Characterization Of Glutathione Peroxidase Research Articles

Characterization of Glutathione Peroxidase 4 in Rat Oocytes, Preimplantation Embryos, and Selected Maternal Tissues during Early Development and Implantation.

This study aimed to describe glutathione peroxidase 4 (GPx4) in rat oocytes, preimplantation embryos, and female genital organs. After copulation, Sprague Dawley female rats were euthanized with anesthetic on the first (D1), third (D3), and fifth days of pregnancy (D5). Ovaries, oviducts, and uterine horns were removed, and oocytes and preimplantation embryos were obtained. Immunohistochemical, immunofluorescent, and Western blot methods were employed. Using immunofluorescence, we detected GPx4 in both the oocytes and preimplantation embryos. Whereas in the oocytes, GPx4 was homogeneously diffused, in the blastomeres, granules were formed, and in the blastocysts, even clusters were present mainly around the cell nuclei. Employing immunohistochemistry, we detected GPx4 inside the ovary in the corpus luteum, stroma, follicles, and blood vessels. In the oviduct, the enzyme was present in the epithelium, stroma, blood vessels, and smooth muscles. In the uterus, GPx4 was found in the endometrium, myometrium, blood vessels, and stroma. Moreover, we observed GPx4 positive granules in the uterine gland epithelium on D1 and D3 and cytoplasm of fibroblasts forming in the decidua on D5. Western blot showed the highest GPx4 levels in the uterus and the lowest levels in the ovary. Our results show that the GPx4 is necessary as early as in the preimplantation development of a new individual because we detected it in an unfertilized oocyte in a blastocyst and not only after implantation, as was previously thought.

Stress-Based Production, and Characterization of Glutathione Peroxidase and Glutathione S-Transferase Enzymes From Lactobacillus plantarum.

More attention has been recently directed toward glutathione peroxidase and s-transferase enzymes because of the great importance they hold with respect to their applications in the pharmaceutical field. This work was conducted to optimize the production and characterize glutathione peroxidase and glutathione s-transferase produced by Lactobacillus plantarum KU720558 using Plackett-Burman and Box-Behnken statistical designs. To assess the impact of the culture conditions on the microbial production of the enzymes, colorimetric methods were used. Following data analysis, the optimum conditions that enhanced the s-transferase yield were the De Man-Rogosa-Sharp (MRS) broth as a basal medium supplemented with 0.1% urea, 0.075% H2O2, 0.5% 1-butanol, 0.0125% amino acids, and 0.05% SDS at pH 6.0 and anaerobically incubated for 24 h at 40°C. The optimum s-transferase specific activity was 1789.5 U/mg of protein, which was ~12 times the activity of the basal medium. For peroxidase, the best medium composition was 0.17% urea, 0.025% bile salt, 7.5% Na Cl, 0.05% H2O2, 0.05% SDS, and 2% ethanol added to the MRS broth at pH 6.0 and anaerobically incubated for 24 h at 40°C. Furthermore, the optimum peroxidase specific activity was 612.5 U/mg of protein, indicating that its activity was 22 times higher than the activity recorded in the basal medium. After SDS-PAGE analysis, GST and GPx showed a single protein band of 25 and 18 kDa, respectively. They were able to retain their activities at an optimal temperature of 40°C for an hour and pH range 4–7. The 3D model of both enzymes was constructed showing helical structures, sheet and loops. Protein cavities were also detected to define druggable sites. GST model had two large pockets; 185Å3 and 71 Å3 with druggability score 0.5–0.8. For GPx, the pockets were relatively smaller, 71 Å3 and 32 Å3 with druggability score (0.65–0.66). Therefore, the present study showed that the consortium components as well as the stress-based conditions used could express both enzymes with enhanced productivity, recommending their application based on the obtained results.

Hepatoprotective efficacy of Nigella sativa seeds dietary supplementation against lead acetate-induced oxidative damage in rabbit - Purification and characterization of glutathione peroxidase.

Lead (Pb) is a toxic ubiquitous environmental pollutant that induces hepatotoxicity in both animals and humans. The ability of Nigella saliva seeds (NSS) in ameliorating lead acetate (PbAc)-induced hepatic oxidative damage was investigated using a rabbit model. Forty New Zealand rabbits were given feed and water ad libitum. They were allocated randomly into four groups: control; PbAc (5g/L drinking water); NSS (20g/kg diet) and NSS+PbAc groups. After two months, liver samples were collected and analyzed for malondialdehyde (MDA), glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx) contents. Purification and characterization of GPx were also evaluated. PbAc exposure significantly (p<0.05) increased MDA (lipid peroxidation biomarker) and reduced the GSH levels and the GST and GPx activities. Concurrently supplemented NSS significantly (p<0.05) decreased MDA levels and restored the GSH, GST, and GPx contents successfully. Electrophoretically, the homogeneous GPx preparation from the liver had a specific activity of 30.44 U/mg protein and a yield of 1.31%. The Km values for cumene hydroperoxide were 4.76μM in control, PbAc and NSS+PbAc groups, and 4.09μM in NSS group. The GPx reaction had a temperature optimum 40°C, pH optimum 8 and molecular weight 21 kDa. The obtained data indicated the potent efficacy of NSS against PbAc-induced oxidative stress; that was mediated through induction and activation of antioxidants, particularly GPx and scavenging free radicals. Moreover, the purified hepatic GPx is characterized as a selenoprotein (Se-GPx).

