RESUMO
L-lysine α-oxidase (LO) is an L-amino acid oxidase with antitumor, antimicrobial and antiviral properties. Pharmacokinetic (PK) studies were carried out by measuring LO concentration in plasma and tissue samples by enzyme immunoassay. L-lysine concentration in samples was measured spectrophotometrically using LO. After single i.v. injection of 1.0, 1.5, 3.0 mg/kg the circulating T1/2 of enzyme in mice varied from 51 to 74 min and the AUC0-inf values were 6.54 ± 0.46, 8.66 ± 0.59, 9.47 ± 1.45 µg/ml × h, respectively. LO was distributed in tissues and determined within 48 h after administration with maximal accumulation in liver and heart tissues. Mean time to reach the maximum concentration was highest for the liver-9 h, kidney-1 h and 15 min for the tissues of heart, spleen and brain. T1/2 of LO in tissues ranged from 7.75 ± 0.73 to 26.10 ± 2.60 h. In mice, plasma L-lysine decreased by 79% 15 min after LO administration in dose 1.6 mg/kg. The serum L-lysine levels remained very low from 1 to 9 h (< 25 µM, 17%), indicating an acute lack of L-lysine in animals for at least 9 h. Concentration of L-lysine in serum restored only 24 h after LO administration. The results of LO PK study show that it might be considered as a promising enzyme for further investigation as a potential anticancer agent.
Assuntos
Aminoácido Oxirredutases/farmacocinética , Trichoderma/enzimologia , Aminoácido Oxirredutases/administração & dosagem , Animais , Proteínas Fúngicas/administração & dosagem , Proteínas Fúngicas/farmacocinética , Lisina/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
Synergism effects of cisplatin and L-lysine-alpha-oxidase (LO), while sequential (no interval) administration of drugs depends on the tumor model and duration of treatment. Synergism is identified at intraperitoneal daily (during 3 days) administration of cisplatin to experimental animals in single doses of 1.5 or 3.0 mg/kg and intravenously 5-fold after 48 h administration of LO and also administered intravenously in cumulative doses of 300-600 E / kg discretely, the first dose--doubled. Synergism of cisplatin and LO is showed by significant (p < 0.05) therapeutic gain against cisplatin at such indicators as increased survival of mice with P388 tumor and increased inhibition of primary tumor melanoma B16.
Assuntos
Aminoácido Oxirredutases/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , Leucemia P388/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Aminoácido Oxirredutases/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Cisplatino/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Análise de Sobrevida , Resultado do TratamentoRESUMO
L-Amino acid oxidases (L-ÐÐÐ, EC 1.4.3.2) comprise a group of flavoproteins, catalyzing oxidative deamination of L-alpha amino acids to the corresponding alpha-keto acids, NH3 and Ð2Ð2. In most cases these enzymes present homodimeric molecules with a molecular mass of 100-150 kDa, which were shown to possess antiviral, antifungal and antitumor activity. L-lysine alpha-oxidase (LO) holds an outstanding place among this group of enzymes and its biological role may differ significantly from the other L-AAO, because it cleaves an essential amino acid - L-lysine without significant action on the other amino acids. Although much research has examined LO effects in the organism, the molecular basis of these effects is yet to be identified. To fill this gap, the present work addressed one of hypothetical mechanisms of LO biological action using the enzyme from Trichoderma cf. aureoviride Rifai ÐÐÐF-4268D and rat pheochromocytoma PC-12 as a model cell line. Using flow cytometry a dose-dependent cytotoxicity of LO was shown. The significant growth of intracellular reactive oxygen species levels, detected by 2,7-dichlorodihydrofluorescein assay, implies generation of peroxide as one of the molecular mechanisms of LO cytotoxic action, although this does not rule out other probable ways of LO action in the organizm.
