Mitochondrial Protection by the Thioredoxin-2 and Glutathione Systems in an in Vitro Endothelial Model of Sepsis Damon A
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Glutathione Reductase (Ec 1.6.4.2)
Enzymatic Assay of GLUTATHIONE REDUCTASE (EC 1.6.4.2) PRINCIPLE: ß-NADPH + GSSG Glutathione Reductase> ß-NADP + 2 GSH Abbreviations used: ß-NADPH = ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form ß-NADP = ß-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form GSSG = Glutathione, Oxidized Form GSH = Glutathione, Reduced Form CONDITIONS: T = 25°C, pH = 7.6, A340nm, Light Path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 100 mM Potassium Phosphate Buffer with 3.4 mM Ethylenediaminetetraacetic Acid (EDTA), pH 7.6 at 25°C (Prepare 200 ml in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Sigma Prod. No. P-5379 and Ethylenediaminetetraacetic Acid, Dipotassium Salt, Sigma Stock No. ED2P. Adjust to pH 7.6 at 25°C with 1 M KOH.) B. 30 mM Glutathione Substrate Solution (GSSG) (Prepare 5 ml in deionized water using Glutathione, Oxidized Form, Disodium Salt, Sigma Prod. No. G-4626.) C. 0.8 mM ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Solution (ß-NADPH) (Prepare 5 ml in cold Reagent A using ß-Nicotinamide Adenine Dinucleotide Phosphate, Tetrasodium Salt, Sigma Prod. No. N-1630.) D. 1.0% (w/v) Bovine Serum Albumin (BSA) (Prepare 100 ml in Reagent A using Albumin, Bovine, Sigma Prod. No. A-4503. This solution should be kept cold.) SPGLUT01 Page 1 of 3 Revised: 08/03/95 Enzymatic Assay of GLUTATHIONE REDUCTASE (EC 1.6.4.2) REAGENTS: (continued) E. Glutathione Reductase Enzyme Solution (Immediately before use, prepare a solution containing 0.30 - 0.60 unit/ml of Glutathione Reductase in cold Reagent D.) PROCEDURE: Pipette (in milliliters) the following reagents into suitable cuvettes: Test Blank Deionized Water 0.65 0.65 Reagent A (Buffer) 1.50 1.50 Reagent B (GSSG) 0.10 0.10 Reagent C (ß-NADPH) 0.35 0.35 Reagent D (BSA) 0.30 0.40 Mix by inversion and equilibrate to 25°C. -
Glutathione Reductase Human, Recombinant Expressed in Escherichia Coli
Glutathione Reductase human, recombinant expressed in Escherichia coli Catalog Number G9297 Storage Temperature –20 °C CAS RN 9001-48-3 Precautions and Disclaimer EC 1.8.1.7 (formerly 1.6.4.2) This product is for R&D use only, not for drug, Synonyms: GR, NADPH:oxidized glutathione household, or other uses. Please consult the Material oxidoreductase, glutathione-disulfide reductase Safety Data Sheet for information regarding hazards and safe handling practices. Product Description Glutathione reductase (GR) is an ubiquitous Storage/Stability flavoenzyme involved in protecting cells from stress. The product ships on wet ice and storage at –20 °C is GR catalyzes the reduction of oxidized glutathione recommended. The product is stable at –20 °C for at (GSSG) to glutathione (GSH). It is an essential least 2 years. component of the glutathione redox cycle, which maintains adequate levels of reduced cellular GSH. References GSH serves as an antioxidant, reacting with free 1. Lopez-Mirabal, H.R., and Winther, J.R., Redox radicals and organic peroxides. Glutathione is also an characteristics of the eukaryotic cytosol. Biochim. electron donor for glutathione peroxidases and a Biophys. Acta, 1783, 629-640 (2008). substrate for glutathione S-transferases contributing to 2. Qiao, M., et al., Increased expression of glutathione the detoxification and elimination of toxic electrophilic reductase in macrophages decreases 1,2 metabolites and xenobiotics. artherosclerotic lesion formation on low-density lipoproetin receptor-deficient mice. Arterioscler. Glutathione reductase is a homodimeric enzyme Thromb. Vasc. Biol., 27, 1375-1382 (2007). containing 1 FAD molecule and 1 NADPH binding 3. Worthington, D.J., and Rosemeyer, M.A., 3 domain per subunit. -
Table S4. List of Enzymes Directly Involved in the Anti-Oxidant Defense Response
