Total Glutathione Reductase Activity Research Articles

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice.

Glutathione reductase (GR) is one of important antioxidant enzymes in plants. This enzyme catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) with the accompanying oxidation of NADPH. Previously, we showed that salt-stress-responsive GR3 is a functional protein localized in chloroplasts and mitochondria in rice. To learn more about the role of GR3 in salt-stress tolerance, we investigated the response to 100mM NaCl treatment in wild-type rice (WT); GR3 knockout mutant of rice (gr3); and the functional gr3-complementation line (C1). Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitously expressed in all tissues except the sheath at heading stage. GR3 promoter-GUS was expressed in the vascular cylinder and cortex of root tissues in rice seedlings, vascular tissue of nodes, embryo and aleurone layer of seeds, and young flowers. Under both normal and salt-stress conditions, total GR activity was decreased by 20% in gr3. Oxidative stress, indicated by malondialdehyde content, was greater in gr3 than the WT under salt stress. As compared with the WT, gr3 was sensitive to salt and methyl viologen; it showed inhibited growth, decreased maximal efficiency of photosystem II, decreased GSH and GSSG contents, and the ratio of GSH to GSSG. Conversely, the gr3-complementation line C1 rescued the tolerance to methyl viologen and salinity and recovered the growth and physiological damage caused by salinity. These results reveal that GR3 plays an important role in salt stress tolerance by regulating the GSH redox state in rice.

Differential Responses of Antioxidative System to Soil Water Shortage in Barley (<i>Hordeum vulgare</i> L.) Genotypes

Drought is one of the major factors limiting the yield and quality of crops in the world. The activity of antioxidative system to tolerate the drought stress is significant in plants. In the present study, the activities and isoform profiles of catalase (CАТ), ascorbate peroxidase (APX), glutathione reductase (GR), and superoxide dismutase (SOD) were analyzed in four barley genotypes grown under soil water restriction. Drought stress caused increase in the activities of CАТ and SOD in all studied genotypes, while APX activity decreased. The total GR activity increased substantially in genotypes K 2778 and St.Garabag 7 and decreased in No. 77 local and St.Pallidum 596 genotypes under conditions of severe water stress. No detectable differences were observed in the isoenzyme pattern (the appearance of a new isoenzymes and disappearance of another one) between control plants and those subjected to soil drought. However, intensification of corresponding isoforms in electrophoretic spectra was observed in stressed barley leaves relative to watered ones. The obtained results possibly suggest that antioxidant protection in barley plants under drought conditions could be attributed mainly to SOD and CAT.

The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis

Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that approximately 65% of the total GR activity is attributed to GR1, whereas approximately 35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E(GSH)) in the cytosol. Using this tool, it is shown that E(GSH) in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.

Antioxidant defence enzyme activities in hepatopancreas, gills and muscle of Spiny cheek crayfish ( Orconectes limosus) from the River Danube

The aim of our study was to determine the activity of antioxidant defence (AD) enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione- S-transferase (GST) in the hepatopancreas, the gills and muscle of Spiny cheek crayfish ( Orconectes limosus) from the River Danube and to compare tissue specificities of investigated enzymes. Our results indicated that both specific and total SOD activities in the hepatopancreas were lower compared to the gills and muscle. Total SOD activity in the gills was lower with respect to that in muscle. CAT and GSH-Px (both specific and total) activities were higher in the hepatopancreas compared to those in the gills and muscle. In the gills the specific and total GR activities were higher than in the hepatopancreas and muscle. The specific and total GST activities were higher in the hepatopancreas compared with the gills and muscle. Our study represents the first comprehensive report of AD enzymes in tissues of O. limosus caught in the River Danube. The noted tissue distributions of the investigated AD enzyme activities most likely reflected different metabolic activities and different responses to environmental conditions in the examined tissues.

Glutathione dependent enzyme activities in the foot of three freshwater mussel species in the Sava river, Serbia

We investigated activities of glutathione peroxidase (GSH-Px), glutathione reductase (GR), and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the foot of three freshwater mussel species: Unio pictorum (Up), Unio tumidus (Ut), and Sinanodonta woodiana (Sw) from the Sava River. Specific and total GSH-Px activity was lower in Sw than in Up and Ut. Total GR activity was higher in Up than in Sw. Specific GST activity was higher in Up than in Ut. Total GST activity was higher in Up than in Ut and Sw. Electrophoretic analysis of proteins shows species specifities between the investigated mussel species. Our study represents the first comprehensive report of the investigated glutathione-dependent enzyme activities in the foot of three freshwater mussel species from the Sava River, Serbia. .

