Further electrophoretic studies of erythrocyte glutathione peroxidase.

A new, inexpensive technique has been devised for the detection of glutathione peroxidase after electrophoresis. Surveys of several racial groups have revealed new variant phenotypes at polymorphic frequencies in African and Lebanese populations.     

Age-associated changes in erythrocyte glutathione peroxidase activity: correlation with total antioxidant potential

Oxidative stress is believed to play a central role in aging and age-associated diseases. It leads to oxidative changes in human red blood cells (RBCs) in vivo and in vitro. In this study, we evaluated the oxidative damage to the erythrocytes during aging in the humans using RBC as a model, by measuring the cytosolic antioxidant enzyme glutathione peroxidase (GPx) activity. GPx activity was found to be significantly decreased as a function of human age and positively correlated with total antioxidant capacity, while negatively correlated with SOD activity. Thus, results of the present study showed involvement of oxidative stress as one of the risk factors, which can initiate and/or promote human aging.     

Changes in erythrocyte glutathione peroxidase and glutathione reductase in alloxan diabetes

Glutathione peroxidase and glutathione reductase activities were measured in erythrocytes from control, diabetic and insulin-treated diabetic rats. A significant decrease in the activity of glutathione peroxidase and an increase in the glutathione reductase activity were found with increase in the time of diabetes which may result in the alteration in the activity of the pentose phosphate pathway by the modulation of the levels of NADPH. Insulin administration reverses the change in the activity of glutathione peroxidase but does not reverse the glutathione reductase activity during diabetes. The overall changes may be due to changes in the levels of insulin, triiodothyronine and thyroxine.     

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.     

Inhibitory effect of selenium and change of glutathione peroxidase activity on rat glioma

The inhibitory effect of selenium (Se) and change of glutathione peroxidase activity during the development of brain tumors was investigated in Wistar rats. Four rat groups classified to match by age and weight were fed a diet containing 0, 0.5, 2.0, and 4.0 μg Se/g. After 6 wk, the rats were injected with 3×10⁶ C₆ cells into the right frontal lobe parenchyma. Survival was significantly longer in the 0.5 and 2.0 μg Se/g groups than in the Se-free and 4.0 μg Se/g groups. The activity of glutathione peroxidase after development of tumors was significantly higher in the high Se group at 18 and 30 d.     

Effect of GP120 on glutathione peroxidase activity in cortical cultures and the interaction with steroid hormones

GP120 (the protein component of the HIV viral coat) is neurotoxic and may contribute to the cell loss associated with AIDS-related dementia. Previously, it has been shown in rat cortical mixed cultures that gp120 increased the accumulation of hydrogen peroxide and superoxide, two reactive oxygen species (ROS). We now demonstrate that gp120 increased activity of the key antioxidant glutathione peroxidase (GSPx), presumably as a defensive mechanism against the increased ROS load. Both estrogen and glucocorticoids (GCs), the adrenal steroid released during stress, blunted this gp120 effect on GSPx activity. The similar effects of estrogen and of GCs are superficially surprising, given prior demonstrations that GCs exacerbated and estrogens protected against gp120 neurotoxicity. We find that these similar effects of estrogen and GCs on GSPx regulation arose, in fact, from very different routes, which are commensurate with these prior reports. Specifically, estrogen has demonstrated antioxidant properties that may prevent the ROS increase (therefore acting as a neuroprotective agent) and rendered unnecessary the compensatory GSPx increased activity. To verify this we have added H2O2 to estrogen + gp120-treated cells, and GSPx activity was increased. However, with addition of H2O2 to GCs + gp120-treated cells there was no increase in activity. GCs appeared to decrease enzyme production and or activity and therefore under insult conditions ROS levels rose in the cell resulting in increased neurotoxicity. Overexpression of GSPx enzyme via herpes vector system reversed the GCs-induced loss of enzyme and eliminated the GCs exacerbation of gp120 neurotoxicity.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Inhibitory effect of phospholipid hydroperoxide glutathione peroxidase on the activity of lipoxygenases and cyclooxygenases