Table S4. List of Enzymes directly involved in the anti-oxidant defense response. Gene Name Gene Symbol Classification/Pathway 6-phosphogluconate dehydrogenase 6PGD NADPH regeneration/Pentose Phosphate Glucose-6-phosphate dehydrogenase G6PD NADPH regeneration/Pentose Phosphate Isocitrate Dehydrogenase 1 IDH1 NADPH regeneration/Krebs Isocitrate Dehydrogenase 2 IDH2 NADPH regeneration/Krebs Malic Enzyme 1 ME1 NADPH regeneration/Krebs Methylenetetrahydrofolate dehydrogenase 1 MTHFD1 NADPH regeneration/Folate Methylenetetrahydrofolate dehydrogenase 2 MTHFD2 NADPH regeneration/Folate Nicotinamide Nucleotide Transhydrogenase NNT NADPH regeneration/NAD Catalase CAT Antioxidants/Catalses/free radical detoxification Glutamate-cysteine ligase catalytic subunit GCLC Antioxidants/Glutathione synthesis Glutamate-cysteine ligase modifier subunit GCLM Antioxidants/Glutathione synthesis Glutathione peroxidase1 GPx1 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione peroxidase2 GPx2 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione peroxidase3 GPx3 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione peroxidase4 GPx4 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione peroxidase5 GPx5 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione peroxidase6 GPx6 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione peroxidase7 GPx7 Antioxidants/Glutathione Peroxidases/free radical detoxification Glutathione S-transferase -
Inhibitory Effects of Some Flavonoids on Thioredoxin Reductase Purified from Chicken Liver ABSTRACT INTRODUCTION
Brazilian Journal of Poultry Science Revista Brasileira de Ciência Avícola Inhibitory Effects of Some Flavonoids on ISSN 1516-635X 2019 / v.21 / n.2 / 001-008 Thioredoxin Reductase Purified from Chicken http://dx.doi.org/10.1590/1806-9061-2018-0982 Liver Original Article Author(s) ABSTRACT Türkoğlu E.AI https://orcid.org/0000-0001-7850-6456 Thioredoxin reductases (TrxRs) are selenocysteine-containing Kuzu MII https://orcid.org/0000-0002-1375-7673 flavoenzymes that reduce Trxin NADPH-dependent manner. In the Ayasan TIII https://orcid.org/0000-0001-7397-6483 view of the direct vital role of TrxR in a wide range of biochemical and IV Inci H https://orcid.org/0000-0002-9791-0435 physiological processes, methods to inhibit this enzyme are clinically Eratak SVV https://orcid.org/0000-0003-3788-8704 important. TrxR has recently emerged as a new candidate in anticancer I Department of Pharmaceutical Biotechnology, drug investigations because of overexpression in tumorous cells. In this Faculty of Pharmacy, University of Health Sciences, Istanbul 34668, Turkey. study, TrxR from chick liver was purified 94.6-fold with a yield of 4.86% II Deparment of Chemistry, Faculty of Science and a specific activity of 0.19 EU/mg. K and V values of TrxR for and Letters, Ağrı İbrahim Çeçen University, Ağrı M max 04100, Turkey. DTNB were calculated as 0.9 mM and 0,03 EU/mL, respectively. Then, III East Mediterranean Agricultural Research Institute, Karatas Road, Adana 01321, Turkey. the effects of the flavonoids hesperidin, naringenin, chlorogenic acid, IV Department of Animal Science, Faculty of ferulic acid, naringin, 3,4-dihydoxybenzoic acid, and ellagic acid on the Agriculture, Bingöl University, Bingöl 12000, Turkey. -
Genomic Insights Into the Uncultured Genus &Lsquo
The ISME Journal (2014) 8, 2463–2477 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Genomic insights into the uncultured genus ‘Candidatus Magnetobacterium’ in the phylum Nitrospirae Wei Lin1,2,7, Aihua Deng3,7, Zhang Wang4, Ying Li2,5, Tingyi Wen3, Long-Fei Wu2,6, Martin Wu4 and Yongxin Pan1,2 1Biogeomagnetism Group, Paleomagnetism and Geochronology Laboratory, Key Laboratory of the Earth’s Deep Interior, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, China; 2France-China Bio-Mineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, China; 3CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; 4Department of Biology, University of Virginia, Charlottesville, VA, USA; 5State Key Laboratory of Agro-Biotechnology and Laboratoire International Associe Franco-Chinois de Bio-Mineralisation et Nano-Structures, College of Biological Sciences, China Agricultural University, Beijing, China and 6Laboratoire de Chimie Bacte´rienne, Aix-Marseille Universite´, CNRS, Marseille Cedex 20, France Magnetotactic bacteria (MTB) of the genus ‘Candidatus Magnetobacterium’ in phylum Nitrospirae are of great interest because of the formation of hundreds of bullet-shaped magnetite magneto- somes in multiple bundles of chains per cell. These bacteria are worldwide distributed in aquatic environments and have important roles in the biogeochemical cycles of iron and sulfur. However, except for a few short genomic fragments, no genome data are available for this ecologically important genus, and little is known about their metabolic capacity owing to the lack of pure cultures. Here we report the first draft genome sequence of 3.42 Mb from an uncultivated strain tentatively named ‘Ca. -
Glutathione Reductase Assay Kit
Glutathione Reductase Assay Kit Item No. 703202 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd · Ann Arbor, MI · USA TABLE OF CONTENTS GENERAL INFORMATION GENERAL INFORMATION 3 Materials Supplied Materials Supplied 4 Safety Data 4 Precautions 5 If You Have Problems Item Number Item Quantity 4 Storage and Stability 703210 GR Assay Buffer (10X) 1 vial 4 Materials Needed but Not Supplied INTRODUCTION 5 Background 703212 GR Sample Buffer (10X) 1 vial 5 About This Assay 703214 GR Glutathione Reductase (control) 1 vial PRE-ASSAY PREPARATION 6 Reagent Preparation 7 Sample Preparation 703216 GR GSSG 1 vial ASSAY PROTOCOL 9 Plate Set Up 703218 GR NADPH 3 vials 11 Performing the Assay 400014 96-Well Solid Plate (Colorimetric Assay) 1 plate ANALYSIS 12 Calculations 13 Performance Characteristics 400012 96-Well Cover Sheet 1 cover RESOURCES 14 Interferences If any of the items listed above are damaged or missing, please contact our 16 Troubleshooting Customer Service department at (800) 364-9897 or (734) 971-3335. We cannot 17 References accept any returns without prior authorization. 18 Plate Template 19 Notes WARNING: THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE. 19 Warranty and Limitation of Remedy ! GENERAL INFORMATION 3 Safety Data INTRODUCTION This material should be considered hazardous until further information becomes available. Do not ingest, inhale, get in eyes, on skin, or on clothing. Wash Background thoroughly after handling. Before use, the user must review the complete Safety Glutathione (GSH) is a tripeptide widely distributed in both plants and Data Sheet, which has been sent via email to your institution. -
Emerging Players in the Regulation of Protein S-Nitrosation in Plants
plants Review Thioredoxins: Emerging Players in the Regulation of Protein S-Nitrosation in Plants Tereza Jedelská , Lenka Luhová and Marek Petˇrivalský * Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitel ˚u27, 78371 Olomouc, Czech Republic; [email protected] (T.J.); [email protected] (L.L.) * Correspondence: [email protected] Received: 17 August 2020; Accepted: 22 October 2020; Published: 24 October 2020 Abstract: S-nitrosation has been recognized as an important mechanism of ubiquitous posttranslational modification of proteins on the basis of the attachment of the nitroso group to cysteine thiols. Reversible S-nitrosation, similarly to other redox-based modifications of protein thiols, has a profound effect on protein structure and activity and is considered as a convergence of signaling pathways of reactive nitrogen and oxygen species. This review summarizes the current knowledge on the emerging role of the thioredoxin-thioredoxin reductase (TRXR-TRX) system in protein denitrosation. Important advances have been recently achieved on plant thioredoxins (TRXs) and their properties, regulation, and functions in the control of protein S-nitrosation in plant root development, translation of photosynthetic light harvesting proteins, and immune responses. Future studies of plants with down- and upregulated TRXs together with the application of genomics and proteomics approaches will contribute to obtain new insights into plant S-nitrosothiol metabolism and its regulation. Keywords: denitrosation; -
DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents
molecules Review DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents Maria Valeria Raimondi 1,*,† , Ornella Randazzo 1,†, Mery La Franca 1 , Giampaolo Barone 1 , Elisa Vignoni 2, Daniela Rossi 2 and Simona Collina 2,* 1 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, via Archirafi 32, 90123 Palermo, Italy; [email protected] (O.R.); [email protected] (M.L.F.); [email protected] (G.B.) 2 Drug Sciences Department, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, via Taramelli 12, 27100 Pavia, Italy; [email protected] (E.V.); [email protected] (D.R.) * Correspondence: [email protected] (M.V.R.); [email protected] (S.C.); Tel.: +390-912-389-1915 (M.V.R.); +390-382-987-379 (S.C.) † These Authors contributed equally to this work. Academic Editors: Simona Collina and Mariarosaria Miloso Received: 25 February 2019; Accepted: 20 March 2019; Published: 22 March 2019 Abstract: Dihydrofolate reductase inhibitors are an important class of drugs, as evidenced by their use as antibacterial, antimalarial, antifungal, and anticancer agents. Progress in understanding the biochemical basis of mechanisms responsible for enzyme selectivity and antiproliferative effects has renewed the interest in antifolates for cancer chemotherapy and prompted the medicinal chemistry community to develop novel and selective human DHFR inhibitors, thus leading to a new generation of DHFR inhibitors. This work summarizes the mechanism of action, chemical, and anticancer profile of the DHFR inhibitors discovered in the last six years. New strategies in DHFR drug discovery are also provided, in order to thoroughly delineate the current landscape for medicinal chemists interested in furthering this study in the anticancer field. -
Free Radical Reduction by Thioredoxin Reductase at the Surface of Normal and Vitiliginous Human Keratinocytes*
Free Radical Reduction by Thioredoxin Reductase at the Surface of Normal and Vitiliginous Human Keratinocytes* Karin U. Schallreuter, M .D ., Mark R. Pittelkow, M.D., and John M. Wood, Ph.D. Departm ents of Dermatology (KUS) an d Biochemistry (JMW) , University of Minneso ta School of Medicine, Minnea polis, and · Department of Dermatology, Mayo Clinic (MRP), Roches ter, Minnesota, U.S.A. Cell cultures of human keratinocytes contain m embrane ulated by calcium concentrations of the cell culture m edium. associated thioredoxin reductase that is extremely active in Stratified keratinocytes are half as active in medium con reducin g radicals on the outer plasma membrane. This en taining 2 mM Ca + + compared with 0.1 mM Ca + + con zyme activity was confirmed by its purification from cul centration. (4) Product inhibition of the enzyme occurs tures of stratified human keratinocytes by affinity column with oxidized coenzyme NADP + (i. e., 87% inhibition of chro m atography. The enzym e was assayed both in vivo enzyme activity over 30 min). The enzyme is heat stable and in vitro usin g a spin-labeled quaternary ammonium at temperatures of70°C for 10 min. It is inactivated at 75°C . compound as the substrate, under saturating conditions in A comparative study of thioredoxin reductase activity on free radical substrate. Specific activities were determined stratified differentiated and undifferentiated rapidly grow by monitoring the sequential decrease in the amplitude of ing celI s was performed . Also, enzyme activity was quan the electron spin resonance signal per unit of cell protein . titated for cultured keratinocytes isolated from vitiliginous The following properties were found: (1) Cultures of adult and normal skin of the same donor. -
Microbiology Letters
RESEARCH LETTER A thioredoxin reductase-like protein from the thermophile, Thermus scotoductus SA-01, displaying iron reductase activity Phillip Armand Bester, Derek Litthauer, Lizelle A. Piater & Esta van Heerden Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa Downloaded from https://academic.oup.com/femsle/article/302/2/182/647366 by guest on 29 September 2021 Correspondence: Esta van Heerden, Abstract Department of Microbial, Biochemical and Food Biotechnology, University of the Free The transition metal iron is an important element for the sustenance of life – it can State, Bloemfontein 9300, South Africa. function either as an electron acceptor or as a donor and serves as a cofactor in Tel.: 12751 401 2472; fax: 12751 444 3219; many enzymes activities. The cytoplasmic NAD(P)H-dependent ferric reductase e-mail: [email protected] in Thermus scotoductus SA-01 shares high sequence and structural similarity to prokaryotic thioredoxin reductases. Here we report the sequence of the ferric Received 9 June 2009; accepted 3 November reductase (which is typically annotated as a thioredoxin reductase-like protein) 2009. and a comparative kinetic study with the thioredoxin reductase from SA-01. Final version published online December 2009. Structurally, the most noteworthy difference, immediately apparent from the protein sequence, is the absence of the disulphide redox centre in the ferric DOI:10.1111/j.1574-6968.2009.01852.x reductase. This is the first report relating the attributes of such a redox protein to Editor: Christiane Dahl its ability to reduce a ferric substrate. Keywords ferric reductase; thioredoxin reductase; thermophilic; Thermus scotoductus SA-01. -
