RESUMO
BACKGROUND: Browning is the key problem hindering the industrialization of pear wine. The use of high-yield glutathione Saccharomyces cerevisiae in the fermentation of pear wine can inhibit browning. Glutathione reductase (GR) can ensure the reduction of glutathione. Spore immobilization of enzymes is a new technology. It is a new attempt to apply spore-immobilized GR in combination with high-yield glutathione S. cerevisiae to inhibit browning of pear wine. RESULTS: Saccharomyces cerevisiae spore immobilization enzyme technology was used to immobilize GR in the spores of mutant S. cerevisiae dit1∆, osw2∆ and chs3∆ and wild-type S. cerevisiae. The enzyme activity of GR immobilized by chs3∆ spores was the highest of 3.08 U mg-1 min-1. The chs3∆ spore-immobilized GR had certain resistance to ethanol, citric acid, sucrose, glucose and proteinase K. Electron microscopy analysis showed that the spore wall of chs3∆ had moderate size holes, which might be the main reason why it immobilized GR with the highest enzyme activity. And the GR was immobilized between the prespore membrane and mannoprotein layer of the spore wall. When chs3∆ spore-immobilized GR (chs3∆-GR) was added to Dangshan pear wine fermented by high-yield glutathione S. cerevisiae JN32-9, the presence of chs3∆-GR could further protect amino acids, polyphenols and glucose from oxidation, thereby reducing the browning of the pear wine during storage by 47.32%. CONCLUSION: GR immobilized by S. cerevisiae spores was effective in inhibiting the browning of pear wine. The method was simple, green and effective and did not increase the production cost of pear wine. © 2024 Society of Chemical Industry.
Assuntos
Fermentação , Glutationa Redutase , Pyrus , Saccharomyces cerevisiae , Esporos Fúngicos , Vinho , Vinho/análise , Pyrus/química , Glutationa Redutase/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Reação de Maillard , Frutas/química , Frutas/microbiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Understanding functional states of individual redox enzymes is important because electron-transfer reactions are fundamental to life, and single-enzyme molecules exhibit molecule-to-molecule heterogeneity in their properties, such as catalytic activity. Zero-mode waveguides (ZMW) constitute a powerful tool for single-molecule studies, enabling investigations of binding reactions up to the micromolar range due to the ability to trap electromagnetic radiation in zeptoliter-scale observation volumes. Here, we report the potential-dependent fluorescence dynamics of single glutathione reductase (GR) molecules using a bimodal electrochemical ZMW (E-ZMW), where a single-ring electrode embedded in each of the nanopores of an E-ZMW array simultaneously serves to control electrochemical potential and to confine optical radiation within the nanopores. Here, the redox state of GR is manipulated using an external potential control of the Au electrode in the presence of a redox mediator, methyl viologen (MV). Redox-state transitions in GR are monitored by correlating electrochemical and spectroscopic signals from freely diffusing MV/GR in 60 zL effective observation volumes at single GR molecule average pore occupancy, ⟨n⟩ â¼ 0.8. Fluorescence intensities decrease (increase) at reducing (oxidizing) potentials for MV due to the MV-mediated control of the GR redox state. The spectroelectrochemical response of GR to the enzyme substrate, i.e., glutathione disulfide (GSSG), shows that GSSG promotes GR oxidation via enzymatic reduction. The capabilities of E-ZMWs to probe spectroelectrochemical phenomena in zL-scale-confined environments show great promise for the study of single-enzyme reactions and can be extended to important technological applications, such as those in molecular diagnostics.