Photoinhibition of photosystem II in tobacco plants overexpressing glutathione reductase and poplars overexpressing superoxide dismutase

We studied photoinhibition in two cultivars of tobacco (Nicotiana tabacum L.) expressing the bacterial gor gene in the cytosol and in four lines of poplar (Populus tremula×P. alba) expressing the FeSOD gene of Arabidopsis thaliana in the chloroplast. The respective total activities of glutathione reductase (EC 1.6.4.2) in leaves of gor tobaccos and superoxide dismutase (EC 1.15.1.1) in the FeSOD poplars were 5–8 times higher than in the respective untransformed control plants. Leaves of control and transformed plants were subjected to high‐light stress at 20°C, and photoinhibition of photosystem II (PSII) was measured by oxygen evolution and chlorophyll fluorescence. The leaves were illuminated both in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis. In both cases, the time course of loss of PSII activity was identical in plants overproducing superoxide dismutase (SOD) and in the untransformed controls, suggesting that the ability to convert superoxide to hydrogen peroxide is not a limiting factor in protection against photoinhibition, or in the repair of photoinhibitory damage or that the site of O2− production is not accessible to the transgene product. The rate constant of photoinhibition, measured in lincomycin‐treated leaves, was smaller in glutathione reductase (GR) overproducing tobacco cv. Samsun than in the respective wild‐type, but this difference was not seen in cv. Bel W3. The steady‐state level of PSII activity measured when the PSII repair cycle was allowed to equilibrate with photoinhibitory damage under high light was not higher in the GR overproducing cv. Samsun, suggesting that the repair of photoinhibitory damage was not enhanced in plants overproducing GR in the cytosol.

Regulatory Mechanism of Glutathione Reductase Activity in Human Red Cells

Abstract The mechanism by which glutathione reductase (GR) activity is regulated in relation to flavin metabolism was studied in red cells of normal adults, cord blood, and patients with severe metabolic disorders using spectrophotometric, fluorometric, and radiochemical methods. The increased activity of GR in red cells of adult patients with severe hepatic cirrhosis, chronic uremia, and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is related to the increased per cent saturation of GR with flavin adenine dinucleotide (FAD), and increased red cell flavin level. The per cent saturation of GR with FAD correlates with (1) the degree of clinical severity in hepatic cirrhosis or chronic uremia, and (2) total flavin level in red cells. Thus, the increased GR activity in these patients may be regulated actively or passively by the increased flavin level in red cells. This suggests that the increased requirement for enhanced activity of the pentose phosphate pathway may affect GR metabolism secondarily, leading to the association of GR with FAD through an increased uptake of riboflavin. In contrast to the results in adult red cells, cord erythrocytes show an unexpected metabolic pattern of GR and flavin metabolism. Although the total GR activity of cord red cells is considerably higher than in adult red cells, cord red cell GR is only partly saturated with FAD even in the presence of a significantly increased flavin level in cord red cells. Thus, cord red cells may have an additional control mechanism of GR activity.

Studies on glutathione reductase and regeneration of reduced glutathione in normal human adult and cord red cells

A comparative study of the kinetic characteristics of glutathione reductase and regeneration of reduced glutathione in normal adult and cord red cells was performed. After treatment of intact cord red cells with methyl phenylazoformate, a striking initial delay and a significantly decreased regeneration of reduced glutathione at low temperatures were observed in cord red cells in spite of increased glutathione reductase activity assayed in cord hemolysates.The Michaelis constants of glutathione reductase for reduced nicotinamide adenine dinucleotide phosphate and oxidized glutathione in cord hemolysates were similar to that in adult hemolysates. Maximum glutathione reductase activity was obtained at pH 7.2 and pH 6.8 in cord blood and in adult red cells, respectively.The active form constituted 79% and the inactive form 21% of glutathione reductase in cord hemolysates compared to 68 and 32%, respectively, in adult cells at 37°. Total glutathione reductase activity in cord hemolysates was 1.5 times higher than in adult hemolysates.The functionally lower activity of glutathione reductase in cord blood suggests that the enzyme may be a different one from that of adult red cells.