Assuntos
Aminoácido Oxirredutases/toxicidade , Proteínas Fúngicas/toxicidade , Trichoderma/enzimologia , Animais , Sobrevivência Celular , Células PC12 , RatosRESUMO
L-lysine-alpha-oxidase from Trichoderma sp. fungi inhibits the synthesis of DNA, RNA and protein in cultivated cells of the Burkitt lymphoma (strain P3HRj) in vitro, which results in the blocking of the cells' transfer from phase S to phase G2/M of the mitotic cycle.
Assuntos
Aminoácido Oxirredutases/toxicidade , Linfoma de Burkitt/patologia , Linfoma de Burkitt/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , DNA de Neoplasias/biossíntese , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Fatores de Tempo , Trichoderma/enzimologiaRESUMO
Activity of L-amino acid oxidases was studied using several procedures. Optimal concentrations of L-lysine-alpha-oxidase, suitable for each procedure, were established involving highly purified preparations of the enzyme from Trichoderma sp. Estimation of the enzymatic activity carried out by means of calculation of the reduced cofactor accumulated led to two-fold exceeding of the results. The most sensitive procedure was based on evaluation of ammonium content in the reaction catalyzed by glutamate dehydrogenase and the procedure where peroxidase and o-dianizidine were used.
Assuntos
Aminoácido Oxirredutases/análise , Proteína-Lisina 6-Oxidase/análise , Cinética , Lisina/metabolismo , Métodos , Estereoisomerismo , Especificidade por SubstratoRESUMO
Homogeneous preparations of L-lysine-alpha-oxidase were obtained from Trichoderma sp cultivated by using a surface technique and a fermenter set. The homogeneous enzyme preparations were similar in molecular mass, isoelectric point and substrate specificity. There was the single difference in the absorbance spectra, which may occur due to the presence of cofactor FAD in various oxidation states. The findings suggest that cultivation of Trichoderma sp in the fermenter set did not alter properties of L-lysine-alpha-oxidase produced.
Assuntos
Aminoácido Oxirredutases/metabolismo , Trichoderma/enzimologia , Fermentação , Ponto Isoelétrico , Peso Molecular , Análise Espectral , Especificidade por SubstratoRESUMO
L-lysine-alpha-oxidase, isolated from a strain of Trichoderma sp. exhibited enzymological properties suitable for a chemotherapeutic drug used in experimental oncology.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Antineoplásicos , Fungos Mitospóricos/enzimologia , Trichoderma/enzimologia , Aminoácido Oxirredutases/farmacologia , Antineoplásicos/isolamento & purificação , Cromatografia em Gel , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Peso Molecular , Especificidade da Espécie , Especificidade por SubstratoRESUMO
An improved and relatively rapid procedure is developed for isolation and purification of a new antitumor enzyme L-lysyl-alpha-oxidase from Trichoderma sp. The method involves four steps, instead of six steps described previously, with a yield of 22.4%. The purified enzyme preparation was homogeneous as shown by polyacrylamide gel disc electrophoresis and ultracentrifugation. Physico-chemical and antitumor properties of the enzyme are under study.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Fungos Mitospóricos/enzimologia , Trichoderma/enzimologia , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese DescontínuaRESUMO
The conjugates of L-lysine alpha-oxidase and monoclonal antibodies ICO-80 towards CD-5 receptor were produced using glutaraldehyde. The cytotoxic effect of conjugates on Yurkat cells line appeared to be lower in comparison with the native enzyme. Negligible decrease of conjugate biological activity may be explained by the large molecular weight of conjugate, which is several times higher than the molecular weight of the native enzyme. Such conjugates can not penetrate into the cells. So they catalyze the hydrogen peroxide formation, the main damaging agent, probably only outside the cells. We suppose also that the free native enzyme penetrates into the cell and activates there the oxidative deamination of L-lysine and correspondingly the hydrogen peroxide formation. This may be the proper explanation for the higher cytotoxic effect of L-lysine alpha-oxidase on Yurkat cell line.