Assuntos
Glutationa Redutase , Glutationa , Nanotecnologia , Imagem Individual de Molécula , Difusão , Fluorescência , Dissulfeto de Glutationa , Glutationa Redutase/química , Oxirredução , Imagem Individual de Molécula/métodosRESUMO
A new glutathione reductase gene (psgr) coding for glutathione reductase (GR) from an Antarctic bacterium was cloned and overexpressed into Escherichia coli (E. coli). A sequence analysis revealed that PsGR is a protein consisting of 451 amino acids, and homology modeling demonstrated that PsGR has fewer hydrogen bonds and salt bridges, which might lead to improved conformational flexibility at low temperatures. PsGR possesses the flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs. Recombinant PsGR (rPsGR) was purified using Ni-NTA affinity chromatography and was found to have a molecular mass of approximately 53.5 kDa. rPsGR was found to be optimally active at 25 °C and a pH of 7.5. It was found to be a cold-adapted enzyme, with approximately 42% of its optimal activity remaining at 0 °C. Moreover, rPsGR was most active in 1.0 M NaCl and 62.5% of its full activity remained in 3.0 M NaCl, demonstrating its high salt tolerance. Furthermore, rPsGR was found to have a higher substrate affinity for NADPH than for GSSG (oxidized glutathione). rPsGR provided protection against peroxide (H2O2)-induced oxidative stress in recombinant cells, and displayed potential application as an antioxidant protein. The results of the present study provide a sound basis for the study of the structural characteristics and catalytic characterization of cold-adapted GR.
Assuntos
Adaptação Fisiológica , Temperatura Baixa , Glutationa Redutase/metabolismo , Psychrobacter/enzimologia , Tolerância ao Sal , Sequência de Aminoácidos , Bioensaio , Genes Bacterianos , Glutationa Redutase/química , Glutationa Redutase/isolamento & purificação , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oxirredução , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
The Hg-C bond of MeHgCl, a ubiquitous environmental toxicant, is notoriously inert and exceedingly difficult to cleave. The cleavage of the Hg-C bond of MeHgCl at low temperature, therefore, is of significant importance for human health. Among various bis(imidazole)-2-selones Ln Se (n=1-4, or 6), the three-spacer L3 Se shows extraordinarily high reactivity in the degradation of various mercury alkyls including MeHgCl because of its unique ability to coordinate through κ2 -fashion, in which both the Se atoms simultaneously attack the Hg center of mercury alkyls for facile Hg-C bond cleavage. It has the highest softness (σ) parameter and the lowest HOMO(Ln Se)-LUMO(MeHgX) energy gap and, thus, L3 Se is the most reactive among Ln Se towards MeHgX (X=Cl or I). L3 Se is highly efficient, more than L1 Se, in restoring the activity of antioxidant enzyme glutathione reductase (GR) that is completely inhibited by MeHgCl; 80 % GR activity is recovered by L3 Se relative to 50 % by L1 Se. It shows an excellent cytoprotective effect in liver cells against MeHgCl-induced oxidative stress by protecting vital antioxidant enzymes from inhibition caused by MeHgCl and, thus, does not allow to increase the intracellular reactive oxygen species (ROS) levels. Furthermore, it protects the mitochondrial membrane potential (ΔΨm ) from perturbation by MeHgCl. Major Hg-responsive genes analyses demonstrate that L3 Se plays a significant role in MeHg+ detoxification in liver cells.
Assuntos
Antioxidantes/farmacologia , Glutationa Redutase/metabolismo , Substâncias Perigosas/análise , Mercúrio/análise , Mercúrio/toxicidade , Compostos Organometálicos/química , Espécies Reativas de Oxigênio/metabolismo , Selênio/química , Antioxidantes/química , Citoproteção , Glutationa Redutase/química , Substâncias Perigosas/química , Humanos , Mercúrio/química , Espécies Reativas de Oxigênio/química , Selênio/análiseRESUMO
Glutathione reductase (GR, E.C. 1.8.1.7), a flavoenzyme, is responsible for recycling of oxidized glutathione disulfide. This study was performed in two main sections. In the first GR was purified from bovine liver by affinity column chromatography and the purification rate and specific activity of the enzyme were calculated as 1832-fold and 141 EU/mg protein, respectively. The subunit molecular weight of the enzyme was determined as 55 kDa by means of SDS-PAGE. The second section isolated natural components of Arum rupicola Boiss. var. rupicola using column chromatography. The isolation protocol for this plant was performed with a series of different-sized columns with hexane-ethyl acetate. According to the thin-layer chromatography plate, seven substances (R1-R7) were isolated. Our study's aim was to find new activators or inhibitors for GR activity. With this aim, all isolated substances were tested for GR activity. R6 showed competitive inhibition, while R4 had noncompetitive inhibition of GR activity. R1 played a role as an activator of GR activity. The inhibitory activity percentage vs. concentration graph was plotted. Values of IC50 for R4 and R6 were calculated as 0.193 mg/mL and 3.98 µg/mL, respectively, from the equation of this graph.
