Purification and characterisation of the soluble glutathione S-transferase isoenzymes in rat kidney derived NRK cells

Glutathione S-transferase (GST) enzymes are toxicologically important from many points of view. Rat kidney derived established NRK cells were mass cultured for the isolation of GST isoenzymes. These were enriched by affinity chromatography and separated by chromatofocusing and HPLC. Exactly the same major GST subunits were found in NRK cells as in the rat kidney. Strong evidence was also found for the presence of an aberrant form of GST 7-7, as was described in rat kidney. A very good correlation between the NRK GST and rat kidney, and especially cis-platinum treated kidney, was found. It is concluded that NRK cells can be considered as a valuable alternative tool for in vitro research of rat kidney phenomena, especially when toxicological interactions are investigated.     

Alterations in glutathione peroxidase activity following reperfusion injury to rat liver

Prevention of cellular damage after warm ischemia is of major importance in liver transplantation. In this study, we determined the extent to which lipid peroxides contribute to the pathogenesis of hepatic cell damage induced by transient warm ischemia with subsequent reperfusion. In addition, the function and immunohistochemical features of glutathione peroxidase, a potent physiological lipid peroxide scavenger of the liver, was assessed. Reperfusion following 15 or 30 minutes of warm ischemia resulted in a significant elevation in serum and liver lipid peroxidase (LPO) levels. In addition, necrosis of the hepatic periportal area accompanied with remarkable rises in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were observed. In contrast, 30 min of ischemia without reperfusion caused minimal hepatocellular damage. The adverse changes after ischemia/reperfusion were minimized by pretreatment with superoxide dismutase (SOD). These results indicate that increased lipid peroxidation by production of radicals after reperfusion caused the liver cell damage. After ischemia/reperfusion, liver glutathione peroxidase (GSH-PO) activity was significantly decreased and its location altered in the damaged liver. These findings suggest that GSH-PO contributes significantly to the protection against hepatic reperfusion injuries.     

Reduction of thymine hydroperoxide by phospholipid hydroperoxide glutathione peroxidase and glutathione transferases

Thymine hydroperoxide (5-hydroperoxymethyluracil), a model compound representing products of oxidative damage to DNA, is a substrate for glutathione peroxidase and some isoforms of glutathione transferase. In this paper, we show that selenium-dependent human phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx) exhibits about four orders of magnitude higher activity on thymine hydroperoxide than that of other human enzymes such as selenium-dependent glutathione peroxidase and various representatives of glutathione transferases. The results indicate that Se-PHGPx may be an important enzyme in repairing oxidatively damaged DNA.     

A new kinetic model for the mode of action of soluble and membrane-immobilized glutathione peroxidase from bovine erythrocytes--effects of selenium

Kinetic studies on the oxidative reaction of glutathione by hydrogen peroxide were performed using soluble and membrane-bound ox erythrocyte glutathione peroxidase of various types. The effects of organic and inorganic selenium on the glutathione peroxidase activity were also examined. The kinetic behaviour of the enzyme was investigated using a coupled reaction within a relatively large range of hydrogen peroxide and glutathione concentrations. Non-parallel double-reciprocal plots were obtained which suggested that a sequential ordered rather than a ping-pong mechanism was involved. Similar results were obtained with soluble and membrane-bound enzyme, whatever the type of crosslinking used. Crosslinking was performed on a nylon support using various alkylating agents and bifunctional molecules. With all three types of immobilized enzyme thus obtained, a slight but significant increase in the Km was observed. The effects of selenium were then studied. Using soluble enzyme, a slight increase in the activity was observed in the presence of inorganic selenium (sodium selenite) but not with organic selenium (seleno-L-methionine). Inorganic selenium alone was also found to have a slight effect on the membrane-bound enzyme. An increase in the catalytic efficiency was observed when glutathione peroxidase was bound using lysine as the bifunctional agent and either glutaraldehyde or triethyloxonium tetrafluoroborate as the reticulation agent, after a three-month period of incubation.     

