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Introduction

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 an L-amino acid oxidase (BmooLAAO-I) from Bothrops moojeni venom, with bactericidal activity over S. aureus and E.colli and anti-Leishmania activity (L. amazonensis, L. brasiliensis, L. donovani and L. major).In Addition to an anti-tumor effect upon breast adenocarcinoma as well as upon human T leukemia. The L-amino acid oxidase is constituted of two subunits, and it has the function of doing the oxidativae deamination of L-amino acids in the process of digestion of the proteins, generating poisonous by-products. Starting from this mechanism, the potential parasiticide effect appears. Inicial assays were done on procariotic cells such as bacteria to identify its toxicity. Leishmaniase is a tropical disease of great importance for developing countries as Brazil. Where it is endemic, and it has the dog as its largest reservoir. The transmission of the disease is made through the bite of mosquitos of the genus Phlebotomus, that inoculate promastigotas forms present in their saliva in the moment of the bite. In the man the disease can be fatal when it assumes the visceral form, where the parasite harms liver and spleen. The resistence development to several drugs commonly used to combat this disease is stimulating new researches. Snake venoms are under constant studies. In Brazil, the genus Bothrops is one of the most frequent in several areas. Just like leishmaniase cancer represents also an important disease with high mortality and dificult treatment. So new agents are always under development.

Methods

Purification: 300μg of crude venom was applied in a Sepharose-IDA (3,0 x 68,0cm) colun and the peak with Laao profile was recromatographed in Sepharose-IDA and than submited to a gel filtration with Sephadex G-100 (0,9 x 180 cm) and the peak with Laao profile was recromatographed under the same conditions. To verify the purity of the enzim it was done in HPLC with reverse phase C-4 (0,46 x 15 cm) and with SDS-poliacrilame eletrophoresis gel.

Anti-bacteria: E. coli and S aureus was assayed as previously described (Paramo et al., 1998). These bacteria, harvested from fresh agar plates and adjusted to 4x105 CFU/ml, were utlized as a target for determining bactericidal activity. For that, 4x105 cells wew incubated with varying amounts of LaaoBmoo for 30 min at 37˚C, in PBS plus 1% peptone. Surviving bacteria wew counted by dilution plate technique.

Anti-leishmania: The leishmanidal assay was done following steps: promastigotas forms of L. major, L. amazonensis, L. braziliensis e L. donovani (Fig. 02) were cultured in Schneider’s and 1x106 cells/well were counted and incubated with various concentration of LaaoBmoo in 96-wells microtiter plates for 24hs at 26˚C. After that cells were incubated with trypan blue and counted in optical microscope for viable cells.

Anti-tumor: Cultures from human breast adenocarcinoma and human linphocitic leukemia was cultured in RPMI medium (sigma) supplemented with 10% fetal bovine serum, 20mM de L-glutamin and 20μM gentamicin at 5%CO2 and 37˚C. 5 x 105 cells were incubated with various concentrations of LaaoBmoo for 24hs and than use a colorimetric assay with tetrazolium is used to measure the toxicity (Mosman 1983).

Figure 1: LAAO from B.moojeni in polyacrilamide gel eletrophoresis. 1 and 3 - 12% SDS and crude venom, 2 and 4 - LAAOmoo, MW - molecular weight marker (Sigma).

Figure 2: Antibacteria assay with LAAO from B.moojeni.

Figure 3: LAAO leishmanicidal activity.

Figure 4: LAAO antitumor activity. (A) Human breast adenocarcinoma (SK-BR-3)

(B) Human T Leukemia Cells (Jurkat)

This study showed the citotoxicity of LaaoBmoo from B.moojeni over protozoary and bacteria with very positive results. The enzim activty is very high in low concentrations both in procariots and eucariotic cells as a toxic agent. Researchs with HIV replication showed activity as anti-viral not related with hidrogen peroxide. New aproachs to see the potencial of practical use of this enzim are need.

Looking for anti-tumoral effects of this flavoenzim the sensibility of three tumor cell lines of human cancer was tested. The results showed high activity in killing tumor cells. In contrast when we tested this fraction over cell culture from normal tissue the toxcicity is not so high. The toxic effect is not related with hidrogen peroxide generated in enzimatic oxidative deamination. Other paths like apoptosis maybe are involved to increase anti-tumor effects.

SBTX 2004

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