Assuntos
Arum/química , Glutationa Redutase , Fígado/enzimologia , Extratos Vegetais/farmacologia , Animais , Bovinos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Glutationa Redutase/química , Glutationa Redutase/efeitos dos fármacos , Glutationa Redutase/isolamento & purificação , Glutationa Redutase/metabolismo , Fígado/química , Extratos Vegetais/químicaRESUMO
Microfluidic systems are important for performing precise reagent manipulations and reducing material consumption in biological assays. However, optical detection methods limit analyses to fluorescent or UV-active compounds and traditional 2D fluidic designs have limited degrees of freedom. This article describes a microfluidic device that has three inputs and performs 2592 distinct enzyme reactions using only 150 µL of reagent with quantitative characterization. This article also introduces imaging self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (iSAMDI-MS) to map reaction progress, by immobilization of the product onto the floor of the microfluidic channel, into an image that is used for calculating the Michaelis constant ( Km). This approach expands the scope of imaging mass spectrometry, microfluidic detection strategies, and the design of high-throughput reaction systems.
Assuntos
Glutationa Redutase/química , Ensaios de Triagem em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Cinética , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.
Assuntos
Arabidopsis/fisiologia , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Poaceae/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Plantas Tolerantes a Sal/genética , Clonagem Molecular/métodos , Ativação Enzimática , Melhoramento Genético/métodos , Glutationa Redutase/genética , Poaceae/genéticaRESUMO
This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed-type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox-active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.
Assuntos
Glutationa Redutase , Perileno/análogos & derivados , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Antracenos , Domínio Catalítico , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/química , Glutationa Redutase/isolamento & purificação , Perileno/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a cluster of oxidative stress-inducible genes in cells. Here, we aimed to investigate whether trehalose (Tre) protects primary rat proximal tubular (rPT) cells against cadmium (Cd)-induced oxidative stress via Nrf2 antioxidant pathway. Data showed that Tre treatment inhibited Nrf2 nuclear translocation and restored the decline in Kelch-like ECH-associated protein 1 (Keap1) protein level in Cd-exposed rPT cells. Moreover, Cd-activated Nrf2 target genes, including phase II detoxifying enzymes, that is, NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1, direct antioxidant proteins, that is, glutathione peroxidase, superoxide dismutase, catalase, and glutathione biosynthesis-related proteins, that is, glutamatecysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase, were all downregulated by co-treatment with Tre. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced oxidative stress in rPT cells by inhibiting the Nrf2-Keap1 signaling pathway.
Assuntos
Cádmio/toxicidade , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Túbulos Renais Proximais/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trealose/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Cádmio/química , Intoxicação por Cádmio/dietoterapia , Intoxicação por Cádmio/metabolismo , Intoxicação por Cádmio/patologia , Intoxicação por Cádmio/prevenção & controle , Catalase/antagonistas & inibidores , Catalase/química , Catalase/metabolismo , Células Cultivadas , Suplementos Nutricionais , Regulação para Baixo , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/química , Heme Oxigenase-1/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/agonistas , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/química , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/metabolismo , Substâncias Protetoras/uso terapêutico , Ratos , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Trealose/uso terapêuticoRESUMO
Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H2O2, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H2O2. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171-246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu+2 and Cd+2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.