Identification of cDNAS encoding plastid-targeted glutathione peroxidase

OBJECTIVE: Helicobacter pylori (H. pylori) is a major factor in determining the risk for development of gastric adenocarcinoma through the intermediate steps of atrophic gastritis and intestinal metaplasia. Because H. pylori infection is highly prevalent in asymptomatic populations and only a few people develop cancer, additional factors may influence the risk for development of cancer, once infection is established. Some factors may pertain to differences among bacterial strains. Because infection by H. pylori strains possessing cagA (cytotoxin-associated gene A), a gene encoding a high-molecular-weight immunodominant antigen (CagA), is associated with enhanced induction of gastritis, the aim of our study was to evaluate potential differences in the prevalence and intensity of atrophy and intestinal metaplasia between CagA-positive and CagA-negative H. pylori-infected patients. METHODS: Eighty H. pylori-infected patients among 120 consecutive dyspeptic patients referred for upper gastrointestinal endoscopy were studied. Six bioptic specimens were taken from the gastric antrum: five for histological examination, and one for urease test. The H. pylori status was determined by histology, CLO test, and serology (in a standardized ELISA) for serum IgG and IgA directed to H. pylori. The CagA status was determined by Western blotting to detect serum IgG antibodies to CagA. Gastritis was classified according to the Sydney System. A score from 0 to 3 was assigned to each of the following morphological variables: atrophy, intestinal metaplasia, and mononuclear and neutrophilic cell infiltration. The association between CagA status and histological features was assessed by means of the chi2 test for trend. RESULTS: Among the 80 H. pylori-infected patients 53 (66%) were CagA seropositive and 27 (34%) were CagA seronegative. The mean age of the two groups was similar. CagA-positive patients had significantly higher scores for atrophy (p = 0.006), intestinal metaplasia (p = 0.01), and mononuclear (p < 0.001) and polymorphonuclear (p = 0.002) cell infiltration than did CagA-negative patients. No differences in contrast, were found for H. pylori density. CONCLUSION: Infection with CagA-positive H. pylori strains is associated with an increased prevalence and intensity of antral atrophy and intestinal metaplasia, in addition to higher degrees of gastritis. Our results seem to suggest that the CagA status could be a helpful parameter to define a subgroup of H. pylori-infected patients at increased risk of developing gastric adenocarcinoma.     

Radioimmunoassay of glutathione peroxidase in human serum

Glutathione peroxidase (GSHPx) was purified from human serum and used for immunization of rabbits. Antiserum bound up to 75% of added 125I-GSHPx after precipitation with a second antibody. Human serum, but not sera from eight animal species, inhibited the binding of labelled GSHPx, indicating that the antiserum did not react with GSHPx from these species. GSHPx could be measured in less than 10 microliters of human serum by radioimmunoassay. In sera with widely varying selenium concentrations (0.1-2.9 mumol/l) the amount of GSHPx protein (0.3-6.3 mg/l) was strongly correlated with GSHPx activity (r = 0.94) and it was also correlated with serum selenium concentrations (r = 0.64). This indicates that GSHPx protein may be a valuable biological marker of selenium status. In samples with serum selenium concentrations of 0.8-1.2 mumol/l, the concentration of GSHPx was 3.3 (0.4) mg/l (mean (S.D.)), or 0.04 (0.005) mumol/l. This corresponded to 0.16 (0.02) mumol/l of GSHPx selenium and 16% (2.8)% of total serum selenium. The data suggest that the method can be used to measure the proportion of serum selenium that is located in GSHPx. Following storage of serum at room temperature, both serum GSHPx protein and activity declined, but addition of glutathione protected both GSHPx protein and activity.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 degrees C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected 32Pi was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

Purification, characterization and differentiation-dependent expression of a perchloric acid soluble protein from rat kidney

We have recently reported the presence of a novel perchloric acid soluble protein in rat liver (PSP1) that inhibits cell-free protein synthesis in a rabbit reticulocyte system. While studying the perchloric acid soluble proteins from different tissues of rats, we found that the kidney protein cross-reacted with antibody against the PSP1. In this investigation, we have purified a perchloric acid soluble protein from the rat kidney and studied its characterization and expression. The protein extracted from the postmitochondrial supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. By immunoscreening with the rabbit antisera against the PSP1, we detected a cDNA that contained an open reading frame of 411 bp, encoding a 137 amino-acid protein with a molecular mass of 14,149 daltons. The deduced amino acid sequence was completely identical with that of PSP1 from rat liver. The perchloric acid soluble protein from rat kidney (K-PSP1) also inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. Immunohistochemistry showed that the expression of K-PSP1 increased from fetal 17th day to postnatal 4th week, and it remained almost the same until the 7th week of postnatal age. Furthermore, the expression of K-PSP1 in the kidney of the nephrotic rat model was shown to be differentiation dependent. On the other hand, the expression of K-PSP1 in renal tumor cells was downregulated as compared with intact tissue. These results suggest that the expression of K-PSP1 is regulated in a differentiation-dependent manner in the kidney.     

Purification of peroxidase isoenzymes from turnip roots

Simple reproducible procedures for purification of the main soluble (S) and ionically bound (IB) cationic peroxidase isoenzymes from turnip roots were established. The procedures included ammonium sulfate precipitation of the isoenzymes, chromatographic separation of the main isoenzymes using cellulose phosphate columns and purification to homogeneity by hydrophobic interaction chromatography on phenyl Sepharose columns. The specific activity of the phenyl Sepharose purified S and IB isoenzymes were 2760 and 896 units/mg protein with 140 and 4.8 fold increase over the crude extract and 38 and 13% recovery. The pH maxima and K(m) for phenol and H2O2 of purified S and IB were determined.     

