RESUMO
D-Amino acid oxidase (DAAO) specifically catalyzes the oxidative deamination of neutral and polar D-amino acids and finally yields byproducts of hydrogen peroxide. Our previous work demonstrated that the spinal astroglial DAAO/hydrogen peroxide (H2 O2 ) pathway was involved in the process of pain and morphine antinociceptive tolerance. This study aimed to report mouse strain specificity of DAAO inhibitors on antinociception and explore its possible mechanism. DAAO inhibitors benzoic acid, CBIO, and SUN significantly inhibited formalin-induced tonic pain in Balb/c and Swiss mice, but had no antinociceptive effect in C57 mice. In contrast, morphine and gabapentin inhibited formalin-induced tonic pain by the same degrees among Swiss, Balb/c and C57 mice. Therefore, mouse strain difference in antinociceptive effects was DAAO inhibitors specific. In addition, intrathecal injection of D-serine greatly increased spinal H2 O2 levels by 80.0% and 56.9% in Swiss and Balb/c mice respectively, but reduced spinal H2 O2 levels by 29.0% in C57 mice. However, there was no remarkable difference in spinal DAAO activities among Swiss, Balb/c and C57 mice. The spinal expression of glutathione (GSH) and glutathione peroxidase (GPx) activity in C57 mice were significantly higher than Swiss and Balb/c mice. Furthermore, the specific GPx inhibitor D-penicillamine distinctly restored SUN antinociception in C57 mice. Our results reported that DAAO inhibitors produced antinociception in a strain-dependent manner in mice and the strain specificity might be associated with the difference in spinal GSH and GPx activity.
Assuntos
Analgésicos/administração & dosagem , Variação Biológica da População , D-Aminoácido Oxidase/antagonistas & inibidores , Nociceptividade/efeitos dos fármacos , Analgésicos/farmacocinética , Animais , D-Aminoácido Oxidase/metabolismo , Glutationa/análise , Glutationa/metabolismo , Glutationa Peroxidase/análise , Glutationa Peroxidase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Hydrogen sulfide (H2S) has been focused as a biological mediator, which modulates signal transduction and protects cells and tissues from oxidative stress. H2S is also expected as a neuroprotectant because it has a neuroprotective activity. Endogenous H2S is mainly generated from L-cysteine. However, it is difficult to use L-cysteine as a neuroprotectant because of its neurotoxicity. In 2013, a novel biogenesis pathway of H2S from D-cysteine has been identified. In this pathway, D-amino acid oxidase (DAO) converts D-cysteine to 3-mercaptopyruvate (3MP), followed by the generation of H2S from 3MP by 3-mercaptopyrvate sulfurtransferase. DAO is especially abundant in cerebellum among various brain regions and mediates efficient generation of H2S from D-cysteine in the cerebellar tissues. In addition, D-cysteine has more potent neuroprotective activity in cerebellar primary neurons than L-cysteine. Cerebella Purkinje cells (PCs) are characterized by the highly-branched dendrites and are important for cerebellar functions. The dendritic shrinkage and degeneration of PCs are frequently observed in patients and model mice of cerebellar ataxias. We revealed that D-cysteine enhanced dendritic development of primary cultured PCs, but L-cysteine impaired the dendritic development. This effect of D-cysteine was inhibited by DAO inhibitors and reproduced by 3MP and a H2S donor, suggesting that this enhancement of dendritic development is caused by the production of H2S from D-cysteine. Taken together, D-cysteine would be available as a neuroprotectant against cerebellar ataxias, which are accompanied with dendritic shrinkage of cerebellar PCs.
Assuntos
Cisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Neurônios/citologia , Fármacos Neuroprotetores/metabolismo , Animais , Células Cultivadas , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Humanos , Camundongos , Neurogênese , Estresse Oxidativo , Células de Purkinje/citologiaRESUMO
d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.
Assuntos
Senescência Celular/fisiologia , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aminoácidos/metabolismo , Arginina/metabolismo , Senescência Celular/efeitos dos fármacos , Coenzimas/metabolismo , D-Aminoácido Oxidase/antagonistas & inibidores , Dano ao DNA/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Oxirredução , Interferência de RNA , Riboflavina/farmacologia , Serina/metabolismo , TransfecçãoRESUMO
Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound 2: [2-(2,5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on], we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2: and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.