Assuntos
Gastrópodes/genética , Gastrópodes/imunologia , Glutationa Redutase/genética , Imunidade Inata , Sequência de Aminoácidos , Animais , Antioxidantes/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Gastrópodes/efeitos dos fármacos , Gastrópodes/microbiologia , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/fisiologia , Masculino , Metais Pesados/toxicidade , Estresse Oxidativo , Filogenia , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária , Vibrio parahaemolyticus/fisiologia , Poluentes Químicos da Água/toxicidadeRESUMO
The objective of this study was to evaluate the antioxidant effects of propolis, caffeic acid phenethyl ester (CAPE; active compound in propolis), and pollen on biochemical oxidative stress biomarkers in rat kidney tissue inhibited by Nω -nitro-L-arginine methyl ester (L-NAME). The biomarkers evaluated were paraoxonase (PON1), oxidative stress index (OSI), total antioxidant status (TAS), total oxidant status (TOS), asymmetric dimethylarginine (ADMA), and nuclear factor kappa B (NF-κB). TAS levels and PON1 activity were significantly decreased in kidney tissue samples in the L-NAME-treated group (P < 0.05). The levels of TAS and PONI were higher in the L-NAME plus propolis, CAPE, and pollen groups compared with the L-NAME-treated group. TOS, ADMA, and NF-κB levels were significantly increased in the kidney tissue samples of the L-NAME-treated group (P < 0.05). However, these parameters were significantly lower in the L-NAME plus propolis, CAPE, and pollen groups (P < 0.05) compared with rats administered L-NAME alone (P < 0.05). Furthermore, the binding energy of CAPE within catalytic domain of glutathione reductase (GR) enzyme as well as its inhibitory mechanism was determined using molecular modeling approaches. In conclusion, experimental and theoretical data suggested that oxidative alterations occurring in the kidney tissue of chronic hypertensive rats may be prevented via active compound of propolis, CAPE administration.
Assuntos
Ácidos Cafeicos/farmacologia , Nefropatias/etiologia , Simulação de Dinâmica Molecular , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Própole/farmacologia , Animais , Antioxidantes , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/toxicidade , Arildialquilfosfatase/metabolismo , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Meia-Vida , Hipertensão/metabolismo , Hipertensão/patologia , Nefropatias/metabolismo , Masculino , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Álcool Feniletílico/química , Álcool Feniletílico/metabolismo , Álcool Feniletílico/farmacologia , Pólen/química , Pólen/metabolismo , Própole/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-DawleyRESUMO
We report, herein, the purification to homogeneity and the biochemical and kinetic characterization of HcTrxR3, a new isoform of thioredoxin reductase (TrxR) from Haemonchus contortus. HcTrxR3 was found to have a relative molecular weight of 134,000, while the corresponding value per subunit obtained under denaturing conditions, was of 67,000. By peptide mass spectrophotometric analysis, HcTrxR3 was determined to have 99% identity with the H. contortus HcTrxR1 although, and most importantly, they are different in their amino acid sequence in two amino acid positions: 48 (isoleucine instead of leucine) and 460 (leucine instead of proline). The enzyme catalyzes NADPH-dependent reduction of DTNB and, unexpectedly, it follows the pattern of glutathione reductases (GR) performing the reduction of oxidized glutathione (GSSG) to reduced glutathione using NADPH as the reducing cofactor. Hence, it is important to highlight this enzyme's new and unexpected condition that makes so special and one our main finding. Enzyme Kcat values for DTNB, GSSG and NADPH were 12, 3 and 8 s-1, respectively. HcTrxR3 developed, into specific TrxR substrates: ebselen and sodium selenite, with activity at 0.5 and 0.068 (U/mg), respectively; and 0.044 (U/mg) for S-nitrosoglutathione through its GR activity. The enzyme was inhibited by gold compound auranofin (AU), a selective inhibitor of thiol-dependent flavoreductases. Although HcTrxR3 has both TrxR and GR activity as thioredoxin glutathione reductase (TGR) does, it is a TrxR because it has no glutaredoxin domain and it does not develop any hysteretic behavior as does TGR. The importance of this new enzyme is potential to further clarify the detoxification and haemostasis redox mechanism in H. contortus. Likewise, this enzyme could also be a protein model to recognize more differences between TrxR and GR.
