Purification and properties of a recombinant sulfur analog of murine selenium‐glutathione peroxidase

Rocher, Corinne; Lalanne, Jean‐Louis; Chaudière, Jean

doi:10.1111/j.1432-1033.1992.tb16862.x

Cited by 159 publications

(95 citation statements)

References 28 publications

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“…Although the specific activity of the recombinant PRxVI was very low, it was not due to the purification procedure because the activity in the cell homogenates expressing PRxVI was also very low. However, the activity of PRxVI was 34 times that of the C47S mutant and four times that of a mutant GPx with amino-acid substitution of the U residue with cysteine (C), referred to as an U47C mutant GPx ( [28]. Optimal pH was within an alkaline range (around pH 8.0) and consistent with the report by Fisher et al [9].…”

Section: R E S U L T Ssupporting

confidence: 79%

Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role

Fujii¹,

Fujii²,

Taniguchi³

2001

Eur J Biochem

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A family of proteins with thioredoxin (TRx)-dependent peroxidase activity, referred to as peroxiredoxins (PRx), has been identified in many species. The sixth member of this family, PRxVI, contains only one conserved cysteine residue, while other members contain additional cysteines. We have isolated a cDNA for rat PRxVI and constructed a large scale baculovirus system to produce the recombinant protein. The protein was purified by a simple two-step procedure utilizing ion-exchange and gel-filtration chromatography. The purified PRxVI exhibited a low level of glutathione-dependent peroxidase but not TRx-dependent activity. PRxVI expression was the highest in lung, followed by brain, kidney, heart, testis, etc. as judged by Northern and Western blot analyses using a rabbit antibody to the purified PRxVI. Immunohistochemical analyses showed strong staining in the epithelium of the bronchus and bronchioles in lung and in the epithelial cells of kidney tubules. In addition, Sertoli cells in testis and islet of Langerhans cells in pancreas were strongly stained. The developmental changes of PRxVI expression in lung and kidney were low in the prenatal stage but induced postnatally. Moreover, intraperitoneal administration of chloroform induced PRxVI mRNA in kidney. When the distribution and the induced expression of PRxVI under conditions of oxidative stress are considered, a physiological role of it as an antioxidative enzyme is indicated.

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Section: R E S U L T Ssupporting

confidence: 79%

Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role

Fujii¹,

Fujii²,

Taniguchi³

2001

Eur J Biochem

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“…Indeed, the selenol group is mainly in its anion selenolate form at physiological pH while the thiol of a Cys residue is only partly ionized, making Se-Cys more reactive than Cys. Consistently, sitedirected replacement of Se-Cys by Cys considerably decreases the activity of selenoGPXs (Rocher et al 1992;Maiorino et al 1995). In the case of NS-GPX, the sulfenic acid forms a disulfide with a second cysteine of the enzyme (striped bar in Figure 3) and this disulfide can be reduced in a second step (Navrot et al 2006;Herbette et al 2007).…”

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confidence: 79%

The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

et al. 2008

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Thiol/selenol peroxidases are ubiquitous nonheme peroxidases. They are divided into two major subfamilies: peroxiredoxins (PRXs) and glutathione peroxidases (GPXs). PRXs are present in diverse subcellular compartments and divided into four types: 2-cys PRX, 1-cys PRX, PRX-Q , and type II PRX (PRXII). In mammals, most GPXs are selenoenzymes containing a highly reactive selenocysteine in their active site while yeast and land plants are devoid of selenoproteins but contain nonselenium GPXs. The presence of a chloroplastic 2-cys PRX, a nonselenium GPX, and two selenium-dependent GPXs has been reported in the unicellular green alga Chlamydomonas reinhardtii. The availability of the Chlamydomonas genome sequence offers the opportunity to complete our knowledge on thiol/selenol peroxidases in this organism. In this article, Chlamydomonas PRX and GPX families are presented and compared to their counterparts in Arabidopsis, human, yeast, and Synechocystis sp. A summary of the current knowledge on each family of peroxidases, especially in photosynthetic organisms, phylogenetic analyses, and investigations of the putative subcellular localization of each protein and its relative expression level, on the basis of EST data, are presented. We show that Chlamydomonas PRX and GPX families share some similarities with other photosynthetic organisms but also with human cells. The data are discussed in view of recent results suggesting that these enzymes are important scavengers of reactive oxygen species (ROS) and reactive nitrogen species (RNS) but also play a role in ROS signaling. L IFE in an oxygen-rich environment has to deal with the danger of oxidative stress. During normal cell metabolism, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced, essentially by respiratory and photosynthetic electron transfer chains. These highly reactive molecules can react with many cell components and damage DNA, proteins, and lipids. Thus, their concentration has to be strictly controlled. For this purpose, aerobic organisms are equipped with nonenzymatic (ascorbate, glutathione, tocopherol, and carotenoid) or enzymatic (catalase, ascorbate peroxidase, superoxide dismutase, glutathione peroxidase, and peroxiredoxin) antioxidant systems to remove ROS from the cells. To control their concentrations, ROS and RNS have to be sensed. It has been established in many organisms that ROS, and especially hydrogen peroxide, are signaling molecules that diffuse across membranes and induce specific signal transduction pathways. Furthermore, ROS can also be produced on purpose by cells in response to several stimuli to function as second messengers inside the cell. Finally, ROS and RNS can control enzyme activities by triggering several post-translational modifications such as disulfide bond formation, thiol oxidation to sulfenic/sulfinic/sulfonic acid, glutathionylation, nitrosylation, or carbonylation.Thiol/selenol peroxidases have emerged, during recent years, as important scavengers of ROS/RNS but they ...

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“…Substitution of native Sec by Cys in mouse cellular GPx Sec (38) and in pig heart PHGPx Sec (9) led to a dramatic reduction of the enzymatic activity, about 1000-and 250-fold, respectively. Consistently, the naturally Cys-containing cit-PHGPx displays catalytic activity of only 0.2% of that of the pig heart PHGPx Sec (10).…”

Section: Discussionmentioning

confidence: 99%

Substituting Selenocysteine for Catalytic Cysteine 41 Enhances Enzymatic Activity of Plant Phospholipid Hydroperoxide Glutathione Peroxidase Expressed in Escherichia coli

Hazebrouck¹,

Camoin²,

Faltin³

et al. 2000

Journal of Biological Chemistry

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The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding citPHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys 41 by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is seleniumdependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.

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Purification and properties of a recombinant sulfur analog of murine selenium‐glutathione peroxidase

Cited by 159 publications

References 28 publications

Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role

Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role

The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

Substituting Selenocysteine for Catalytic Cysteine 41 Enhances Enzymatic Activity of Plant Phospholipid Hydroperoxide Glutathione Peroxidase Expressed in Escherichia coli

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