Purification and properties of bovine thioredoxin system

Martı́nez-Galisteo, Emilia; Padilla, C. Alicia; García-Alfonso, C.; López-Barea, Juán; Bárcena, José Antonio

doi:10.1016/0300-9084(93)90131-b

Cited by 45 publications

(34 citation statements)

References 31 publications

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“…Cys-SSG was used as the initiating disulfide substrate to produce thioredoxin-disulfide, and thioredoxin and thioredoxin reductase concentrations were adjusted so that one or the other was limiting for the assays of their respective activities. Bovine thioredoxin was assayed by a turbidometric assay using insulin as the substrate and DTT as the reductant for the oxidized thioredoxin (22).…”

Section: Methodsmentioning

confidence: 99%

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,

et al. 2006

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Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time-and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogs. Keywordsgliotoxin; glutaredoxin; mixed disulfides; sporidesmin; thioltransferase Glutaredoxin (GRx, also known as thioltransferase) is a member of the thiol-disulfide oxidoreductase enzyme family, which also includes thioredoxin and their corresponding reductase enzymes GSSG reductase and thioredoxin reductase, respectively. Mammalian NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 October 24. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptGRx1 is a 12 kDa cytosolic protein that has been characterized in vitro as a specific catalyst for the reduction of protein-glutathionyl mixed disulfides (protein-SSG) (1-6). The reaction catalyzed by GRx1 is also selective for GSH as the reducing substrate (4). This thioldisulfide interchange reaction is likely crucial for maintaining intracellular thiol status (2-7) under a variety of physiological and pathophysiological processes like aging, cardiovascular and neurodegenerative di...

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Section: Methodsmentioning

confidence: 99%

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,

et al. 2006

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“…Activity Assay-Thiol-disulfide reductase activity was assayed by measuring the turbidity increase at 650 nm because of insulin reduction (33) and was expressed as a ratio of the slope of the linear part of the turbidity curve to the lag time (34). Oxidase activity was assayed by the oxidative reactivation of reduced and denatured RNase A (27).…”

Section: Methodsmentioning

confidence: 99%

The C-terminal (331–376) Sequence of Escherichia coli DnaJ Is Essential for Dimerization and Chaperone Activity

Shi

Hong

Wang

2005

Journal of Biological Chemistry

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DnaJ, an Escherichia coli Hsp40 protein composed of 376 amino acid residues, is a chaperone with thioldisulfide oxidoreductase activity. We present here for the first time a small angle x-ray scattering study of intact DnaJ and a truncated version, DnaJ (1-330), in solution. The molecular weight of DnaJ and DnaJ (1-330) determined by both small angle x-ray scattering and size-exclusion chromatography provide direct evidence that DnaJ is a homodimer and DnaJ (1-330) is a monomer. The restored models show that DnaJ is a distorted -shaped dimeric molecule with the C terminus of each subunit forming the central part of the , whereas DnaJ (1-330) exists as a monomer. This indicates that the deletion of the C-terminal 46 residues of DnaJ impairs the association sites, although it does not cause significant conformational changes. Biochemical studies reveal that DnaJ (1-330), while fully retaining its thiol-disulfide oxidoreductase activity, is structurally less stable, and its peptide binding capacity is severely impaired relative to that of the intact molecule. Together, our results reveal that the C-terminal (331-376) residues are directly involved in dimerization, and the dimeric structure of DnaJ is necessary for its chaperone activity but not required for the thiol-disulfide oxidoreductase activity.Hsp40 proteins collaborate specifically with Hsp70 proteins and some other factors to participate in a wide range of cellular processes essential to cell survival, such as assisting the folding, assembly, disassembly, and translocation of newly synthesized proteins, by suppressing aggregation or mediating degradation of misfolded proteins (1, 2). The Hsp40 family proteins are divided into three subtypes (3) (see Scheme 1). They all contain a J domain responsible for binding to the ATPase domain and stimulating the ATPase activity of Hsp70. Type I Hsp40 proteins, such as Escherichia coli DnaJ, yeast Yjd1, and human Hdj-2, have a Gly/Phe-rich region, which has been suggested to assist J domain interactions with Hsp70 (4). The Gly/Phe-rich region is followed by a conserved cysteine-rich region forming a zinc finger domain and a poorly conserved C-terminal domain. The peptide binding site(s) has been found to locate within these two domains (5-7). Type II Hsp40 proteins, such as yeast Sis1 and mammalian Hdj-1, also contain the J, a Gly/Phe-rich region, and C-terminal domains but without the zinc finger domain (8, 9). Both type I and type II Hsp40 proteins act as molecular chaperones to bind and deliver nonnative proteins to Hsp70 (7, 8, 10), but they are not functionally equivalent (11). Type III Hsp40 proteins, such as yeast YJL162c and virus T antigen, contain only the J domain, and they do not function as molecular chaperones (3).In addition to the DnaK-dependent co-chaperone activity described above, DnaJ also exerts autonomous DnaK-independent chaperone activity (9, 12, 13), which is characterized by its ability to bind with unfolded proteins and prevent them from irreversible aggregation (10, 11). Moreover, DnaJ has ...

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“…The insulin reduction method is based on the capacity of reduced Trx to reduce oxidized insulin, measuring the increase in turbidity at 650 nm for 1 h at 30°C as described by Martínez-Galisteo et al (1993). Trx was reduced by DTT or the NADPH/TR system as described by Laloi et al (2001) using the cytoplasmic NADPH/TR from wheat (Triticum aestivum) and the mitochondrial Trr2 from Saccharomyces cerevisiae.…”

Section: Trx Activitymentioning

confidence: 99%

Mitochondrial and Nuclear Localization of a Novel Pea Thioredoxin: Identification of Its Mitochondrial Target Proteins

et al. 2009

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Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

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Purification and properties of bovine thioredoxin system

Cited by 45 publications

References 31 publications

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,

The C-terminal (331–376) Sequence of Escherichia coli DnaJ Is Essential for Dimerization and Chaperone Activity

Mitochondrial and Nuclear Localization of a Novel Pea Thioredoxin: Identification of Its Mitochondrial Target Proteins

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Purification and properties of bovine thioredoxin system

Cited by 45 publications

References 31 publications

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones,

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones,

The C-terminal (331–376) Sequence of Escherichia coli DnaJ Is Essential for Dimerization and Chaperone Activity

Mitochondrial and Nuclear Localization of a Novel Pea Thioredoxin: Identification of Its Mitochondrial Target Proteins

Contact Info

Product

Resources

About

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,