Purification and characterization of glutathione peroxidase 1 in the red muscle of Pacific bluefin tuna Thunnus orientalis

Glutathione peroxidase GPx1 was purified from the red muscle of Pacific bluefin tuna Thunnus orientalis and its enzymatic properties were characterized. The enzyme was optimally active at pH 7.4 with a specific activity for hydrogen peroxide and a K m of 6.7 μM. cDNA was also isolated and it contained a predicted open reading frame (ORF) encoding a 188 amino acid protein. The phylogenetic tree shows that fish including Pacific bluefin tuna, pufferfish, and zebrafish, but not mammals, possess two genetically distinct types of GPx1, i.e., GPx1a and GPx1b. The purified enzyme was classified as a fish GPx-1b enzyme on the basis of the phylogenetic tree of the GPx1 family.

Localization and characterization of glutathione peroxidase (GPx) in boar accessory sex glands, seminal plasma, and spermatozoa and activity of GPx in boar semen

Boar ejaculate owes its characteristic large volume mainly to accessory sex gland (ASG) secretions. These are main contributors to the protective functions of seminal plasma, especially against oxidative damage. Numerous antioxidants have been detected in ASG secretions, and, respectively, in seminal plasma. However, as regards one key antioxidant protector -- the Se-dependent enzyme glutathione peroxidase (GPx) -- there is no agreement yet among researchers as to its presence in boar seminal plasma. Nevertheless, the beneficial effect of dietary Se supplementation on male fertility has been widely recognized. The aim of the present study was to investigate the localization and characterization of GPx in boar ASGs, seminal plasma, and spermatozoa, as well as to evaluate GPx activity in boar semen. Immunohistochemical assays demonstrated GPx presence in the epithelial cells, vacuole membranes, and vascular endothelium of boar seminal vesicle, prostate and bulbourethral glands. Western blot analysis demonstrated the presence of a monomer form of GPx with MW 20 kDa in lysates from seminal vesicle, prostate, bulbourethral glands, and spermatozoa, but not in seminal plasma. Surprisingly, peroxidase activity detected in seminal plasma from normal ejaculates was nearly three times as high as in spermatozoa. Our findings confirmed the presence of immunoreactive GPx in the boar reproductive tract, while further investigation is still warranted to uncover the exact protein forms involved and their function.

Purification and characterization of glutathione peroxidase from human blood platelets. Age-related changes in the enzyme

Platelet glutathione peroxidase (GPx) is known to play a pivotal role in controlling the level of lipid hydroperoxides, especially those resulting from the 12-lipoxygenase activity. GPx was purified fromm the cell cytosol by more than 700-fold using an exchange chromatography, FPLP, gel filtration and covalent fixation. Isoelectric focusing revealed a peak activity at pH 5.1. The molecular mass of enzyme was found between 90 and 100 kDa by gel filtration, and was approximating at 23kDa by SDS-PAGE. A polyclonal antibody raised against commercial bovine erthrocyte GPx recognized the human platelet enzyme. It is concluded that human platelet GPx is likely a homotetramer of 92 kDa as described for most sources. We have also found that the decreased platelet GPx activity observed in platelets from elederly people is associated with a lower content of the immunoreactive enzyme.

Recent Developments in Trace Element Metabolism and Function: Newer Roles of Selenium in Nutrition

Until recently, studies of the function of selenium focused on the selenoenzyme, glutathione peroxidase. However, the recognition that several metabolic effects of selenium are not associated with glutathione peroxidase has forced a re-evaluation of the function of this enzyme and the element. Hepatic glutathione peroxidase contains a significant percentage of the regulated selenium in the rat and is more sensitive to selenium deficiency than other selenoproteins. Thus, in addition to its enzymatic activity, it might have a storage function for the element. Another selenoprotein, designated selenoprotein P, has been found in rat plasma and has been quantitated. Its function is not yet known, but it has been postulated to be a transport protein for selenium and a defense against oxidant stress. Understanding the nutritional effects of selenium will require better characterization of glutathione peroxidase, selenoprotein P and other selenoproteins.