The partially purified phospholipid hydroperoxide glutathione peroxidase (PHGPx) from A431 cells was used to systematically compare the inhibitory effect on the enzyme activity of various lipoxygenases and cyclooxygenases. Under the standard assay system, platelet 12-lipoxygenase, 15-lipoxygenase, and cyclooxygenase-2 were the most sensitive to the inhibition by PHGPx. 5-Lipoxygenase and cyclooxygenase-1 were less sensitive to the inhibition by PHGPx than platelet 12-lipoxygenase and cyclooxygenase-2, respectively, and the difference was approximately 10-fold. Reduction of 12(S)-hydroperoxyeicosatetraenoic acid to 12(S)-hydroxyeicosatetraenoic acid by PHGPx was observed in the presence of glutathione (GSH), and the inhibitory effect of PHGPx on 12-lipoxygenase-catalyzed arachidonate metabolism was reversed by the addition of exogenous lipid hydroperoxide. The results indicate that PHGPx directly reduced lipid hydroperoxides and then down-regulated the activity of arachidonate oxygenases. Moreover, a high-level expression of PHGPx mRNA and its 12-lipoxygenase-inhibitory activity was observed in cancer cells and endothelial cells, and these results suggest that PHGPx may play a significant role in the regulation of reactive oxygen species formation in these cells.     

Plasma and erythrocyte selenium and glutathione peroxidase activity in preterm infants at risk for bronchopulmonary dysplasia

The purpose of this study was to examine the relationships between selenium status, as measured by plasma and erythrocyte selenium and glutathione peroxidase (GPx) activity, and other postnatal factors, including selenium intake, gestational age, and oxygen dependence in preterm infants at risk for bronchopulmonary dysplsia. Eighteen preterm infants of 30 wk gestational age or less were included. At postnatal wk 1 and 4, selenium concentrations and GPx activity were measured and oxygen dependence and daily selenium intakes were determined from the medical chart. Plasma and erythrocyte selenium concentrations decreased from wk 1 to wk 4, whereas erythrocyte GPx activity increased. Increased selenium intakes during wk 1 were associated with increased erythrocyte GPx activity at both time-points, as well as a decreased need for supplemental oxygen on d 28. Preterm infants display increasing erythrocyte GPx activity despite declines in plasma and erythrocyte selenium. GPx activity might be enhanced by very early selenium supplementation.     

77Se NMR characterization of 77Se-labeled ovine erythrocyte glutathione peroxidase

Lambs, maintained on a selenium-deficient diet supplemented with 94 atom % Na2 27SeO3, have been used as a source of 77Se-enriched erythrocyte glutathione peroxidase. After 5 months on this diet, the percentage of selenium in the enzyme derived from the supplement had reached 88%. From each monthly bleeding of two sheep, approximately 20 mg of 77Se-enriched glutathione peroxidase could be isolated in pure form. Although attempts to observe 77Se NMR signals from the native enzyme labeled with 6,6'-[77Se]diselenobis-(3-nitrobenzoic acid) failed, due to the low solubility of the enzyme, two 77Se resonances were observed after unfolding the enzyme with 8 M urea and reaction with iodoacetamide. These resonances, at 195 and 377 ppm, were from the selenoether alkylamide derivative and from protein cross-linked selenide sulfide species, respectively. Relaxation time measurements on the selenoether at 4.7 and 9.4 teslas enabled an estimate of the chemical shift anisotropy to be made. A value of less than or equal to 262 ppm was determined. Reduction of the denatured selenide sulfide species with dithiothreitol gave an observable 77Se resonance from the Se- moiety at pH 8 and from SeH at pH 4.2. The chemical form of the selenocysteine residue in the resting state enzyme most consistent with formation of the acetamide derivative and the selenide sulfide is Se- or SeH. From the magnitudes of the estimated chemical shift anisotropies, it is predicted that direct observation of selenium in the native enzyme will be feasible if the enzyme concentration can be increased to 0.25 mM tetrameric glutathione peroxidase.     

Population study of erythrocyte glutathione reductase activity

Summary The activity of erythrocyte glutathione reductase (GR) was determined in a group of 87 prisoners from northern Thailand (65 with normal, 22 with deficient erythrocyte G-6-PD) without and with added FAD. The amount of stimulation by FAD was inversely related to the original activity suggesting that FAD stimulation in vivo is one of the main determinants of GR activity. 4 subjects showed insufficient stimulation by FAD. The binding of FAD to GR seemed to be closer to saturation in G-6-PD deficient subjects but the maximal stimulated activity of these subjects was higher than in the group with normal G-6-PD. This suggests that the marked increase of GR activity in G-6-PD deficient erythrocytes is due to increased binding of FAD and to a larger amount of stimulable enzyme. Original GR activity was positively correlated with the socio-economic status, and subjects who had taken riboflavin in the period prior to examination had a higher mean GR activity than those without vitamin intake. GR activity was markedly raised by administration of riboflavin and in G-6-PD deficient subjects glutathione stability was improved. In comparison to central European subjects more than 50 percent of the examined population are GR deficient. This seems to be due to a low dietary supply of riboflavin in most cases. The findings in 4 subjects with insufficient stimulation by added FAD raise the question whether hereditary forms of GR deficiency exist in this population.

