Acetaldehyde does not inhibit glutathione peroxidase and glutathione reductase from mouse liver in vitro

Acetaldehyde, the primary ethanol metabolite, has been implicated in the pathogenesis of alcoholic liver disease, but the mechanism involved is still under investigation. This study aims at the search for direct in vitro effects of different concentrations of acetaldehyde (30, 100 and 300microM) on the activities of glutathione reductase (GR), glutathione peroxidase (GPx) from liver supernatants, and the thiol-peroxidase activity of ebselen. They did not change after pre-incubation with acetaldehyde, which suggests that acetaldehyde does not have any direct effect. Nor were direct effects of acetaldehyde toward thiols, such as dithioerythritol and glutathione (GSH), observed either, even though GSH - measured as non-protein thiols from liver supernatants - were oxidized in the presence of acetaldehyde. In addition, acetaldehyde (up to 300microM) significantly oxidized GSH when incubated in the presence of commercially available gamma-glutamyltranspeptidase (GGT), but not in the presence of glutathione-S-transferase. The interaction between ebselen and GSH was also evaluated in an attempt to better understand the possible link between acetaldehyde and nucleophilic selenol groups. The formation and stability of ebselen intermediaries, produced in the chemical interaction between GSH and ebselen, were not affected by acetaldehyde either. Overall, the acetaldehyde oxidation of hepatic low-molecular thiols depends on mouse liver constituents and GGT is proposed as an important enzyme involved in this phenomenon. Thiol depletion, a phenomenon usually observed in the livers of alcoholic patients, can be related to GSH metabolism, and the involvement of GGT may reflect a molecular mechanism involved in thiol oxidation.     

Purification and characterization of an estrogen-binding peroxidase from human fetuses.

A peroxidase found under two forms with a molecular weight of 220,000 and 170,000 respectively, was purified from human fetuses. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, gel filtration and hydrophobic interaction chromatography. The purification factor approximated 400. These two forms of peroxidase were found to be immunologically identical as shown when utilizing immunodiffusion. They were able to bind estradiol in the presence of H2O2. This bond resisted to denaturation and solvent extraction therefore suggesting a covalent binding of estradiol to the enzyme.     

A supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities

For constructing a bifunctional antioxidative enzyme with both superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, a supramolecular artificial enzyme was successfully constructed by the self-assembly of the Mn(III)meso-tetra[1-(1-adamantyl methyl ketone)-4-pyridyl] porphyrin (MnTPyP-M-Ad) and cyclodextrin-based telluronic acid (2-CD-TeO₃H) through host-guest interaction in aqueous solution. The self-assembly of the adamantyl moieties of Mn(III) porphyrin and the β-CD cavities of 2-CD-TeO₃H was demonstrated by the NMR spectra. In this supramolecular enzyme model, the Mn(III) porphyrin center acted as an efficient active site of SOD and tellurol moiety endowed GPx activity. The SOD-like activity (IC₅₀) of the new catalyst was found to be 0.116 μM and equals to 2.56% of the activity of the native SOD. Besides this, supramolecular enzyme model also showed a high GPx activity, and a remarkable rate enhancement of 27-fold compared to the well-known GPx mimic ebselen was observed. More importantly, the supramolecular artificial enzyme showed good thermal stability.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass approximately = 20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/microg of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite.     

Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzyme

Glutathione peroxidase (GSHPx), (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) was purified to homogeneity from human plasma. This resulted in a 6800-fold purification of the enzyme with a 2.8% yield. The purification process involved ammonium sulfate fractionation, DEAE-cellulose batch and column chromatographies, hydroxyapatite, and Sephadex G-200 and DEAE-Sephadex A-25 chromatographies. The major peak on DEAE-Sephadex A-25 column chromatography was found to be homogeneous on polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Relative mobility in nondenaturing polyacrylamide gel electrophoresis at pH 8.2 was 0.5 for the purified enzyme as detected by both protein staining and enzyme activity compared with 0.38 for erythrocyte GSHPx. The molecular weight of the plasma enzyme as determined by gel filtration was found to be approximately 100,000. SDS-gel electrophoresis of the plasma enzyme gave a subunit molecular weight of approximately 23,000. This suggests that the plasma enzyme exists as a tetramer in its native state, similar to that seen for the erythrocyte enzyme, but with slightly different mobility on SDS-gel electrophoresis. Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein. Utilizing iodinated concanavalin A, it was found that plasma GSHPx, but not the erythrocyte GSPx, is a glycoprotein. Purified plasma enzyme catalyzes both the reduction of tertiary butyl hydroperoxide and hydrogen peroxide. The apparent Km of plasma GSHPx for GSH is 5.3 mM and for tertiary butyl hydroperoxide it is 0.57 mM. Copper, mercury, and zinc strongly inhibit the enzyme activity of plasma GSHPx. Rabbit antibodies directed against the human erythrocyte GSHPx do not precipitate the enzyme activity of the purified plasma enzyme. Radioimmunoassay utilizing erythrocyte GSHPx and anti-erythrocyte GSHPx antibodies showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHPx from plasma.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37°C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H₂O₂ and HbO₂, may be related to the pathogenesis of hemolysis in favism.     

A trifunctional enzyme with glutathione S-transferase,glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Purification and properties of glutathione peroxidase from human placenta.

Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.     

In vitro synthesis of glutathione peroxidase from selenite Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [⁷⁵Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [⁷⁵Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [³H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and ⁷⁵Se was incorporated from [⁷⁵Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the ⁷⁵Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [⁷⁵Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase     

人肝谷胱甘肽过氧化物酶纯化及性质研究

经硫酸铵分级沉淀,离子交换层析和凝胶过滤等步骤,从人肝中获得了ＰＡＧＥ单一条带的谷胱甘肽过氧化物酶,比活提高１２０倍,得率为２５％。凝胶过滤法测得分子量为９０９８０,ＳＤＳ－ＰＡＧＥ测定亚基分子量为２２４２３．原子吸收法测得每分子酶含有四个硒原子。等电聚焦显示该酶等电点为５．０．酶活力的最适ｐＨ为８．５,最适温度为３７℃。动力学实验提示该酶作用机理属于乒乓机制型。     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

New strategies for the isolation and activity determination of naturally occurring type-4 glutathione peroxidase

Type 4 glutathione peroxidase (GPx4) is a widely expressed mammalian selenoenzyme known to play a vital role in cytoprotection against lipid hydroperoxide (LOOH)-mediated oxidative stress and regulation of oxidative signaling cascades. Since prokaryotes are not equipped to express mammalian selenoproteins, preparation of recombinant GPx4 via commonly used bacterial transformation is not feasible. A published procedure for isolating the enzyme from rat testis employs affinity chromatography on bromosulfophthalein–glutathione-linked agarose as the penultimate step in purification. Since this resin is no longer commercially available and preparing it in satisfactory operational form is tedious, we have developed an alternative purification approach based on sequential anion exchange, size exclusion, and cation exchange chromatography. Final preparations were found to be essentially homogeneous in GPx4 (M_r  20 kDa), as demonstrated by SDS–PAGE with protein staining and immunoblotting. Specific enzymatic activity was determined using a novel thin-layer chromatographic approach in which the kinetics of phosphatidylcholine hydroperoxide loss or cholesterol-7α-hydroperoxide loss was monitored. A >400-fold purification of active enzyme has been attained. The relatively straightforward isolation procedure described should prove valuable for further functional studies on GPx4, e.g. how its ability to catalyze LOOH reduction compares with that of other LOOH detoxifying enzymes.     

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.     

Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation ofmicrosomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 μg, was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,100. The turnover number (37°C, pH 7.6) on (β-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-γ-palmitoyl)-l-α-phosphatidylcholine was 91 mol mo⁻¹ s⁻¹. As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK ± CHol cells but not from human liver microsomes. Human cell line microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4–5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes. PHGPx from human liver exhibits similar properties to previously described enzymes with PHGPx activity isolated from pig and rat tissues, but does not inhibit peroxidation of human liver microsomes owing to a high level of PHGPx activity already present in these microsomes.     

Purification,characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The M _r and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H₂O₂ have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.     

Incorporation of glutathione peroxidase active site into polymer based on imprinting strategy

Glutathione peroxidase (GPx, EC 1.11.1.9) is a key enzyme involved in scavenging of reactive oxygen species in biological system. For developing an efficient GPx-like antioxidant, catalytically necessary amino acid derivatives which located near the GPx active center were prepared as functional monomers. Via predetermined imprinting with substrate glutathione (GSH), a polymer-based GPx mimic with a similar structure of catalytic center of natural GPx was developed, and it demonstrated high-catalytic efficiency and substrate specificity. The imprinting polymer (I-PEM) exhibits GPx-like activity about three times higher than that of 2-SeCD, a cyclodextrin-based GPx mimic. The detailed studies on kinetics revealed that not only the substrate binding but also positional arrangement of reacting groups contribute significantly to the catalytic efficiency of the peroxidase model.     

Purification and characterization of diacylglycerol lipase from human platelets.

Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-beta-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectro-focusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2 inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine and histidine residues are required for the enzymatic activity of DGL. DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, even in the presence of 2 mM EGTA. This indicates that DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 microM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50<5 microM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.     

Changes in blood glutathione concentrations, and in erythrocyte glutathione reductase and glutathione S-transferase activity after running training and after participation in contests

Previously sedentary men (n = 23) and women (n = 18) were trained to run a half marathon contest after 40 weeks. Total blood glutathione had increased by 20 weeks of training and had returned to normal after 40 weeks. Erythrocyte glutathione reductase activity had increased by 20 weeks and remained elevated after 40 weeks. This effect was accompanied by decreases in glutathione reductase coefficients, which indicated that increases in the presence of riboflavin may have been responsible for the changes in reductase activity. Erythrocyte glutathione S-transferase activity had increased slightly after 20 weeks of training and a much more marked increase was found after 40 weeks. This may have been indicative of the occurrence of lipid peroxidation in this phase of training. The participants ran a 15-km race after the first 20 weeks of training and a half marathon after 40 weeks. Blood glutathione tended to decrease after the 15-km race and increased after the half marathon. In both cases it had returned to normal values 5 days after the race. Erythrocyte glutathione reductase was elevated 1 day after the races, and had returned to normal after 5 days. This could also have been explained from concurrent changes in the riboflavin content of the erythrocytes. Erythrocyte glutathione S-transferase activity decreased after both races, but was restored 5 days after the half marathon while such was not the case after the 15-km race.