RESUMEN
Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Artritis/genética , Fisura del Paladar/genética , Enfermedades del Tejido Conjuntivo/genética , Matriz Extracelular/genética , Pérdida Auditiva Sensorineural/genética , Miopía/genética , Neoplasias/genética , Desprendimiento de Retina/genética , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/metabolismo , Artritis/enzimología , Artritis/patología , Fisura del Paladar/enzimología , Fisura del Paladar/patología , Colágeno/química , Colágeno/genética , Colágeno/metabolismo , Enfermedades del Tejido Conjuntivo/enzimología , Enfermedades del Tejido Conjuntivo/patología , Elastina/química , Elastina/genética , Elastina/metabolismo , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/química , Matriz Extracelular/enzimología , Regulación de la Expresión Génica , Pérdida Auditiva Sensorineural/enzimología , Pérdida Auditiva Sensorineural/patología , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Miopía/enzimología , Miopía/patología , Neoplasias/enzimología , Neoplasias/patología , Especificidad de Órganos , Desprendimiento de Retina/enzimología , Desprendimiento de Retina/patología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18⯰C to 32⯰C at +3⯰Câ¯day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 µatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32⯰C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32⯰C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30⯰C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.
Asunto(s)
Metabolismo Energético , Gastrópodos/fisiología , Regulación del Desarrollo de la Expresión Génica , Calentamiento Global , Respuesta al Choque Térmico , Modelos Biológicos , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Animales , Acuicultura , Dióxido de Carbono/envenenamiento , Hipoxia de la Célula , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Gastrópodos/clasificación , Gastrópodos/crecimiento & desarrollo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Calor/efectos adversos , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , México , Músculos/fisiología , Filogenia , Estabilidad Proteica , Distribución AleatoriaRESUMEN
A gene encoding 1-aminocyclopropane-1-carboxylic oxidase (ACO), which catalyzes the terminal step in ethylene biosynthesis, was isolated from Agrostis stolonifera. The AsACO gene is composed of 975 bp, encoding 324 amino acids. Three exons interspersed by two introns form AsACO gDNA. A BLAST search of the nucleotide sequence revealed a high level of similarity (79-91%) between AsACO and ACO genes of other plants. A phylogenetic tree was constructed via BLAST in the NCBI, and revealed the highest homology with wheat TaACO. The calculated molecular mass and predicted isoelectric point of AsACO were 36.25 and 4.89 kDa, respectively. Analysis of subcellular localization revealed that AsACO is located in the nucleus and cytoplasm. The Fe(II)-binding cofactors and cosubstrate were identified, pertaining to the ACO family. The expression patterns of AsACO were determined by quantitative real time PCR. AsACO expression was highest in the stem, and was strongly up-regulated in response to ethephon, methyl jasmonate, salicylic acid, and cold temperature, but down-regulated in response to drought and NaCl treatment. The protein encoded by AsACO exhibited ACC oxidase activity in vitro. Taken together, these findings suggest that AsACO contains domains common to the ACO family, and is induced in response to exogenous hormones. Conversely, some abiotic stress conditions can inhibit AsACO expression.
Asunto(s)
Agrostis/enzimología , Agrostis/genética , Aminoácido Oxidorreductasas/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas de Plantas/genética , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , Biología Computacional , ADN Complementario/genética , Vectores Genéticos/metabolismo , Peso Molecular , Filogenia , Hojas de la Planta/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Fracciones Subcelulares/enzimología , Transcripción GenéticaRESUMEN
An L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) was purified to a high degree using sequential CM-Sepharose ion-exchange and phenyl-Sepharose chromatography. When analyzed by mass spectrometry, the purified BmooLAAO-I presented a molecular weight of 64,889 and 130,779 under denaturing and nondenaturing conditions, respectively. BmooLAAO-I is a homodimeric acidic glycoprotein with a pI approximately 4.7, and the N-terminal sequence shows close structural similarity to other snake venom LAAOs. This enzyme was inactivated by freezing or low pH, and secondary structural analysis by circular dichroism revealed 48% alpha-helix, 20% beta-sheet, 12% beta-turn, and 20% random coil structures. BmooLAAO-I exhibited bactericidal, antitumoral, trypanocidal, edematogenic, and platelet-aggregating activities. All of these effects were inhibited by catalase, suggesting that these biological effects are mediated by the production of H(2)O(2). BmooLAAO-I induced typical apoptotic DNA fragmentation in HL-60 cells, which was also inhibited by catalase. These results point to the potential use of BmooLAAO-I as a therapeutic agent for treatment of diseases in which induction of H(2)O(2) production can be beneficial.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Aminoácido Oxidorreductasas/farmacología , Bothrops , Venenos de Crotálidos/enzimología , Fragmentación del ADN/efectos de los fármacos , Aminoácido Oxidorreductasas/química , Animales , Catalasa/farmacología , Cromatografía Liquida , Células HL-60 , Humanos , Peróxido de Hidrógeno/metabolismo , Estructura Secundaria de ProteínaRESUMEN
A supramolecular approach was used for adsorbing a monolayer of adamantane-modified phenylalanine dehydrogenase on beta-cyclodextrin-coated Au electrodes. The enzyme electrode (poised at +200 mV vs. Ag/AgCl) showed a linear amperometric response up to 3 mM L-phenylalanine (L-Phe) with a lower detection limit of 15 microM. The reversible nature of this immobilization approach was confirmed.