Assuntos
Aminoácido Oxirredutases/farmacologia , Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Aminoácido Oxirredutases/química , Enzimas Imobilizadas/farmacologia , Humanos , Células Tumorais CultivadasRESUMO
A simple and relatively sensitive procedure was developed for determination of L-lysine at 3-30 mmole/L concentration. The procedure does not involve the carcinogenic compound o-dianisidine. L-lysine alpha-oxidase catalyzed oxidative deamination of L-lysine with O2 consumption and formation of H2O2, NH3 and alpha-keto-epsilon-aminocaproic acid. Horseradish peroxidase and a non-carcinogenic compound 3,3,5,5'-tetramethylbenzidine dihydrochloride as an electron donor were used in determination of H2O2 formed. The procedure developed enabled also to measure the L-lysine alpha-oxidase activity at the enzyme concentrations of 10-500 ng/ml. The only limitation of the procedure is relatively low pH-values of the reaction medium.
Assuntos
Aminoácido Oxirredutases , Benzidinas , Lisina/análise , Trichoderma/enzimologia , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Cinética , Análise EspectralRESUMO
The goal of the present study was the development of the optimal method of co-immobilization of two enzymes: L-lysine alpha-oxidase from Trichoderma sp. and horseradish peroxidase. Commercial nitrocellulose, nylon and N+ nylon membranes were used as carriers. The immobilization was carried out either by absorbtion or by covalent binding with aldehyde groups. The aldehyde groups were attached to the surface of the carriers by UV-irradiation of membranes in the presence of p-azidotetrafluorobenzaldehyde. The optimal concentrations of reagents, enzymes and reaction conditions were found. The membranes with the co-immobilised L-lysine alpha-oxidase and peroxidase were shown to be useful for the determination of L-lysine concentrations.
Assuntos
Aminoácido Oxirredutases/metabolismo , Enzimas Imobilizadas/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Membranas Artificiais , Azidas/química , Benzaldeídos/química , Indicadores e Reagentes , Cinética , Porosidade , Trichoderma/enzimologia , Raios UltravioletaRESUMO
Some physicochemical properties of L-lysine oxidase from two strains of Trichoderma were studied. Content of metals and cofactors (Se, Zn, Cu, Fe, Co, Mn, Mo), amino acid analysis, secondary structure were estimated. The enzyme molecule from Trichoderma sp was found to contain both FAD and PQQ cofactors.
Assuntos
Aminoácido Oxirredutases/química , Trichoderma/enzimologia , Aminoácidos/análise , Cromatografia em Gel , Dicroísmo Circular , Flavina-Adenina Dinucleotídeo/química , Cofator PQQ , Quinolonas/química , Especificidade da Espécie , Espectrometria de FluorescênciaRESUMO
The conjugation of the drugs with vector molecules enables to obtain therapeutic preparation, which may be transported to the selected target organ. In the present work the methods of conjugation of antineoplastic enzyme L-lysine alpha-oxidase with antibodies were elaborated. Conjugates were worked out through the attachment of amino groups on the antibody surface either with the aldehyde groups which were created in L-lysine alpha-oxidase molecule (0.2% of initial enzymatic activity) or with the aldehyde groups of cross-linking molecules. Maximal (78%) L-lysine alpha-oxidase activity in conjugates was observed when oxidized peroxidase which contained the aldehyde groups was used as crosslinking agent. The glutaraldehyde method yielded 70% of initial enzyme activity.
