Abstract
Glutathione reductase (GR) is a homodimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) using NADPH as a cofactor. The enzyme is a major component of cellular defense mechanisms against oxidative injury. In this study, GR was purified from the liver of the anoxia-tolerant turtle, Trachemys scripta elegans. The overall fold purifications were 13.3- and 12.1-fold with final specific activities of 5.5 and 1.44 U/mg of protein for control and anoxic turtle GR, respectively. SDS-PAGE of purified turtle liver GR showed a single protein band at approximately 55 kDa. Reverse phase HPLC of turtle GR revealed a single peak that had the same retention time as yeast GR. No new isoform of GR was detected in liver of T. s. elegans during anoxia. The K m values of turtle GR for GSSG and NADPH was 44.6 and 6.82 μM, respectively, suggesting a substantially higher affinity of turtle GR toward GSSG than most other vertebrates. Unlike other human GR, NADP+ did not inhibit turtle GR activity. The activation energy of turtle GR, calculated from the slope of the Arrhenius plot, was 32.2 ± 2.64 kJ/mol. Turtle GR had high activity under a broad pH range (having activity between pHs 4 and 10; optimal activity at pH 6.5) and the enzyme maintains activity under the pH drop that occurs under anoxic conditions. The high affinity of turtle GR suggests that turtles have high redox buffering capacity of tissues to protect against oxidative stress encountered during anoxia/reoxygenation.
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References
Ultsch GR Ecology and physiology of hibernation and overwintering among freshwater fishes, turtles, and snakes. Biol Rev 64:435–516, 1989
Jackson DC Hibernating without oxygen: physiological adaptations of the painted turtle. J Physiol 543:731–737, 2002
Li C, Jackson RM Reactive oxygen species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 282: C227–C241, 2002
Sadek HA, Nulton-Persson AC, Szweda PA, Szweda LI Cardiac ischemia/reperfusion, aging, and redox-dependent alterations in mitochondrial function. Arch Biochem Biophys 420:201–208, 2003
Solaini G, Harris DA Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion. Biochem J 390:377–394, 2005
Ramaglia V, Buck LT Time-dependent expression of heat shock protein 70 and 90 in tissues of the anoxic western painted turtle. J Exp Biol 207:3775–3784, 2004
Hermes-Lima M, Storey JM, Storey KB 2001 Antioxidant defenses, animal adaptation to oxygen availability during environmental stress. In: Storey KB, Storey JM (Eds) Cell and molecular responses to stress, Volume 2, Protein adaptations and signal transduction. Elsevier Press, Amsterdam, p. 263–287
Smith CV, Jones DP, Guenthner TM, Lash LH, Lauterburg BH Compartmentation of glutathione: implications for the study of toxicity and disease. Toxicol Appl Pharmacol 140:1–12, 1996
Schirmer RH, Krauth-Siegel RL, Schulz GE 1989 Glutathione reductase. In: Dolphin R, Poulson Avramovie O (Eds) Glutathione: chemical, biochemical and medical aspects. Wiley, D., Chichester, pp. 553–596
Mullineaux PM, Creissen GP 1996 Glutathione reductase: regulation and role in oxidative stress. In: Scandalios J. (Eds) Oxidative stress, Monograph 34. Harbour Press, Cold Spring, New York
Williams CH Jr 1976 Flavin containing dehydrogenases. In: Boyer PD (Ed) The enzymes XIII. Academic Press, New York, pp. 89–173
Holmgren A Pyridine nucleotide-disulfide oxidoreductases. Experientia Suppl 36:149–180, 1980
Untucht-Grau R, Schirmer RH, Schirmer I, Krauth-Siegel RL Glutathione reductase from human erythrocytes: amino acid sequence of the structurally known FAD-binding domain. Eur J Biochem 120:407–419, 1981