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Zusammenfassung In einer Gruppe von 87 Strafgefangenen in Nordthailand (65 mit normaler, 22 mit defizienter G-6-PD) wurde die Aktivität der Glutathion-Reductase (GR) mit und ohne Zusatz von FAD bestimmt. Das Ausmaß der Stimulierung des Enzyms durch FAD verhielt sich reziprok zur ausgangsaktivität. Bei 4 Versuchspersonen war die Stimulierung durch FAD insuffizient. Die Bindung von FAD an GR war bei Versuchspersonen mit G-6-PD-Mangel näher am Sättigungspunkt. Aber auch die gesamte stimulierbare Aktivität der GR war bei Versuchspersonen mit G-6-PD-Mangel größer als bei Gesunden. Die deutliche Erhöhung der GR-Aktivität bei G-6-PD-Mangel scheint zwei Ursachen zu haben: vermehrte Bindung von FAD an das Enzym und Vermehrung der Gesamtmenge an stimulierbarem Enzym. In einer weiteren Gruppe war die Ausgangsaktivität der GR vom sozio-ökonomischen Status der Versuchsperson abhängig. Personen, die vor der Untersuchung Riboflavin-haltige Medikamente eingenommen hatten, hatten höhere Aktivitäten. Die GR-Aktivität wurde durch Einnahme von Riboflavin unter Kontrolle stark erhöht, und bei Versuchspersonen mit G-6-PD-Mangel wurde die Glutathion-Stabilität der Erythrocyten verbessert. Im Vergleich zu Mitteleuropäern sind mehr als die Hälfte der thailändischen Versuchspersonen als GR defizient einzustufen. Dies ist wahrscheinlich auf eine geringere Zufuhr an Riboflavin in der Nahrung zurückzuführen. Die Befunde bei 4 Versuchspersonen mit insuffizienter Stimulierung der GR durch FAD könnte durch einen erblichen Mangel an GR verursacht sein.

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Established and supported by Stiftung Volkswagenwerk.     

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.     

Human catalytic antibodies with glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/micromol and 28.8U/micromol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied.     

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K_M of 2.4 μM for human thioredoxin and a very low K_M for H₂O₂ (?0.7 μM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H₂O₂ or peroxynitrite, were determined by competition kinetics, k₂ = 1.0 × 10⁸ and 1.4 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K_d < 10⁻²³ M⁴ which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.     

Effects of concurrent administration of monensin and selenium on erythrocyte glutathione peroxidase activity and liver selenium concentration in broiler chickens

Different toxic doses of selenium and monensin preparations were administered to broiler chickens. The two substances were given by oral route, alone or concurrently, for variable periods. Erythrocyte glutathione peroxidase. (GSH-Px) activity was found to be elevated after the administration of the drugs. This increase was considerably higher when selenium and monensin were administered concurrently, indicating the occurrence of strong interaction between them. Administration of selenium led to a rapid increase in the liver selenium concentration. This increase, in turn, was enhanced by concurrent application of monensin. Monensin given alone did not have any significant effect on the changes of liver selenium concentration. Further results suggest that administration of monensin increases erythrocyte GSH-Px activity, even in the absence of supplemental selenium or during increased liver selenium concentration.     

Vanadium effect on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase in vitro.

The effect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase has been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 microM). No significant inhibitory effects were found when V(V) was tested with catalase and when either V(IV) or V(V) were assayed with glutathione peroxidase. For the latter, the effect of V on the different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent inhibitory dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with the metal. Besides, no significant binding of V(IV) or V(V) to the enzyme could be demonstrated. The lack of a direct inhibitory effect of V on the activity of the main antioxidant enzymes suggests that many biological and toxicological effects of V may be mediated more by oxidative reactions of the metal or of its complexes with physiologically relevant biomolecules than by a direct modulation of enzymatic activities.