Ferroptosis-Related Flavoproteins: Their Function and Stability
International Journal of Molecular Sciences Review Ferroptosis-Related Flavoproteins: Their Function and Stability R. Martin Vabulas Charité-Universitätsmedizin, Institute of Biochemistry, Charitéplatz 1, 10117 Berlin, Germany; [email protected]; Tel.: +49-30-4505-28176 Abstract: Ferroptosis has been described recently as an iron-dependent cell death driven by peroxida- tion of membrane lipids. It is involved in the pathogenesis of a number of diverse diseases. From the other side, the induction of ferroptosis can be used to kill tumor cells as a novel therapeutic approach. Because of the broad clinical relevance, a comprehensive understanding of the ferroptosis-controlling protein network is necessary. Noteworthy, several proteins from this network are flavoenzymes. This review is an attempt to present the ferroptosis-related flavoproteins in light of their involvement in anti-ferroptotic and pro-ferroptotic roles. When available, the data on the structural stability of mutants and cofactor-free apoenzymes are discussed. The stability of the flavoproteins could be an important component of the cellular death processes. Keywords: flavoproteins; riboflavin; ferroptosis; lipid peroxidation; protein quality control 1. Introduction Human flavoproteome encompasses slightly more than one hundred enzymes that par- ticipate in a number of key metabolic pathways. The chemical versatility of flavoproteins relies on the associated cofactors, flavin mononucleotide (FMN) and flavin adenine dinu- cleotide (FAD). In humans, flavin cofactors are biosynthesized from a precursor riboflavin that has to be supplied with food. To underline its nutritional essentiality, riboflavin is called vitamin B2. In compliance with manifold cellular demands, flavoproteins have been accommo- Citation: Vabulas, R.M. dated to operate at different subcellular locations [1]. -
Mechanism and Structure of Thioredoxin Reductase from <I>Escherichia Coli</I>
SERIAL REVIEW FLAVOPROTEIN STRUCTURE AND MECHANISM 6 Mechanism and structure of thioredoxin reductase from Escherichia coli CHARLES H. WILLIAMS, JR.’ Department of Veterans Affairs Medical Center, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48105, USA The family of flavoproteins with redox-active cys- through a ternary complex; the kinetic evidence for tein/cystine residues that link the pyridine nucleotides such a complex is discussed.-Williams, C. H., Jr. with sulfur-containing substrates is an ever-growing one. Mechanism and structure of thioredoxin reductase In the case of thioredoxin reductase, the second sub- from Escherichia coli. FASEB J. 9, 1267-12 76 strate is the small protein thioredoxin, itself containing (1995) a redox-active disuffide, which in its reduced form is the substrate for ribonucleotide reductase. Like other mem- Key Words: thioredoxin reductase flavoprotein . disulfide reduc- bers of this family, thioredoxin reductase catalysis in- tase pyridine nucleotide thiol-disulfide interchange volves the reduction of the enzyme-bound FAD by NADPH, followed by electron transfer from the reduced THIOREDOXIN REDUCTASE CATALYZES THE REDUCTION of the flavin to the redox-active disulfide, which in turn is 12,000 Mr redox protein thioredoxin by NADPH, as shown responsible for the reduction of thioredoxin. The crystal below, where Tr(S2) is thioredoxin and Tr(SH)2 is reduced structure of the enzyme raises interesting mechanistic thioredoxin.2 questions about how this internal transfer of reducing equivalents takes place. The possible answers to these NADPH + H +Tr(S2) NADP +Tr(SH)2 questions provide the principal theme of this article. Thioredoxin reductase is a member of the pyridine nu- cleotide-disulfide oxidoreductase family of flavoenzymes.