Assuntos
Glutationa Redutase/isolamento & purificação , Haemonchus/enzimologia , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Abomaso/parasitologia , Sequência de Aminoácidos , Animais , Azóis/metabolismo , Ácido Ditionitrobenzoico/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Doenças das Cabras/parasitologia , Cabras , Hemoncose/parasitologia , Hemoncose/veterinária , Concentração de Íons de Hidrogênio , Isoindóis , Cinética , NADP/metabolismo , Compostos Organosselênicos/metabolismo , S-Nitrosoglutationa/metabolismo , Alinhamento de Sequência , Ovinos , Doenças dos Ovinos/parasitologia , Selenito de Sódio/metabolismo , Especificidade por Substrato , Temperatura , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.
Assuntos
Digoxina/química , Dopamina/química , Inibidores Enzimáticos/química , Furosemida/química , Glucosefosfato Desidrogenase/isolamento & purificação , Glutationa Redutase/isolamento & purificação , Fosfogluconato Desidrogenase/isolamento & purificação , Animais , Ligação Competitiva , Ensaios Enzimáticos , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/química , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/química , Concentração de Íons de Hidrogênio , Cinética , Masculino , Peso Molecular , Miocárdio/química , Miocárdio/enzimologia , Fosfogluconato Desidrogenase/antagonistas & inibidores , Fosfogluconato Desidrogenase/química , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , TemperaturaRESUMO
Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems.
Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Glutationa Redutase/biossíntese , Glutationa Redutase/química , Alteromonadaceae/genética , Regiões Antárticas , Proteínas de Bactérias/genética , Glutationa Redutase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of ~53.5 kDa. The PgGR peptide exhibits 54-89% sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal- and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.
Assuntos
Glutationa Redutase/genética , Estresse Oxidativo/fisiologia , Pennisetum/enzimologia , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes de Plantas , Glutationa Redutase/química , Glutationa Redutase/isolamento & purificação , Glutationa Redutase/metabolismo , Dados de Sequência Molecular , Pennisetum/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Conformação ProteicaRESUMO
Comparative efficiency of three extraction solutions, including the universal sodium phosphate buffer (USPB), the Tris-HCl buffer (UTHB), and the specific buffers, were compared for assays of soluble protein, free proline, superoxide radical (O2â-), hydrogen peroxide (H2O2), and the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) in Populus deltoide. Significant differences for protein extraction were detected via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Between the two universal extraction buffers, the USPB showed higher efficiency for extraction of soluble protein, CAT, GR, O2â-, GPX, SOD, and free proline, while the UTHB had higher efficiency for extraction of APX, POD, and H2O2. When compared with the specific buffers, the USPB showed higher extraction efficiency for measurement of soluble protein, CAT, GR, and O2â-, parallel extraction efficiency for GPX, SOD, free proline, and H2O2, and lower extraction efficiency for APX and POD, whereas the UTHB had higher extraction efficiency for measurement of POD and H2O2. Further comparisons proved that 100 mM USPB buffer showed the highest extraction efficiencies. These results indicated that USPB would be suitable and efficient for extraction of soluble protein, CAT, GR, GPX, SOD, H2O2, O2â-, and free proline.