Asunto(s)
Aminoácido Oxidorreductasas/química , Técnicas Biosensibles/instrumentación , Electroquímica/instrumentación , Microelectrodos , Fenilalanina/análisis , beta-Ciclodextrinas/química , Bacillus/enzimología , Técnicas Biosensibles/métodos , Materiales Biocompatibles Revestidos/química , Enzimas Inmovilizadas/química , Diseño de Equipo , Análisis de Falla de Equipo , Oro/química , Complejos Multiproteicos/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A mono-aminated dextran derivative was attached to Bacillus badius phenylalanine dehydrogenase via a carbodiimide-catalyzed reaction. The optimum temperature for the conjugate was 10 degrees C higher than for native enzyme, and its thermostability was improved by 8 degrees C. The activation free energy of thermal inactivation at 45 degrees C was increased by 16.8 kJ/mol. The improved conformational stability of the modified enzyme was confirmed by fluorescence spectroscopy.
Asunto(s)
Aminoácido Oxidorreductasas/química , Bacillus/enzimología , Dextranos/química , Activación Enzimática , Estabilidad de Enzimas , Calor , Desnaturalización ProteicaRESUMEN
The glycine-cleavage complex (GCV) and serine hydroxymethyltransferase represent the two systems of one-carbon transfer that are employed in the biosynthesis of active folate cofactors in eukaryotes. Although the understanding of this area of metabolism in Plasmodium falciparum is still at an early stage, we discuss evidence that genes and transcription products of the GCV are present and expressed in this parasite. The potential role of the GCV and its relevance to the life cycle and pathogenesis of the malaria erythrocytic stages are also considered. According to its expression profile, the GCV seems to be particularly active in gametocytes. The GCV enzyme dihydrolipoamide dehydrogenase has two isoforms encoded by two different genes. It has been demonstrated recently that both genes are functional, with one of them identified as being part of a pyruvate dehydrogenase complex that is present exclusively in the apicoplast of Plasmodium species. The other isoform probably forms part of the Plasmodium GCV. The GCV is the first enzyme complex involved in folate metabolism in this parasite that can be assumed, with a good degree of certainty, to be located in the mitochondria.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Proteínas Portadoras/metabolismo , Eritrocitos/parasitología , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Complejos Multienzimáticos/metabolismo , Plasmodium falciparum/enzimología , Transferasas/metabolismo , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/genética , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dihidrolipoamida Deshidrogenasa/genética , Dihidrolipoamida Deshidrogenasa/metabolismo , Ácido Fólico/química , Regulación Enzimológica de la Expresión Génica , Glicina/metabolismo , Glicina Hidroximetiltransferasa/genética , Malaria Falciparum/parasitología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Transferasas/química , Transferasas/genéticaRESUMEN
It is widely accepted that immunological cross-reactivity of snake venoms is mediated by antibodies that recognize venom components bearing either amino acid sequence homology or similar biological functions. However, here we demonstrate that polyspecific Bothrops antivenom is a source of cross-reactive antibodies that interact with venom proteins of distinctive primary structures and biological functions. The homoserine lactone derivative of the undecapeptide IQRWSLDKYAM (Ile1-Hse11), excised from the l-amino acid oxidase (LAAO) of the Bothrops moojeni venom, was the ligand of an affinity resin used to isolate specific anti-Ile1-Hse11 antibodies which were instrumental in revealing immunological cross-reactivity among unrelated venom proteins. We examined the extent of the cross-reactivity of these antibodies by probing electroblots of venoms from representative snakes of genera Bothrops, Lachesis, Crotalus and Micrurus, and by unambiguous structural characterization of the affinity-purified proteins of B. moojeni venom recovered from an agarose-anti-Ile1-Hse11 column. Our results indicate that all venoms tested had at least three reactive components toward anti-Ile1-Hse11 antibodies, among which we identified two serine proteases, one phospholipase A2 homologue, and LAAO. We hypothesize that the cross-reactivity of the anti-Ile1-Hse11 antibodies to unrelated venom proteins derives from their mechanism of antigen recognition, whereby complementarity is achieved through reciprocal conformational adaptation of the reacting molecules. Also, we believe these findings have implications both in the development of improved antivenoms and the preparation of immunochemical reagents for diagnostic and scientific investigation purposes in the field of snake venoms.