Assuntos
Proteínas de Transporte/metabolismo , D-Aminoácido Oxidase/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Esquizofrenia/tratamento farmacológico , Sítio Alostérico/efeitos dos fármacos , D-Aminoácido Oxidase/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neuralgia/tratamento farmacológicoRESUMO
In silico prediction of the new drug-target interactions from existing databases is of important value for the drug discovery process. Currently, the amount of protein targets that have been identified experimentally is still very small compared with the entire human proteins. In order to predict protein-ligand interactions in an accurate manner, we have developed a support vector machine (SVM) model based on the chemical-protein interactions from STITCH. New features from ligand chemical space and interaction networks have been selected and encoded as the feature vectors for SVM analysis. Both the 5-fold cross validation and independent test show high predictive accuracy that outperforms the state-of-the-art method based on ligand similarity. Moreover, 91 distinct pairs of features have been selected to rebuild a simplifier model, which still maintains the same performance as that based on all 332 features. Then, this refined model is used to search for the potential D-amino acid oxidase inhibitors from STITCH database and the predicted results are finally validated by our wet experiments. Out of 10 candidates obtained, seven D-amino acid oxidase inhibitors have been verified, in which four are newly found in the present study, and one may have a new application in therapy of psychiatric disorders other than being an antineoplastic agent. Clearly, our model is capable of predicting potential new drugs or targets on a large scale with high efficiency.
Assuntos
D-Aminoácido Oxidase/antagonistas & inibidores , Desenho de Fármacos , Proteínas/metabolismo , Antineoplásicos/farmacologia , Simulação por Computador , Bases de Dados Factuais , Descoberta de Drogas/métodos , Humanos , Ligantes , Transtornos Mentais/tratamento farmacológico , Modelos Teóricos , Terapia de Alvo Molecular , Máquina de Vetores de SuporteRESUMO
D-Amino acid oxidase (DAAO), a FAD-dependent peroxisomal flavoenzyme that catalyzes oxidation of D-amino acids to hydrogen peroxide, is distributed in the spinal cord almost exclusively expressed within astrocytes. The present study aims to explore potential contributions of spinal DAAO to the development of bone cancer pain and morphine tolerance to analgesia. Tibia inoculation of carcinoma cells produced mechanical allodynia (but not heat hyperalgesia), in synchronous with induction of DAAO expression and DAAO enzymatic activity, as well as activation of spinal astrocytes marked by GFAP. Subcutaneous and intrathecal injection of the specific DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked mechanical allodynia in a dose- and time-dependent manner in tumor-bearing rats, with maximum inhibition of 40-50 %. Multi-daily intrathecal injections of the DAAO gene silencer siRNA/DAAO also yielded anti-allodynic effects by approximately 40 % and the analgesia remained for at least 6 days. Subcutaneous injection of CBIO suppressed the production of spinal hydrogen peroxide and GFAP expression. 7-Day multiple bi-daily injections of CBIO produced anti-allodynia without inducing self-tolerance to analgesia or cross-tolerance to morphine, and concurrent injections of CBIO with morphine produced apparent additive anti-allodynia and completely prevented morphine tolerance in behaviors and spinal expression of µ-opioid receptors. Our results provide the first evidence that spinal DAAO contributes to the development of morphine tolerance to analgesia and bone cancer pain accounting for 40-50 % pain status, probably via production of hydrogen peroxide leading to activation of astrocytes. The unique characterizations of DAAO inhibitors make them a potential for the treatment of cancer pain when they are administered alone or in combination with morphine.
Assuntos
Neoplasias Ósseas/enzimologia , Carcinoma 256 de Walker/enzimologia , D-Aminoácido Oxidase/metabolismo , Dor/tratamento farmacológico , Analgesia/métodos , Analgésicos Opioides/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Astrócitos/patologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/fisiopatologia , Carcinoma 256 de Walker/patologia , Carcinoma 256 de Walker/fisiopatologia , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/genética , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Hiperalgesia/prevenção & controle , Injeções Espinhais , Isoxazóis/farmacologia , Morfina/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Dor/metabolismo , Dor/fisiopatologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologia , Medula Espinal/patologia , Tíbia/efeitos dos fármacos , Tíbia/enzimologia , Tíbia/fisiopatologiaRESUMO
A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.