Assuntos
Antioxidantes/isolamento & purificação , Glutationa Redutase/química , Estresse Oxidativo , Antioxidantes/química , Ascorbato Peroxidases/química , Ascorbato Peroxidases/isolamento & purificação , Soluções Tampão , Catalase/química , Catalase/isolamento & purificação , Glutationa Redutase/isolamento & purificação , Peróxido de Hidrogênio/química , Peroxidase/química , Peroxidase/isolamento & purificação , Superóxido Dismutase/química , Superóxido Dismutase/isolamento & purificaçãoRESUMO
TiO2 nanoparticles used as vectors for the delivery of drugs have shown greater effectiveness. However, TiO2 nanoparticles can cause oxidative stress in liver and kidney, so we analyzed if a previous or simultaneous quercetin treatment could counteract this in rats. Five groups of male Wistar rats (200-250 g) were included: (1) healthy controls, (2) TiO2 group, (3) quercetin group, (4) preventive group: quercetin for 5 days prior to exposure of TiO2, and (5) therapeutic group: TiO2 (5 mg/kg, i.v.) plus quercetin single dose for 5 days (5 mg/kg/day, i.p.). Hepatic and renal function tests were made. Five animals from each group were sacrificed (0, 14 and 28 days), and liver and kidney tissue were obtained. Malondialdehyde (MDA), reduced/oxidized glutathione, and activity of glutathione peroxidase/reductase were measured, as well as the level of gene expression by q-PCR. There were no significant changes in serum ALT and AST activities. More damage was observed at 14 versus 28 days, because TiO2 was excreted in urine. Quercetin indeed showed a renal protective effect by increasing glutathione reductase and peroxidase levels and reducing MDA levels. On the other hand, TiO2 liver damage was less pronounced with quercetin as therapeutic treatment. TiO2 induces significantly the glutathione reductase expression and it can be down-regulated by quercetin. Biochemical tests in serum and urine showed a better effect of quercetin administered in the therapeutic group. Care should be taken with the dose and time of administration of quercetin, because this antioxidant could also have a pro-oxidant effect.
Assuntos
Antioxidantes/uso terapêutico , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Quercetina/uso terapêutico , Titânio/toxicidade , Animais , Antioxidantes/administração & dosagem , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Sistemas de Liberação de Medicamentos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa/agonistas , Glutationa/antagonistas & inibidores , Glutationa/química , Glutationa/metabolismo , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/química , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Injeções Intraperitoneais , Injeções Intravenosas , Rim/metabolismo , Rim/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiopatologia , Masculino , Nanopartículas Metálicas/administração & dosagem , Oxirredução , Quercetina/administração & dosagem , Distribuição Aleatória , Ratos Wistar , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/tratamento farmacológico , Insuficiência Renal/fisiopatologia , Insuficiência Renal/prevenção & controle , Titânio/administração & dosagemRESUMO
Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618-12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an "electrophilic activation" mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic activation serves as a compensatory mechanism in the absence of the more electrophilic Sec residue. We present an argument for the importance of S-electrophilicity in Cys orthologs of selenoenzymes.
Assuntos
Drosophila melanogaster/enzimologia , Homocisteína/química , Selenocisteína/química , Tiorredoxina Dissulfeto Redutase/química , Animais , Biocatálise , Dissulfetos/química , Ativação Enzimática , Glutationa Redutase/química , Mutação , Oligopeptídeos/química , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
BACKGROUND: Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances. SCOPE OF REVIEW: This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family. MAJOR CONCLUSIONS: It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid-base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions. GENERAL SIGNIFICANCE: Glutathione-dependent enzymes are excellent study objects for structure-function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione.
Assuntos
Glutationa/metabolismo , Animais , Catálise , Glutationa/química , Glutationa Peroxidase/química , Glutationa Peroxidase/metabolismo , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Humanos , Lactoilglutationa Liase/química , Lactoilglutationa Liase/metabolismo , Modelos Moleculares , Oxirredução , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMO
Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells.