geram dados para a identificação de novos inibidores naturais dos efeitos tóxicos do envenenamento ofídico, além de servirem como uma ferramenta na compreensão do mecanismo de ação das nucleotidases presentes em venenos de serpentes.

Malta-Neto, N.R.1; Stabeli, R.G.5; Marcussi, S.1,2; Nomizo, A.4; Sant’Ana, C.D.4; Giglio, J. R.2; Sampaio, S. V.4; Oliveira, E.B.2; Soares, A.M.1,4.
(smarcussi@rbi.fmrp.usp.br)

L-Amino acid oxidases (LAAO, EC 1.4.3.2) are flavoenzymes which catalyze the stereospecific oxidative deamination of an L-amino acid substrate to a corresponding α-ketoacid with the production of hydrogen peroxide and ammonia, via an imino acid intermediate. These enzymes are widely distributed in many different organisms such as bacteria, fungi, green algae and venomous snakes and are involved in the utilization of nitrogen sources. The present investigation reports isolation and biochemical characterization of L-amino acid oxidase (BmooLAAO-I) from Bothrops moojeni venom,
anti-Tumor effect upon murine melanoma, human breast adenocarcinoma, human leukemia T as well as upon peripheral human mononuclear cells. The citotoxic assay was done by using the MTT method described by Mosmann. It still shows the capacity of BmooLAAO-I to induce apoptosis in human leukemia cells compared to paclitaxel. It was tested in flux citometer after incubation with anexin V and propidium iodide. BmooLAAO-I is an acidic glycoprotein with about 130kDa arranged in two 66kDa monomers. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. BmooLAAO-I also displays dose dependent and anti-Tumor activities. The mechanism by its acts seems to be oxidative effects of hydrogen peroxide. Other mechanism involved in citotoxicity is apoptosis induction. SV-LAAOs are therefore interesting multifunctional enzymes, not only for a better understanding of the ophidian envenomation mechanism, but also due to their biotechnological potential as model for therapeutic agents.

Zhao QT, Xie K, Zhang J, Miao JY.

Hemorrhagic snake venom specially induces apoptosis of VEC (vascular endothelial cells). Five apoptosis-inducing proteins had been purified and characterized from crude snake venom. Two of these are L-amino acid oxidase (LAO), the others belong to metalloprotease/disintegrin family. LAO catalyzes H2O2 production by oxidizing some plasma membrane proteins of VEC, disintegrins interfere with binding of integrins with their ligands. The expression of p53 and bcl-2 increases during VEC apoptosis induced by snake venom, moreover, the mRNA of bcl-2 is spliced into two fragments. It has been proved that one of adhesion-dependent signal molecules, alphavbeta3, and one of phospholipid signal molecules, PC-PLC (phosphatidylcholine-specific phospholipase C), are involved in above apoptosis-inducing signal transudation pathway. These results throw light on finding out specific component from protein is snake venom. This component is able to induce tumor vascular endothelial cells apoptosis. This review summarized progress of research on hemorrhagic snake venoms.