Assuntos
Analgesia/métodos , D-Aminoácido Oxidase/antagonistas & inibidores , Dor/fisiopatologia , Medula Espinal/enzimologia , Animais , Astrócitos/enzimologia , Astrócitos/fisiologia , D-Aminoácido Oxidase/genética , Regulação para Baixo , Células Epiteliais/enzimologia , Formaldeído/farmacologia , Injeções Espinhais , Túbulos Renais/enzimologia , Masculino , Dor/induzido quimicamente , Dor/enzimologia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Medula Espinal/fisiopatologia , TransgenesRESUMO
BACKGROUND AND PURPOSE: Spinal reactive oxygen species (ROS) are critically involved in chronic pain. D-Amino acid oxidase (DAAO) oxidizes D-amino acids such as D-serine to form the byproduct hydrogen peroxide without producing other ROS. DAAO inhibitors are specifically analgesic in tonic pain, neuropathic pain and cancer pain. This study examined the role of spinal hydrogen peroxide in pain and the mechanism of the analgesic effects of DAAO inhibitors. EXPERIMENTAL APPROACH: Formalin-induced pain behaviours and spinal hydrogen peroxide levels were measured in rodents. KEY RESULTS: Formalin injected into the paw increased spinal hydrogen peroxide synchronously with enhanced tonic pain; both were effectively prevented by i.t. fluorocitrate, a selective astrocyte metabolic inhibitor. Given systemically, the potent DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked spinal DAAO enzymatic activity and specifically prevented formalin-induced tonic pain in a dose-dependent manner. Although CBIO maximally inhibited tonic pain by 62%, it completely prevented the increase in spinal hydrogen peroxide. I.t. catalase, an enzyme specific for decomposition of hydrogen peroxide, completely depleted spinal hydrogen peroxide and prevented formalin-induced tonic pain by 65%. Given systemically, the ROS scavenger PBN (phenyl-N-tert-butylnitrone) also inhibited formalin-induced tonic pain and increase in spinal hydrogen peroxide. Formalin-induced tonic pain was potentiated by i.t. exogenous hydrogen peroxide. CBIO did not increase spinal D-serine level, and i.t. D-serine did not alter either formalin-induced tonic pain or CBIO's analgesic effect. CONCLUSIONS AND IMPLICATIONS: Spinal hydrogen peroxide is specifically and largely responsible for formalin-induced pain, and DAAO inhibitors produce analgesia by blocking spinal hydrogen peroxide production rather than interacting with spinal D-serine.
Assuntos
Peróxido de Hidrogênio/metabolismo , Dor/metabolismo , Coluna Vertebral/metabolismo , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Formaldeído , Peróxido de Hidrogênio/farmacologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Masculino , Camundongos , Dor/induzido quimicamente , Dor/tratamento farmacológico , Dor/fisiopatologia , Ratos , Ratos Wistar , Serina/metabolismo , Coluna Vertebral/efeitos dos fármacosRESUMO
D-Serine selectively causes necrosis of S(3) segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S(3), thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of d-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to approximately 30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[(14)C]serine or [(14)C]AIB (0.36 mmol/l) together with [(3)H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[(14)C]serine and [(14)C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated d-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents d-serine-induced tubulopathy by inhibition of D-serine uptake in S(3) segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.
Assuntos
Ácidos Aminoisobutíricos/uso terapêutico , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Serina/toxicidade , Aminoácidos/sangue , Animais , D-Aminoácido Oxidase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Glucose/metabolismo , Glutationa/metabolismo , Glicosúria/induzido quimicamente , Peróxido de Hidrogênio/metabolismo , Injeções Intraperitoneais , Insulina , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Alça do Néfron/efeitos dos fármacos , Alça do Néfron/metabolismo , Masculino , Oxirredução , Ratos , Ratos Wistar , Serina/administração & dosagem , Serina/metabolismoRESUMO
Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.
Assuntos
D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/química , Saccharomycetales/enzimologia , Cefalosporinas/análise , Cefalosporinas/farmacocinética , Estabilidade Enzimática , Temperatura Alta , Cinética , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , TermodinâmicaRESUMO
When D-propargylglycine was injected intraperitoneally into mice, polyuria, glycosuria, and aminoaciduria were observed as has been previously reported in rats. The urine of the mice treated with D-propargylglycine contained twice as much protein as that of the control mice. Polyacrylamide gel electrophoresis showed a new protein of approximately 62 kDa in the urine of the D-propargylglycine-treated mice. Protein sequencing revealed that this protein was serum albumin. Since the above-mentioned symptoms suggested dysfunction of the renal proximal tubules, the activity of urinary N-acetyl-beta-D-glucosaminidase, a marker enzyme of injury to the proximal tubules, was measured. The urinary enzyme activity was 2.6 times higher in the D-propargylglycine-treated mice than in the control mice. Light- and electron-microscopy showed degenerative and necrotic cells in the straight part of the proximal tubules of the treated mice. However, none of these symptoms was observed in D-propargylglycine-treated mutant mice, lacking D-amino-acid oxidase. These results indicate that D-propargylglycine itself is not nephrotoxic but its metabolite produced by the D-amino-acid oxidase reaction is nephrotoxic and injures proximal tubular cells, resulting in an impairment of the reabsorption of water, glucose, amino acids, and proteins.
