RESUMO
Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 µM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.
Assuntos
Sulfato de Cobre/química , Glutationa Redutase/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Ácido Ascórbico/química , Glutationa Redutase/antagonistas & inibidores , Radical Hidroxila/química , Cinética , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/química , Proteólise , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidoresRESUMO
In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.
Assuntos
NADPH Oxidases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células CACO-2 , Membrana Celular/enzimologia , Sistema Livre de Células/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , NADPH Oxidase 1 , Neutrófilos/metabolismo , Oniocompostos/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Apoptosis of HL60 cells by maltol was analyzed in relation to the maltol/iron-mediated generation of reactive oxygen species. Addition of maltol with FeSO(4) induced an apoptotic cell death as judged by flow cytometry analysis and DNA fragmentation on electrophoresis, but maltol or iron alone did not affect the cells. Treatment of HL60 cells with maltol/iron complex caused an effective inactivation of aconitase the most sensitive enzyme to reactive oxygen species. Maltol/iron-mediated apoptosis and the inactivation of aconitase was prevented by TEMPOL, the scavenger of reactive oxygen species. These findings suggest that maltol/iron complex can generate reactive oxygen species by the redox cycling, resulting in an apoptosis of HL60 cells. Cytotoxicity of maltol can be explained by the prooxidant properties of this compound.
Assuntos
Apoptose/efeitos dos fármacos , Ferro/toxicidade , Pironas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Aconitato Hidratase/antagonistas & inibidores , Aconitato Hidratase/metabolismo , Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Células HL-60 , Humanos , Ferro/farmacologia , Pironas/farmacologia , Marcadores de SpinRESUMO
The prooxidant and apoptosis-inducing effects of Sho-saiko-to, a traditional Sino-Japanese herbal medicine and its active ingredients were analyzed. Among the components of Sho-saiko-to, wogon, the extract of Scutellaria and licorice root extract induced apoptosis of HL60 cells and increased the intracellular levels of reactive oxygen species. Lower concentrations (5 to 20 muM) of baicalein, the principal flavonoid in the Scutellaria root extract, showed induction of cell apoptosis and elevated the intracellular reactive oxygen species. However, the increase in the concentrations of baicalein rather inhibited the induction of apoptosis and the elevated levels of reactive oxygen species in cells. Induction of baicalein-mediated apoptosis was inhibited by addition of Tempol, the scavenger of reactive oxygen species. Glycyrrhetinic acid, an ingredient of licorice root extract, also induced apoptosis followed by increase in the intracellular reactive oxygen species. The effect of Sho-saiko-to on cell differentiation can be explained by the action of two ingredients, baicalein and glycyrrhetinic acid, which cause apoptosis and increase in reactive oxygen species in cells.
Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas , Flavanonas/farmacologia , Ácido Glicirretínico/farmacologia , Fragmentação do DNA , Glycyrrhiza/química , Células HL-60 , Humanos , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Espécies Reativas de Oxigênio/metabolismo , Scutellaria/químicaRESUMO
Effects of eugenol compounds on the production of nitric oxide (NO) in RAW264.7 macrophages were analyzed in relation to the anti-inflammatory action of these compounds. Eugenol and isoeugenol inhibited lipopolysaccharide (LPS)-dependent production of NO, which was due to the inhibition of protein synthesis of inducible nitric oxide synthase (iNOS). Isoeugenol showed the most effective inhibitory effect and eugenol was less effective. LPS-dependent expression of cyclooxygenase-2 (COX-2) protein was also inhibited markedly by isoeugenol, and less effectively by eugenol. Anti-inflammatory action of eugenol compounds may be explained by the inhibition of NO production and COX-2 expression, the pro-inflammatory mediators.
Assuntos
Eugenol/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Eugenol/química , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Capsaicin induces apoptosis in some types of cells, but its mechanism remains obscure. In this study, peroxynitrite, a powerful oxidant generated from the reaction of superoxide and nitric oxide (NO) in biological system, was demonstrated to be responsible for capsaicin-mediated apoptosis in C6 glioma cells. Capsaicin-induced apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and also identified by Annexin V staining and comet assay. Capsazepine and ruthenium red, the vanilloid receptor 1 (VR1/TPRV1) antagonists, did not inhibit capsaicin-induced apoptosis. Exposure to capsaicin not only promoted the generation of superoxide and iNOS, but also markedly suppressed the expression of SODs. Nitrite and nitrate, the NO metabolites accumulated in the medium, and the nitrotyrosine was also increased in proteins of C6 glioma cells exposed to capsaicin. Pretreatment of cells with 4 microM ebselen (a peroxynitrite scavenger) showed effective inhibitory effect on the capsaicin-induced apoptosis. These results suggest that peroxynitrite can act as a potential mediator in the capsaicin-induced apoptosis in C6 glioma cells.
Assuntos
Apoptose/fisiologia , Capsaicina/toxicidade , Ácido Peroxinitroso/fisiologia , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Azóis/farmacologia , Western Blotting/métodos , Contagem de Células/métodos , Linhagem Celular Tumoral , Ensaio Cometa/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma , Marcação In Situ das Extremidades Cortadas/métodos , Isoindóis , Camundongos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Compostos Organosselênicos/farmacologia , Fenantridinas/metabolismo , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Ikappa-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Ikappa-Balpha was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Ikappa-Balpha.