Asunto(s)
Aminoácido Oxidorreductasas/inmunología , Anticuerpos Monoclonales/inmunología , Bothrops , Reacciones Cruzadas/inmunología , Venenos de Serpiente/inmunología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Aminoácido Oxidorreductasas/química , Animales , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , L-Aminoácido Oxidasa , Análisis de Secuencia de Proteína , Venenos de Serpiente/química , Venenos de Serpiente/toxicidadRESUMEN
An extracellular L-glutamate oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G. The enzyme had a molecular weight of approximately 120,000 and consisted of two identical subunits, each with a molecular weight of 61,000. The isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.0-7.4. GLOD showed the maximum activity at 37ºC. The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among 21 amino acids tested for substrate specificity, L-glutamate was almost exclusively oxidized. D-glutamate and L-aspartate were oxidized but only to extents of 0.79 percent and 0.53 percent, respectively.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Aminoácido Oxidorreductasas/metabolismo , Aminoácido Oxidorreductasas/química , Streptomyces/enzimología , Cromatografía , Medios de Cultivo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Peso Molecular , Especificidad por Sustrato , TemperaturaRESUMEN
Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic DeltaGo' (at least -5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP(+)-dependent beta-oxidation of palmitoyl-CoA in the glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.
Asunto(s)
Difosfatos/metabolismo , Microcuerpos/metabolismo , Piruvato Ortofosfato Diquinasa/metabolismo , Trypanosoma cruzi/metabolismo , Alanina-Deshidrogenasa , Aminoácido Oxidorreductasas/química , Animales , Western Blotting , Carbonatos/química , Detergentes/farmacología , Digitonina/química , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/metabolismo , Glucólisis , Concentración de Iones de Hidrógeno , Iones , Modelos Biológicos , NADP/química , Octoxinol , Oxígeno/metabolismo , Polietilenglicoles/farmacología , Piruvato Carboxilasa/química , Piruvatos/química , Fracciones Subcelulares/metabolismo , TermodinámicaRESUMEN
The isolation and biochemical/enzymatic characterization of an L-amino acid oxidase, Balt-LAAO-I, from Bothrops alternatus snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI approximately 5.37, homodimeric, Mr approximately 123,000, whose N-terminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Balt-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Balt-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Aminoácido Oxidorreductasas/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bothrops , Venenos de Crotálidos/enzimología , Agregación Plaquetaria/efectos de los fármacos , Aminoácido Oxidorreductasas/química , Secuencia de Aminoácidos , Animales , Antibacterianos/química , L-Aminoácido Oxidasa , Ratones , Datos de Secuencia MolecularRESUMEN
In this paper are presented structural analysis and expression studies of one genomic clone encoding a 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) from papaya. Using RT-PCR amplification of ACC oxidase cDNAs from ripe papaya, a product of 800 bp was obtained, which after sequence analysis was found to code for a protein highly homologous to ACC oxidase proteins. This PCR product was used as a probe for screening a genomic library, and two different groups of clones were obtained as indicated by restriction mapping. One clone (CPACCO-1) was selected for further study and fully sequenced. Comparison of this sequence with the PCR product and other cloned ACC oxidase genes revealed that CPACCO-1 encoded the transcript in four exons interrupted by three introns. Southern blot analysis showed one or two major bands hybridized to the PCR probe, suggesting that the ACC oxidase gene is present in one or two copies in the papaya genome. By northern blot analysis it was found that the ACC oxidase transcripts appear in the pulp earlier than in the peel, suggesting a developmental regulation. A wounding experiment revealed the highest expression of this gene by 2 h. Transcriptional regulation by ethylene could be due to the presence of a putative GCC box in the promoter region.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Carica/enzimología , Frutas/enzimología , Aminoácido Oxidorreductasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Frutas/crecimiento & desarrollo , Expresión Génica , Datos de Secuencia Molecular , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.
Asunto(s)
Aminoácido Oxidorreductasas/química , Azospirillum brasilense/enzimología , Cianobacterias/enzimología , NADP/química , Catálisis , Ditionita/química , Ferredoxinas/química , Ácido Glutámico/química , Glutaminasa/química , Glutamina/química , Ácidos Cetoglutáricos/química , Oxidación-Reducción , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Espectrofotometría , Sulfitos/química , Sales de Tetrazolio/química , VolumetríaRESUMEN
We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Venenos de Crotálidos/enzimología , Agkistrodon , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Células HL-60 , Humanos , L-Aminoácido Oxidasa , Conformación ProteicaRESUMEN
Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use of biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.