Glutathione peroxidase activity and glutathione concentration in genetically dystrophic mice

The case of a 6-year-old boy is reported who was delivered out of a frontal position by application of the vacuum cup to the left part of the forehead. His left eye exhibits a pronounced somewhat irregular astigmatism. There are some vertical descemet tears. Because of the application of the vacuum pump close to the left bulbus, an increase of the tissue tension and consequently a deformation of the bulbus occurred by the vacuum in the bulbar region. The described lesions parallel the vertical descement tears which occur in forceps-delivery. Owing to the greater horizontal corneal diameter, a more pronounced extension of the cornea in the horizontal meridian occurs, which would explain the vertical course of the descemet tears. The obstetric contraindication for the application of the vacuum extraction in case of a frontal or facial position of the fetus is justified also from the ophthalmological point of view because of the risk of occurrence of irreversible corneal lesions.     

Effect of in vivo hyperoxia on the glutathione system in neonatal rat lung

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

Kinetic study of the reaction of glutathione peroxidase with peroxynitrite

Glutathione peroxidases and their mimics, e.g., ebselen or diaryl tellurides, efficiently reduce peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) to nitrite and protect against oxidation and nitration reactions. Here, we report the second-order rate constant for the reaction of the reduced form of glutathione peroxidase (GPx) with peroxynitrite as (8.0 +/- 0.8) x 10(6) M-1 s-1 (per GPx tetramer) at pH 7.4 and 25 degreesC. The rate constant for oxidized GPx is about 10 times lower, (0.7 +/- 0.2) x 10(6) M-1 s-1. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. The data support the conclusion that GPx can exhibit a biological function by acting as a peroxynitrite reductase.     

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl-coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6-hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase-positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6-hydroxydopamine-injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6-hydroxydopamine-induced toxicity.     

Selenium concentration and activity of glutathione peroxidase in lysate of human erythrocytes

The concentration of selenium in human red cells was measured by a fluorometric determination method after wet digestion of the biological material. In the red cells from the same blood samples the activity of the selenoenzyem glutathione peroxidase (EC 1.11.1.9) was determined with tertiary butyl hydroperoxide as acceptor substrate. The results show that only 10% of the total selenium content of human red cells is fixed to the enzyme. No correlation between the enzyme activity and the selenium concentration could be found. On the basis of these results can be presumed that the protective effect of selenium in glutathione peroxidase against oxidant damage of cells is not the only biological function of this element.     

Overexpression of glutathione S-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress

Transgenic tobacco seedlings that overexpress a cDNA encoding an enzyme with both glutathione S-transferase (GST) and glutathione peroxidase (GPX) activity had GST- and GPX-specific activities approximately twofold higher than wild-type seedlings. These GST/GPX overexpressing seedlings grew significantly faster than control seedlings when exposed to chilling or salt stress. During chilling stress, levels of oxidized glutathione (GSSG) were significantly higher in transgenic seedlings than in wild-types. Growth of wild-type seedlings was accelerated by treatment with GSSG, while treatment with reduced glutathione or other sulfhydryl-reducing agents inhibited growth. Therefore, overexpression of GST/GPX can stimulate seedling growth under chilling and salt stress, and this effect could be caused by oxidation of the glutathione pool.     

Selenium-dependent and selenium-non-dependent glutathione peroxidase in patients with Balkan endemic nephropathy

We studied the activity of erythrocyte selenium (Se)-dependent, Se-non-dependent glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in uremic patients (UP) in clinically healthy members from families affected with Balkan nephropathy (HMF/BEN) and in healthy volunteers from endemic settlements (control group). The SOD activity was not significantly different in the groups studied and the Se-non-dependent GSH-Px activity in HMF/BEN and UP was not different from the control group. However, the activity of Se-dependent GSH-Px in UP was lower compared with the control group, whereas the mean value of the Se-dependent GSH-Px activity in HMF/BEN was not significantly different when compared with the other two investigated groups.     

Protection of PC12 cells glutathione peroxidase in L-DOPA induced cytotoxicity

L-DOPA may cause side-effects during the treatment of Parkinson's disease. We investigated the role of glutathione peroxidase (GSHPx) in cellular defense against L-DOPA cytotoxicity. A line of PC12 cells overexpressing GSHPx with plasmid pRc/CMV-GSHPx was established and stable transfectants overexpressing GSHPx were used for this study. GSHPx activity was found to be 1.5-fold higher in GSHPx-transfectants than in mock-controlled transfectants. Transfectants over expressing GSHPx were also significantly more resistant to exposure to either L-DOPA or t-butyl hydroperoxide than mock-transfected cells. Results suggested that L-DOPA may cause neuronal cell death by an oxidative pathway and GSHPx may play an important role in cellular defense against oxidative stress.