Assuntos
Antioxidantes/farmacologia , Cinamatos/farmacologia , Macrófagos/efeitos dos fármacos , Espécies Reativas de Nitrogênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular , Depsídeos , Relação Dose-Resposta a Droga , Proteínas I-kappa B/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Fosforilação/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Tirosina/metabolismo , Ácido RosmarínicoRESUMO
Hinokitiol (alpha-thujaplicin, 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one), one of the tropolone compounds purified from the woods of Chamaecyparis and Thujopsis (hinoki and hiba), produced reactive oxygen species as a complex with transition metals. Hinokitiol/iron complex inactivated aconitase, the most sensitive enzyme to reactive oxygen, whereas it did not affect aldolase and glyceraldehyde 3-phosphate dehydrogenase. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species and superoxide dismutase, suggesting that the hinokitiol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of hinokitiol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2'-deoxyguanosine in DNA. Cytotoxic effect of hinokitiol can be explained by its prooxidant properties: hinokitiol/transition metal complex generates reactive oxygen species causing inactivation of aconitase and production of hydroxyl radical resulting in the formation of DNA base adduct.
Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Monoterpenos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tropolona/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina , Aconitato Hidratase/antagonistas & inibidores , Aconitato Hidratase/metabolismo , Ácido Ascórbico , Sulfato de Cobre , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Compostos Ferrosos , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Oxidantes/farmacologia , Tropolona/farmacologia , Leveduras/enzimologiaRESUMO
Curcumin, a well-known antioxidant in a principal ingredient of turmeric, acted as a prooxidant causing a copper-dependent DNA damage and the induction of apoptosis. Treatment of DNA from plasmid pBR322 and calf thymus with curcumin plus copper ion caused strand scission and the formation of 8-hydroxy-2(')-deoxyguanosine in DNA. Addition of catalase protected DNA from the curcumin-dependent injuries, indicating that hydroxyl radical may participate in the DNA damage. Flow cytometry analysis showed that curcumin caused an apoptotic cell death of HL60 cells in a dose- and time-dependent manner. Curcumin-mediated apoptosis was closely related to the increase in intracellular reactive oxygen species. On the contrary, capsaicinoids, which have a ortho-methoxy phenolic structure without beta-diketone in the side chain, did not produce 8-hydroxy-2(')-deoxyguanosine. Capsaicin further did not induce apoptosis of HL60 cells, but rather protected cells from prooxidant-induced apoptosis. Curcumin can generate reactive oxygen species as a prooxidant in the presence of transition metals in cells, resulting in DNA injuries and apoptotic cell death. The prooxidant action of curcumin may be related to the conjugated beta-diketone structure of this compound.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Cobre/toxicidade , Curcumina/farmacologia , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , 8-Hidroxi-2'-Desoxiguanosina , Animais , Bovinos , Desoxiguanosina/química , Citometria de Fluxo , Células HL-60 , Humanos , Radical Hidroxila/farmacologia , Oxidantes/farmacologia , Plasmídeos/genética , Espécies Reativas de Oxigênio , TimoRESUMO
Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg2+, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg2+ implies the identical binding sites of Fe2+ and Mg2+, but the cleavage sites were discriminated from the cofactor Mg2+-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe2+ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg2+ concentrations are responsible for the control of cellular respiration and glycolysis.
Assuntos
Ferro/farmacologia , Piruvato Quinase/metabolismo , Animais , Ácido Ascórbico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Coelhos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg(2+), Mn(2+ )or Fe(2+) ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.
Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Isocitrato Desidrogenase/efeitos dos fármacos , Espécies Reativas de Oxigênio/farmacologia , Sítios de Ligação/genética , Meios de Cultura , Eletroforese em Gel Bidimensional , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Glucosefosfato Desidrogenase/efeitos dos fármacos , Glucosefosfato Desidrogenase/metabolismo , Isocitrato Desidrogenase/isolamento & purificação , Isocitrato Desidrogenase/metabolismo , Espectrometria de Massas , Peso Molecular , Oxirredução , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2'-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO(4) was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.
Assuntos
DNA/química , Desoxiguanosina/análogos & derivados , Oxidantes/farmacologia , Pironas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Aconitato Hidratase/antagonistas & inibidores , Animais , Ácido Ascórbico/química , Bovinos , Sulfato de Cobre/química , Óxidos N-Cíclicos/farmacologia , Adutos de DNA/síntese química , Desoxiguanosina/síntese química , Ferro/química , Oxirredução/efeitos dos fármacos , Marcadores de Spin , Superóxidos/síntese químicaRESUMO
The vanilloid receptor 1(VR1) is a nonselective cation channel that is activated by pungent vanilloid compound, extracellular protons, or noxious heat. mRNA of VR1 and vanilloid receptor 1-like receptor (VRL1) were expressed in PC12 cells, and only VRI mRNA was detected in glioma and A10 cell lines. VRI protein was demonstrated in PC12 cells by immunocytochemistry and Western blotting. Capsaicin (CPS), the VRI receptor agonist, led to an increase in intracellular calcium ion, and this effect was blocked by pretreatment with VR1 receptor antagonist capsazepin (CPZ). Treatment of PC12 cells with low concentration of CPS (5-50 microM) increased reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS) was expressed after CPS treatment for 24 h. These CPS-induced changes are inhibited by pretreatment of CPZ. These findings suggest that CPS-induced iNOS expression through the VR1 and/or VRL1-mediated pathway, and this may explain the CPS-mediated physiological and pathological effects in neuron system.