Gastrointestinal glutathione peroxidase

The gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the GPx family. In rodents, it is exclusively expressed in the gastrointestinal tract, in humans also in liver. It has, therefore, been discussed to function as a primary barrier against the absorption of ingested hydroperoxides. A vital function of GI-GPx can be deduced from the unusual stability of its mRNA under selenium-limiting conditions, the presence of low amounts of GI-GPx protein in selenium deficiency where cGPx is absent, and the fast reappearance of the GI-GPx protein upon refeeding of cultured cells with selenium compared to the slower reappearance of cGPx protein. Furthermore, the Secis efficiency of GI-GPx is low when compared to cGPx and PHGPx. It is, however, almost independent of the selenium status of the cells tested. All these characteristics rank GI-GPx high in the hierarchy of selenoproteins and point to a role of GI-GPx which might be more crucial than that of cGPx, at least in the gastrointestinal system.     

Blood glutathione peroxidase

Biological Trace Element Research - Manual and automated assays for the determination of glutathione peroxidase activity in bovine, sheep, pig, and human blood samples are described. The...     

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.     

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.     

Isolation and purification of glutathione peroxidase

Electrophoretically homogeneous glutathione peroxidase (EC 1.11.1.9) preparation from rat liver with a specific activity of 1.46 U/mg of protein and a yield of 7.2% was obtained using the purification procedure developed. The K _M values for reduced glutathione and hydrogen peroxide were 0.033 and 0.208 mM, respectively. The enzymatic reaction had the following characteristics: the temperature optimum, 32°C; the pH optimum, 7.4; and the activation energy, 29.1 kJ/mol. The molecular weight of the enzyme was 88 kDa.     

Reaction of cyanide with glutathione peroxidase

An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 mM KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from [¹⁴C]iodoacetate in equimolar ratio to enzyme selenium. These observations suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and S-cyanoglutathione; spontaneous oxidation of the selenol to a labile oxidized form of GSH peroxidase leads to irreversible inactivation.     

Isolation and purification of glutathione peroxidase

Electrophoretically homogeneous glutathione peroxidase (EC 1.11.1.9) preparation from rat liver with a specific activity of 1.46 U/mg of protein and a yield of 7.2% was obtained using the purification procedure developed. The K(M) values for reduced glutathione and hydrogen peroxide were 0.033 and 0.208 mM, respectively. The enzymatic reaction had the following characteristics: the temperature optimum, 32 degrees C; the pH optimum, 7.4; and the activation energy, 29.1 kJ/mol. The molecular weight of the enzyme was 88 kDa.     

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.     

Phylogenetic distribution of glutathione peroxidase.

1. The enzyme glutathione peroxidase (E.C.1.11.1.9), known to be a selenoprotein from mammalian sources, was detected in the following vertebrates: fish, frog, salamander, and turtle. 2. Among invertebrates, the enzyme was detected in crayfish and snail but not in insects or earthworm. 3. No plant tissues or microorganisms showed any evidence of the enzyme activity. 4. The presence of the enzyme activity in so many animal groups implies the widespread occurrence of genetic information for the specific assimilation of the selenium atom.     

Developmental study of rat brain glutathione peroxidase and glutathione reductase

Glutathione peroxidase and glutathione reductase activities were measured in whole rat brains at selected ages from birth to adulthood. On a wet weight basis glutathione peroxidase activity increased 70% during development and glutathione reductase activity increased 160%. On a protein basis glutathione peroxidase declined slightly in activity during the first two weeks of life and then maintained the 14-day activity into adulthood while glutathione reductase showed a 30% increase in activity. While less than the developmental changes in many enzymes involved in aerobic glycolysis or catecholamine metabolism, these increases do suggest a role in CNS metabolism.     

Nanoparticulate glutathione peroxidase mimics based on selenocystine-pullulan conjugates

We synthesized nanoparticulate glutathione peroxidase (GPx) mimics in which selenocystine (SeCyst) was conjugated to a hydrophilic linear polysaccharide, pullulan (Pul). The SeCyst ester-conjugated Pul derivatives (SeCyst-Pul) in phosphate buffer (pH 7) were treated with a sonicator to spontaneously form particulate materials. Dynamic light scattering measurements revealed that the SeCyst-Pul conjugates could form particulate materials with diameters between 100 and 300 nm. Distinctive endothermic peaks were observed for the SeCyst-Pul aggregate solutions based on a differential scanning calorimetric analysis. The tryptophan (Trp) fluorescence intensity of SeCyst benzyl ester-tryptophanyl-Pul (SeCyst-Bz-Trp-Pul) mostly decreased in comparison to those of the Trp-Pul (its precursor) and free Trp, which indicates that the Trp residues come close to each other during the aggregation of the conjugates. Formation of SeCyst-Pul aggregates could be induced by the hydrophobic interactions between the SeCyst esters and the amino acid residues on Pul. The GPx-like activity of SeCyst-Bz-Trp-Pul aggregates for the reduction of H2O2 was enhanced nearly 20-fold higher than that of free SeCyst. The double-reciprocal plots of the SeCyst-Bz-Trp-Pul aggregate-catalyzed reduction yielded parallel lines by varying the substrate concentrations, indicating a "ping-pong" mechanism that is similar to those of the natural GPxs. The enhanced GPx activity of the SeCyst-Bz-Trp-Pul aggregate was also supported by higher kinetic parameters, k(cat)/K(m) (GSH) and k(cat)/K(m) H2O2. Overall, the enhanced activity of the SeCyst-Bz-Trp-Pul aggregate would be attributed to a hydrophobic environment that was formed at the vicinity of the SeCyst.     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.     

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.     

Effect of sarcolysine on glutathione peroxidase and glutathione reductase activity in sarcoma C-45

A drop of glutathione peroxidase and glutathione reductase activity was revealed in sarcoma C-45 at the period of its most intensive growth. Repeated sarcolysine injections (1.2 mg/kg, intraperitoneally) caused a sharp fall in the activity of both enzymes with a simultaneous reduction of the ratio of glutathione reductase and glutathione peroxidase activities. The important role of the glutathione enzyme redox system in the realization of antitumour action of the chemotherapeutic drugs is supposed.     

Identification of the binding site of methylglyoxal on glutathione peroxidase: methylglyoxal inhibits glutathione peroxidase activity via binding to glutathione binding sites Arg 184 and 185

Methylglyoxal (MG), a physiological alpha-dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272, 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.     

Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes

Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.     

The amino-acid sequence of bovine glutathione peroxidase

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.