Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase

Yadav, Sudhanshu S.; Elesela, Srikanth; Singh, Neetu; Rathaur, Sushma

doi:10.1016/j.parint.2012.12.008

Cited by 19 publications

(6 citation statements)

References 43 publications

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“…N-ethylmaleimide (NEM), a potent thiol modifier, has been used to inhibit human glutathione reductase [34, 49]. Figure 4D shows that the NEM-treated human glutathione reductase failed to reduce the mitoNEET [2Fe-2S] clusters (spectrum 2).…”

Section: Resultsmentioning

confidence: 99%

“…E. coli succinic semialdehyde dehydrogenase [38] and 2,4-dienoyl-CoA reductase [39], which use NADPH as substrate but do not have a redox active disulfide in the catalytic center, also fail to reduce the mitoNEET [2Fe-2S] clusters (data not shown). Furthermore, modification of the redox active disulfide in human glutathione reductase by thiol modifier NEM [34, 49] completely inhibits the enzyme activity to reduce the mitoNEET [2Fe-2S] clusters (Figures 4 and 5). Taken together, we conclude that the redox active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe-2S] clusters (Figure 8).…”

Section: Discussionmentioning

confidence: 99%

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Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

Landry

Cheng

Ding

2015

Free Radical Biology and Medicine

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Human mitochondrial outer membrane protein mitoNEET is a newly discovered target of type II diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane alpha helix tethered to mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox active [2Fe-2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe-2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe-2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters, indicating that the redox active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe-2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe-2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe-2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

Landry

Cheng

Ding

2015

Free Radical Biology and Medicine

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“…GR was purified from C. trutta gill and liver by using preparation of hemolysate, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. GR has been purified from many different sources (Calberg and Mannervik, 1981;Le Trang et al, 1983;Akkemik et al, 2011;Taser and Ciftci, 2012;Yadav et al, 2013) using various purification procedures. All reported purification procedures involve several chromatographic steps, such as, DEAE-Sephadex, Sephadex G-100, hydroxyapatite (Calberg and Mannervik, 1981), 2',5'-ADP Sepharose 4B (Madamanchi et al, 1992), Sephadex G-75, CM-Cellulose, Sephacryl S-200 (Calberg et al, 1981), Reactive Red-120-Agarose, Sephacryl S-300 (Garcia-Alfonso et al, 1993), fast protein liquid chromatography (FPLC)-anion Exchange and FPLC-hydrophobic interaction chromatography (Madamanchi et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

In vitro effects of some metal ions on glutathione reductase in the gills and liver of Capoeta trutta

Atamanalp

Kırıcı

Beydemır

2017

Regul. Mech. Biosyst.

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Many aquatic environmental problems have arisen in consequence of contamination of water by toxic metals and organic pollutants in the present age of technology. Metals play vital roles in enzyme activities and other metabolic events due to their bioaccumulative and nonbiodegradable properties among aquatic pollutants. The aim of this study was to evaluate the inhibitory effects of some metal ions (Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+) on Capoeta trutta gill and liver glutathione reductase (EC: 1.8.1.7; GR). For this purpose, initially, GR was purified from C. trutta gill and liver. Purification procedure consisted of three steps; preparation of hemolysate, ammonium sulphate precipitation and 2’, 5’-ADP Sepharose 4B affinity chromatography. Using this procedure, C. turtta gill GR, having the specific activity of 19.111 EU/mg proteins, was purified with a yield of 38.8% and 910.05-fold; C. trutta liver GR, having the specific activity of 16.167 EU/mg proteins, was purified with a yield of 21.1% and 734.86-fold. The purity of the enzymes was checked on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and each purified enzyme showed a single band on the gel. In addition, inhibitory effects of some metal ions (Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+) on GR from gill and liver were investigated in vitro. Ki constants and IC50 values for metal ions which showed inhibition effects were determined by Lineweaver-Burk graps and plotting activity % vs. [I]. In conclusion, IC50 values for fish gill GR were 0.000625, 0.153, 0.220, 0.247 and 0.216 mM and Ki constants for fish gill GR were 0.00045 ± 0.00008, 0.128 ± 0.036, 0.182 ± 0.138, 0.482 ± 0.219 and 0.112 ± 0.047 mM for Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+, respectively. IC50 values for fish liver GR were 0.000437, 0.217, 0.185, 0.355 and 0.349 mM and Ki constants for fish liver GR were 0.00025 ± 0.00013, 0.532 ± 0.146, 0.123 ± 0.066, 0.093 ± 0.020 and 0.151 ± 0.084 mM for Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+, respectively. In vitro inhibition rank order was determined as Ag+ > Co2+ > Zn2+ > Ni2+ > Pb2+ for fish gill GR; Ag+ > Cu2+ > Co2+ > Pb2+ > Ni2+ for fish liver GR. From these results, we showed that Ag+ metal ion is the most potent inhibitor of GR enzyme on gill and liver tissues.

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“…The solution was homogenized and 0.2 µm filtered prior to loading onto a column with 2′5′ ADP Sepharose 4B resin. The reductases were eluted using a linear increase to 0.3 mM NADP + in the same tris buffer (Yadav et al, ). The reductase activities of the collected fractions were determined using the above described method.…”

Section: Methodsmentioning

confidence: 99%

Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing

Handlogten¹,

Zhu

Ahuja³

2017

Biotech & Bioengineering

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Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG , IgG , and IgG respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction mitigation strategies targeted at the dominant reduction mechanism. Biotechnol. Bioeng. 2017;114: 1469-1477. © 2017 Wiley Periodicals, Inc.

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Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase

Cited by 19 publications

References 43 publications

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

In vitro effects of some metal ions on glutathione reductase in the gills and liver of Capoeta trutta

Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing

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