Kelner MJ, Montoya MA Structural organization of the human glutathione reductase gene: determination of correct cDNA sequence and identification of a mitochondrial leader sequence. Biochem Biophys Res Commun 269:366–368, 2000
Willmore WG, Storey KB Antioxidant systems and anoxia tolerance in a freshwater turtle Trachemys scripta elegans. Mol Cell Biochem 170:177–185, 1997
Willmore WG, Storey KB Glutathione systems and anoxia tolerance in turtles. Am J Physiol Regul Integr Comp Physiol 273: R219–R225, 1997
Helmerhorst E, Stokes GB Microcentrifuge desalting: a rapid quantitative method for desalting small amounts of protein. Anal Biochem 104:130–135, 1980
DiIlio C, Polidoro G, Arduini A, Muccini A, Federici G Glutathione peroxidase, glutathione reductase, glutathione S-transferase and gammaglutamyl transpeptidase activities in the human early pregnancy placenta. Biochem Med 29:143–148, 1983
Vesterberg O Isoelectric focusing of proteins. Meth Enzymol 22:389–412, 1971
Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254, 1976
Brooks SPJ A simple computer program with statistical tests for the analysis of enzyme kinetics. BioTechniques 13:906–911, 1992
Willmore WG, Storey KB Purification and properties of the glutathione S-transferases from the anoxia-tolerant turtle, Trachemys scripta elegans. FEBS J 272:3602–3614, 2005
Hermes-Lima M, Storey JM, Storey KB Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails. Comp Biochem Physiol B Biochem Mol Biol 120:437–448, 1998
Grundy JE, Storey KB Antioxidant defenses and lipid peroxidation damage in estivating toads, Scaphiopus couchii. J Comp Physiol B 168:132–142, 1998
Pannunzio TM, Storey KB Antioxidant defenses and lipid peroxidation during anoxia stress and aerobic recovery in the marine gastropod, Littorina littorea. J Exp Mar Biol Ecol 221:277–292, 1998
Nieto-Sotelo J, Ho THD Effects of heat shock on the metabolism of glutathione in maize plants. Plant Physiol 82:1031–1035, 1986
Schoner S, Krause H Protective systems against active oxygen species in spinach: response to cold acclimation and excess light. Planta 180:383–389, 1990
Hausladen A, Alscher RG Purification and characterization of glutathione reductase isozymes specific for the state of cold-hardiness of red spruce. Plant Physiol 105:205–213, 1994
Foster JG, Hess JL Responses of superoxide dismutase and glutathione reductase activities on cotton leaf tissues exposed to an atmosphere enriched in oxygen. Plant Physiol 66:482–487, 1980
Tanaka K, Saji H, Kondo N Immunological properties of spinach glutathione reductase and inductive biosynthesis of the enzyme with ozone. Plant Cell Physiol 29:637–642, 1988
Grill EM, Winnaker EL, Zenk MH Photosynthesis: the principle heavy-metal complexing peptides of higher plants. Science 230:674–676, 1985
Cakmak I, Marschner H Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol 98:1222–1227, 1992
Smirnoff N, Colombe SV Drought influences activity of enzymes of the chloroplast hydrogen peroxide scavenging system. J Exp Bot 39:1097–1108, 1988
Tanaka K, Masuda R, Sugimoto T, Omasa K, Sakaki T Water-deficiency-induced changes in the contents of defensive substances against active oxygen in spinach leaves. Agric Biol Chem 54:2629–2634, 1990
Toribio F, Martínez-Lara E, Pascual P, López-Barea J Methods of purification of glutathione peroxidase and related enzymes. J Chromatog B 684:77–97, 1996
Carlberg I, Mannervik B Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem 250:5475–5480, 1975
López-Barea J, Lee C-Y Mouse liver glutathione reductase. Purification, kinetics and regulation. Eur J Biochem 98:487–499, 1979
