[27] Glutathione reductase from bovine brain

doi:10.1016/S0076-6879(02)48646-6

Methods in Enzymology

Volume 348, 2002, Pages 281-288

https://doi.org/10.1016/S0076-6879(02)48646-6 Get rights and content

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Glutathione reductase (GR, NADPH:oxidized-glutathione oxidoreductase, EC1.6.4.2) catalyzes the reduction of glutathione disulfide (GSSG) during the glutathione redox cycling. GSSG is generated by the nonenzymatic reaction of radicals with reduced glutathione (GSH) and is product of the reactions catalyzed by glutathione peroxidases, which use GSH as an electron donor for the reduction of peroxides. Glutathione-dependent disposal of peroxides and radicals appears to be especially important for the brain, as this organ has an intense oxidative metabolism but is disadvantaged regarding the detoxification of reactive oxygen species. Enzymes such as glutathione peroxidases, superoxide dismutases, and catalase are present in brain in lower activities than in other tissues. This is also the case for GR.

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2019, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
In addition to alcohols or water, derived from the reduction of organic peroxides or H2O2, respectively, the usual GPx-catalyzed reaction also produces oxidized glutathione (GSSG), which is a disulfide formed from the oxidation of two GSH molecules. The GSSG generated by the GPx-catalyzed reaction can be reduced by the flavoenzyme glutathione reductase (GR) (Fig. 1B, event b), which uses NADPH as reducing agent [77]. The reduction of GSSG to GSH (2 molecules) is needed to the maintenance of the proper GSH-GSSG redox cycling; the availability of NADPH, generated mainly by the oxidative phase of the pentose phosphate pathway (PPP) (Fig. 1B, event c) represents a crucial event [78].
Methylmercury (MeHg) is a toxic chemical compound naturally produced mainly in the aquatic environment through the methylation of inorganic mercury catalyzed by aquatic microorganisms. MeHg is biomagnified in the aquatic food chain and, consequently, piscivorous fish at the top of the food chain possess huge amounts of MeHg (at the ppm level). Some populations that have fish as main protein's source can be exposed to exceedingly high levels of MeHg and develop signs of toxicity. MeHg is toxic to several organs, but the central nervous system (CNS) represents a preferential target, especially during development (prenatal and early postnatal periods). Though the biochemical events involved in MeHg-(neuro)toxicity are not yet entirely comprehended, a vast literature indicates that its pro-oxidative properties explain, at least partially, several of its neurotoxic effects. As result of its electrophilicity, MeHg interacts with (and oxidize) nucleophilic groups, such as thiols and selenols, present in proteins or low-molecular weight molecules. It is noteworthy that such interactions modify the redox state of these groups and, therefore, lead to oxidative stress and impaired function of several molecules, culminating in neurotoxicity. Among these molecules, glutathione (GSH; a major thiol antioxidant) and thiol- or selenol-containing enzymes belonging to the GSH antioxidant system represent key molecular targets involved in MeHg-neurotoxicity. In this review, we firstly present a general overview concerning the neurotoxicity of MeHg. Then, we present fundamental aspects of the GSH-antioxidant system, as well as the effects of MeHg on this system.
A mechanistic model for thiol redox dynamics in the organogenesis stage rat conceptus
2018, Reproductive Toxicology
Precise control of the glutathione/glutathione disulfide (GSH/GSSG) redox balance is vital for the developing embryo, but regulatory mechanisms are poorly understood. We developed a novel, mechanistic mass-balance model for GSH metabolism in the organogenesis stage (gestational day 10.0–11.13) rat conceptus predicting the dynamics of 8 unique metabolites in 3 conceptal compartments: the visceral yolk sac (VYS), the extra-embryonic fluid (EEF) and the embryo proper (EMB). Our results show that thiol concentrations in all compartments are well predicted by the model. Protein synthesis is predicted to be a major efflux pathway for all amino acid precursors of GSH synthesis and an essential model element. Our model provides quantitative insights in the transport fluxes and enzymatic fluxes needed to maintain thiol redox balances under normal physiological conditions. This is crucial to further elucidate the mechanisms through which chemical exposure can perturb redox homeostasis, causing oxidative stress, and potentially birth defects.
Modulating ROS to overcome multidrug resistance in cancer
2018, Drug Resistance Updates
Citation Excerpt :
Following its interaction with H2O2, GSH is biotransformed to its oxidized form, glutathione disulfide (GSSG) (Pompella et al., 2003; Kuhn and Borchert, 2002). In contrast, glutathione reductase (GR), using NADPH as an electron donor, biotransforms GSSG to GSH (Pompella et al., 2003; Dringen and Gutterer, 2002). The ratio of reduced intracellular glutathione to oxidized glutathione (GSH/GSSH) is frequently used as an index of cellular oxidative stress (Zafrilla et al., 2013; Zitka et al., 2012).