Assuntos
Alcinos/toxicidade , D-Aminoácido Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/toxicidade , Glicina/análogos & derivados , Rim/efeitos dos fármacos , Alcinos/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , D-Aminoácido Oxidase/fisiologia , Glicina/metabolismo , Glicina/toxicidade , Camundongos , Dados de Sequência Molecular , Fator de Necrose Tumoral alfa/biossínteseRESUMO
2-oxo-3-pentynoate has been characterized as an active-site-directed inhibitor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed fashion. The addition of FAD affords no protection from inactivation, whereas the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The inactivation follows first-order kinetics for at least five half-lives. The rate of inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before inactivation occurs. Values of 0.017 +/- 0.0005 min-1 and 44 +/- 7 microM were determined for the limiting rate of inactivation and the apparent dissociation constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptophan 2-monooxygenase treated with 2-oxo-3-pentynoate show that two peptides are alkylated in the absence of indole-3-acetamide but not in its presence. The two peptides were identified by mass spectrometry as residues 333-349 and 503-536. Based upon sequence analysis, cysteine 511 and either cysteine 339 or histidine 338 are the likely sites of modification. In contrast, incubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentynoate results in a loss of 55% or 100%, respectively, of the initial activity. In neither case does a competitive inhibitor affect the rate of inactivation, suggesting that the effect is not due to modification of active-site residues.
Assuntos
Dioxigenases , Inibidores Enzimáticos/química , Ácidos Graxos Insaturados/química , Oxigenases de Função Mista/antagonistas & inibidores , Fragmentos de Peptídeos/química , Alquilação , Sequência de Aminoácidos , Sítios de Ligação , D-Aminoácido Oxidase/antagonistas & inibidores , Ativação Enzimática , Dados de Sequência Molecular , Oxigenases/antagonistas & inibidores , Pseudomonas/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Inactivation of D-amino acid oxidase occurred by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu2+ and Hg2+. It was also sensitive to oxidative inactivation by Fe2+ and H2O2 when both reagents were added in millimolar amounts. When oxidatively inactivated D-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion of D-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of the D-amino acid oxidase activity. Treatment of apo-D-amino acid oxidase from T. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.
Assuntos
Cátions Bivalentes/farmacologia , D-Aminoácido Oxidase/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Fungos Mitospóricos/enzimologia , Sequência de Aminoácidos , Compostos Bicíclicos com Pontes/farmacologia , Cobre/farmacologia , Endopeptidases/metabolismo , Glutamato Desidrogenase/metabolismo , Ferro/farmacologia , Isoenzimas/metabolismo , Mercúrio/farmacologia , Dados de Sequência Molecular , NADP/metabolismo , OxirreduçãoRESUMO
D-Amino acid oxidase from Trigonopsis variabilis was purified to homogeneity by a combination of freeze/thawing, isoelectric precipitation and chromatography on Mono Q. This purification procedure required very little working effort. The homogeneous enzyme exhibited a ratio A280/A450 of about 6.5 and was obtained in high yield (63%) and a good stability. Using D-methionine as a substrate, a specific activity of 120 U/mg was determined colorimetrically at 26 degrees C, corresponding to 185 U/mg polarographically at 37 degrees C. Polyclonal antibodies were raised against the homogeneous protein and Western immunoblot analysis showed that the 39-kDa subunit can undergo defined cleavages at the carboxy terminus of amino acid positions 104, 106 and 108, leading to 27-kDa and 12-kDa fragments as revealed by SDS/PAGE, which are still enzymically active in their native form. The enzyme was inactivated by all sulfhydryl-modifying reagents tested. Inactivation by 5,5'-dithiobis(-2-nitrobenzoate) was correlated with a modification of up to 2 mol/mol protein of the six cysteine residues present in the monomer. Identification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl-modifying reagent monobromobimane. In the presence of a substrate amino acid, under anaerobic conditions, the protein could be protected from modification and, thus, inactivation by this reagent. Peptide mapping by reverse-phase chromatography of endoproteinase Glu-C-digested monobromobimane-labeled enzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid under anaerobic conditions. Isolation and sequencing of the labeled peptide led to the identification of Cys298 as the reactive cysteine residue.
Assuntos
Cisteína/metabolismo , D-Aminoácido Oxidase/metabolismo , Fungos Mitospóricos/enzimologia , Sequência de Aminoácidos , Western Blotting , Cromatografia Líquida de Alta Pressão , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/química , D-Aminoácido Oxidase/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Corantes Fluorescentes , Focalização Isoelétrica , Dados de Sequência Molecular , Mapeamento de Peptídeos , Reagentes de Sulfidrila/farmacologiaRESUMO
The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.