Assuntos
Anticorpos/química , Lisina/química , Animais , Reagentes de Ligações Cruzadas , Portadores de Fármacos , Equidae , Glutaral , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/imunologia , Imunoglobulina G , Camundongos , Oxirredução , Coelhos , EspectrofotometriaRESUMO
During previous decade L-amino acid oxidases (LAAO) attracted the steady interest of researchers due to their poly functional effects on different biological systems. The review summarizes information concerning the sources, structure, phisico-chemical and catalytical properties of LAAO which exhibit antibacterial, antifungal, antiprotozoal, antiviral effects as well as the ambiguous action on platelet aggregation. Special attention is devoted to the elucidation of molecular mechanisms of LAAO action. It is proposed that the unique properties of LAAO) are based on their catalytic reaction, which causes the decrease of L-amino acid levels, including the essential amino acids and formation of hydrogen peroxide. The action of liberated H2O2 on cells involves the synthesis of oxygen reactive species and the development of necrotic and apoptotic pathways of cell death. The presence of carbohydrate moieties in LAAO molecules promotes their attachment to cell's surface and creation of high H2O2 local concentrations. The wide range of LAAO biological effects is undoubtedly connected with their important functional roles in the organism. In particular, it was shown that in the mice brain the LAAO-catalyzed reaction is the single pathway of L-lysine degradation, while in the mice milk LAAO carry out the antibacterial effect and in human leucocytes LAAO take part in fulfilling their defending role. Protector action may be also attributed to the oxidases from the other numerous sources: microscopic fungi, snake venoms and sea inhabitants.
Assuntos
Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Aminoácido Oxirredutases/imunologia , Aminoácidos/imunologia , Animais , Catálise , Humanos , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , OxirreduçãoAssuntos
Aminoácido Oxirredutases/biossíntese , Proteínas Fúngicas/biossíntese , Peróxido de Hidrogênio/metabolismo , Cloreto de Sódio/farmacologia , Trichoderma/efeitos dos fármacos , Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Meios de Cultura , Fibras na Dieta/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/metabolismo , Cinética , Salinidade , Estresse Fisiológico , Trichoderma/metabolismoRESUMO
Intraperitoneal injection of bacterial lipopolysaccharide in a dose of 1 mg/kg was followed by prestimulation of whole blood leukocytes in rats. Activities of peroxide- and lipoperoxide-utilizing antioxidant enzymes glutathione peroxidase, glutathione S-transferase, and catalase increased 1 day after lipopolysaccharide administration, while the content of malonic dialdehyde in the skin remained unchanged.
Assuntos
Catalase/metabolismo , Endotoxemia/enzimologia , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Pele/enzimologia , Animais , Endotoxemia/induzido quimicamente , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Glutationa/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Malondialdeído/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
Full-thickness skin wounds (460 mm(2)) in rats were associated with increased blood chemiluminescence and neutrophil infiltration of the wound tissue and surrounding skin (recorded by myeloperoxidase activity). Activities of glutathione peroxidase and glutathione S-transferase in the skin and wound tissue increased on days 4 and 8. A correlation was revealed between activities of these enzymes and myeloperoxidase activity. Activities of myeloperoxidase and catalase increased in patient's skin excised during plastic surgeries of more than 2.5 h duration.
Assuntos
Antioxidantes/metabolismo , Procedimentos Cirúrgicos Dermatológicos , Pele/enzimologia , Ferimentos e Lesões/metabolismo , Animais , Catalase/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Humanos , Masculino , Peroxidase/metabolismo , Ratos , Ratos Wistar , Pele/metabolismo , Pele/patologia , Pele/fisiopatologia , Fatores de TempoRESUMO
This review summarizes data on the properties of L-lysine alpha-oxidase, an enzyme that belongs to the group of oxidases of L-amino acids. This enzyme acts virtually only on L-lysine with a rather low Km yielding alpha-keto-epsilon-aminocaproic acid. The decrease in the level of the essential amino acid L-lysine and the formation of hydrogen peroxide during the reaction possibly provide the basis for the unique properties of L-lysine alpha-oxidase: cytotoxic, antitumor, antimetastatic, antiinvasive, antibacterial, and antiviral activities, as well as an immunomodulating effect. Native L-lysine alpha-oxidase and its immobilized forms are promising tools for determination of concentration of L-lysine in various biological materials.