The successful treatment of cancer has significantly improved as a result of targeted therapy and immunotherapy. However, during chemotherapy, cancer cells evolve and can acquire “multidrug resistance” (MDR), which significantly limits the efficacy of cancer treatment and impacts patient survival and quality of life. Among the approaches to reverse MDR, modulating reactive oxidative species (ROS) may represent a strategy to kill MDR cancer cells that are mechanistically diverse. ROS in cancer cells play a central role in regulating and inducing apoptosis, thereby modulating cancer cells proliferation, survival and drug resistance. The levels of ROS and the activity of scavenging/anti-oxidant enzymes in drug resistant cancer cells are typically increased compared to non-MDR cancer and normal cells. Consequently, MDR cancer cells may be more susceptible to alterations in ROS levels. Numerous studies suggest that compounds modulating cellular ROS levels can enhance MDR cancer cell death and sensitize MDR cancer cells to certain chemotherapeutic drugs.
In the current review, we discuss the critical and targetable redox-regulating enzymes, including mitochondrial electron transport chain (ETC) complexes, NADPH oxidases (NOXs), enzymes related to glutathione metabolism, glutamate/cystine antiporter xCT, thioredoxin reductases (TrxRs), nuclear factor erythroid 2-related factor 2 (Nrf2), and their roles in regulating cellular ROS levels, drug resistance as well as their clinical significance. We also discuss and summarize the findings in the past decade regarding the efficacy of ROS modulators for the treatment of MDR cancer alone or as sensitizing compounds. Compounds that are efficacious in modulating ROS generation represent a prominent class of drug candidates that warrants evaluation in clinical trials for patients harboring MDR cancers.
A new thioredoxin reductase with additional glutathione reductase activity in Haemonchus contortus
2017, Experimental Parasitology
Citation Excerpt :
Furthermore, HcTrxR3 revealed an unexpected GR activity when catalyzed by NADPH dependent reduction of GSSG to produce GSH (Table 2). Even more noticeable, HcTrxR3 kinetic parameters for GSSG show values similar to those obtained with GRs from different organisms (Dringen and Gutterer, 2002; Savvides et al., 2002). We demonstrated that Ebselen is a direct substrate for HcTrxR3 just as it is for human TrxR (Zhao et al., 2002).
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 K_cat 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.
Effect of organic ligands on lead-induced oxidative damage and enhanced antioxidant defense in the leaves of Vicia faba plants
2014, Journal of Geochemical Exploration
Citation Excerpt :
Enzyme specific activity is expressed as micromoles of guaiacol oxidized (GO)·min− 1·g− 1 protein. Glutathione reductase was assayed according to Dringen and Gutterer (2002) using Glutathione Reductase Assay Kit (Cayman; Cat. 703002).
The biogeochemical behavior of metals varies with their chemical speciation. The present study evaluated the effect of metal speciation on lead-induced oxidative stress to Vicia faba leaves. Lead was applied to V. faba plants in the form of lead nitrate alone or chelated by citric acid (CA) and ethylenediaminetetraacetic acid (EDTA) in hydroponic conditions. The exposure time for all the treatments was 1, 4, 8, 12 and 24 h. The activation of antioxidant enzymes in V. faba leaves varies with Pb speciation and duration of exposure. Application of Pb alone increased the activities of superoxide dismutases (SOD), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX) and glutathione reductase (GR) while reducing that of catalase (CAT) in V. faba leaves. Application of EDTA dose dependently alleviated the Pb-induced activation of SOD, APX, GPOX and GR, and depression of CAT by decreasing Pb accumulation in V. faba leaves. By contrast, CA had no effect on Pb accumulation and antioxidant enzyme activities except that the activation of SOD, APX, GPOX and GR, and reduction of CAT was delayed. The results showed that the physiological responses of V. faba leaves to Pb toxicity vary with its chemical speciation. Therefore, it is proposed that metal speciation must be given consideration in risk assessment and remediation studies.
Short term exposure to multi-walled carbon nanotubes induce oxidative stress and DNA damage in Xenopus laevis tadpoles
2014, Ecotoxicology and Environmental Safety
Citation Excerpt :
The supernatant was also used for antioxidant enzyme activity. GR activity was assayed according to Dringen and Gutterer (2002), using Glutathione Reductase Assay Kit (GRSA-1KT, Sigma, France). The reaction was monitored by following the change in at 340 nm as oxidized glutathione-dependent oxidation of NADPH for 1 min.
The potential impact of Multiwalled Carbon NanoTubes (MWCNTs) was investigated on Xenopus laevis tadpoles exposed to 0.1, 1 and 10 mg/L. Oxidative stress was measured in entire larvae exposed and DNA damage (Comet assay) was carried out in erythrocytes of circulating blood from 2 h to 24 h according to standardized recommendations. Results showed significant H₂O₂ production when larvae were exposed to 1 mg/L and 10 mg/L of MWCNTs after 4 h and 2 h of exposure, respectively. Antioxidant enzyme activities showed significant induction of catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) from only 2 h of exposure to 10 mg/L of MWCNTs. In presence of 1 mg/L of MWCNTs, only GR and CAT activities were significantly induced at 4 h. Enzyme activities do not follow a simple dose-effect relation, but the time of induction is shortened in relation with the tested concentration. The Comet assay results showed significant DNA damages with a dose dependent response. The profiles of DNA damages show fluctuations, in course of time, which are characteristics of oxidative stress response in relation with the continuous balance between damage and compensation process.

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[27] Glutathione reductase from bovine brain

Publisher Summary

Prog. Neurobiol.

J. Biol. Chem.

Brain Res. Rev.

Brain Res.

Curr. Top. Cell. Regul.

Neurosci. Lett.

FEBS Len.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Enzymes

Biochem. Mol. Med.

Physiol. Rev.