Garcia-Alfonso CE, Martinez-Galisteo E, Llobell A, Bàrcena JA, López-Barea J Horse-liver glutathione reductase: purification and characterization. Int J Biochem 25:61–68, 1993
Tamura T, McMicken HW, Smith CV, Hansen TN Mitochondrial targeting of glutathione reductase requires a leader sequence. Biochem Biophys Res Comm 222:659–663, 1996
Krauth-Siegel RL, Blatterspiel R, Saleh M, Schiltz E, Shimer RH, Untucht-Grau R Glutathione reductase from human erythrocytes. The sequences at the NADPH domain and the interface domain. Eur J Biochem 121:259–267, 1982
Moroff G, Kosow DP Characterization of human platelet glutathione reductase. Biochim Biophys Acta 527:327–336, 1978
Öğüs H, Özer N Human jejunal glutathione reductase: purification and evaluation of the NADPH- and glutathione-induced changes in redox state. Biochem Med Metab Biol 45:65–73, 1991
Erat M, Sakiroglu H, Ciftci M Purification and characterization of glutathione reductase from bovine erythrocytes. Prep Biochem Biotech 33:283–300, 2003
Outten CE, Culotta VC Alternative start sites in the Saccharomyces cerevisiae GLR1 gene are responsible for mitochondrial and cytosolic isoforms of glutathione reductase. J Biol Chem 279:7785–7791, 2004
Nakashima K, Miwa S, Yamauchi K Human erythrocyte glutathione reductase. I. Purification and properties. Biochim Biophys Acta 445:309–323, 1976
Worthington DJ, Rosemeyer MA Glutathione reductase from human erythrocytes. Catalytic properties and aggregation. Eur J Biochem 67:231–238, 1976
Carlberg I, Mannervik B Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2′,5′-ADP-Sepharose 4B. Anal Biochem 116:531–536, 1981
Righetti PG, Caravaggio T Isoelectric points and molecular weight of proteins. J Chromat 127:1–28, 1976
Pace CN Conformational stability of globular proteins. Trends Biochem Sci 15:14–17, 1990
Somero GN: Temperature as a selective factor in protein evolution: the adaptational strategy of “compromise”. J Exp Zool 194:175–188, 1975
Somero GN, Low PS Temperature: a “shaping force” in protein evolution. Biochem Soc Symp 41:33–42, 1976
Ultsch GR, Carwile ME, Crocker CE, Jackson DC The physiology of hibernation among painted turtles: the eastern painted turtle, Chrysemys picta picta. Physiol Biochem Zool 72:493–501, 1999
Somero GN Adaptation of enzymes to temperature: searching for basic “strategies”. Comp Biochem Physiol B Biochem Mol Biol 139:321–333, 2004
Lievre V, Becuwe P, Bianchi A, Bossenmeyer-Pourie C, Koziel V, Franck P, Nicolas MB, Dauca M, Vert P, Daval JL Intracellular generation of free radicals and modifications of detoxifying enzymes in cultured neurons from the developing rat forebrain in response to transient hypoxia. Neuroscience 105:287–297, 2001
Arthur PG, Lim SCC, Meloni BP, Munns SE, Chan A, Knuckey NW The protective effect of hypoxic preconditioning on cortical neuronal cultures is associated with increases in the activity of several antioxidant enzymes. Brain Res 1017:146–154, 2004
Reischl E High sulfhydryl content in turtle erythrocytes: is there a relation with resistance to hypoxia? Comp Biochem Physiol B 85:723–726, 1986
Acknowledgments
Thanks to J.M. Storey and A. Ima for editorial revisions to the manuscript. This work was supported by a postgraduate scholarship to W.G.W. and a discovery grant to K.B.S. from the NSERC Canada; K.B.S. holds the Canada Research Chair in Molecular Physiology.
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Willmore, W.G., Storey, K.B. Purification and properties of glutathione reductase from liver of the anoxia-tolerant turtle, Trachemys scripta elegans . Mol Cell Biochem 297, 139–149 (2007). https://doi.org/10.1007/s11010-006-9339-8
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DOI: https://doi.org/10.1007/s11010-006-9339-8