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.     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

The isolation of a fetal rat liver glutathione S-tranferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Y_c (M_r 28 000) and a subunit (M_r 25 500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit ‘Y_fetus’. The enzyme which we term glutathione S-transferase Y_cY_fetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

The isolation of a fetal rat liver glutathione S-transferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated from fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Yc (Mr 28,000) and a subunit (Mr 25,500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit 'Yfetus'. The enzyme which we term glutathione S-transferase YcYfetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Dual method for the determination of peroxidase activity and total peroxides-iodide leads to a significant increase of peroxidase activity in human sera

Peroxidases are very important enzymes, e.g., as preventive antioxidants by removing noxious peroxides from the blood. For this reason we evaluated a colorimetric method which detects the activity of endogenous peroxidases by their reaction with hydrogen peroxide, using tetramethylbenzidine as the chromogenic substrate. This assay design can be easily reversed by change of the variable compound to measure also total peroxides in plasma or serum. An increased total antioxidant status was reported previously by the addition of iodide to human serum. In this study iodide activated the endogenous peroxidases significantly in comparison to control sera and isomolar NaCl as well as horseradish peroxidase. Corresponding to the increased peroxidase activity a concomitant decrease of total peroxides occurred in the same samples. This exchangeable assay design is a beneficial opportunity to screen total peroxide levels as well as peroxidase activity in human sera without time-consuming preparations. The method proved to be simple and is favorable due to its specificity, reproducibility, and low costs. Moreover, we were able to find an explanation for the increased total antioxidant status in the presence of iodide, which is presumably an indirect protective effect via an enhanced activity of enzymatic antioxidants, thereby reducing endogenous peroxides.     

Effects of dietary iron overload on glutathione peroxidase knockout mice

Excess iron (Fe) intake has been associated with an increased risk of cardiovascular disease in humans, presumably the result of increased oxidative stress. Previous work by us has shown that feeding a high-Fe diet to selenium (Se)-deficient weanling mice for 4 wk resulted in elevated plasma cholesterol and triglycerides and increased hepatic thiobarbituric acid reactive substances (TBARS). Here, we report the effect of Fe overload in mice lacking cellular glutathione peroxidase (GPX1 knockout [KO] mice), the selenoenzyme thought to account for much of the antioxidant action of Se. Four groups of 9–13 weanling wild-type (WT) or GPX1 KO mice were randomly assigned, then fed either an Fe-adequate (35 ppm Fe) or high-Fe (1100 ppm Fe) casein-based diet for 4 wk. Iron was added as ferric citrate. Both diets also contained 0.2 ppm Se added as sodium selenite. As expected, liver GPX1 activity was essentially absent in the KO mice. Another Se parameter measured (hepatic thioredoxin reductase activity) did not vary across groups. Although liver Fe was elevated in mice fed the high-Fe diet, liver TBARS was largely unaffected either by mouse genotype or diet fed. Moreover, plasma lipids were not elevated in the Fe-overloaded GPX1 KO mice. Thus, decreased GPX1 activity cannot account for the pro-oxidant hyperlipidemic effects observed earlier in mice fed the high-Fe Se-deficient diet. This suggests that impairment of Se functions other than GPX1 activity may be responsible for the elevated plasma lipids and hepatic TBARS seen in the Fe-overloaded Se-deficient mice.     

The influence of fasting on liver sulfhydryl groups, glutathione peroxidase and glutathione-S-transferase activities in the rat

Sulfhydryl groups, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) are important elements of the antioxidant defence in the organism. The efficacy of their antioxidant action is influenced by many factors. In this work, the effect of fasting on total, protein-bound and nonprotein sulfhydryl groups and on the activity of liver and serum GPx and GST in rats were determined. Male Wistar rats were divided into two groups: non-fasted and 18-hour fasted. In fasted animals liver content of nonprotein sulfhydryl groups (represented predominantly by reduced glutathione; GSH) was diminished by 22% in comparison to non-fasted group, whereas total and protein-bound -SH groups were unaffected. The activity of liver and serum GPx was unchanged in food deprived rats. In these animals the activity of GST in serum was reduced by 26%. Fasting had no significant effect on the activity of GST in the liver. Our results demonstrate that in rats deprived of food for 18 hours liver and serum GPx and GST are not involved in protection against action of reactive oxygen species formed during fasting. The observed drop in the content of liver nonprotein sulfhydryl groups without concomitant rise in the activity of GPx and GST indicates that this effect may be due to augmented degradation of GSH, its potentiated efflux from hepatocytes and formation of conjugates with intermediates arising as a result of reactive oxygen species action.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.     

Oxidative stress related DNA adducts in the liver of female rats fed with sunflower-, rapeseed-, olive- or coconut oil supplemented diets

Both animal and epidemiological studies support an effect of fatty acid composition in the diet on cancer development, in particular on colon cancer. We investigated the modulating effect of supplementation of the diet of female F344 rats with sunflower-, rapeseed-, olive-, or coconut oil on the formation of the promutagenic, exocyclic DNA adducts in the liver, an organ where major metabolism of fatty acids takes place. 1,N(6)-ethenodeoxyadenosine (etheno-dA), 3,N(4)-ethenodeoxycytidine (etheno-dC) and 1,N(2)-propandodeoxyguanosine from 4-hydroxy-2-nonenal (HNE-dGp) were determined as markers for DNA-damage derived from lipid peroxidation products and markers for oxidative stress. 8-Oxo-deoxyguanosine (8-Oxo-dG) was also measured as direct oxidative stress marker. The body weight of the rats was not influenced by the four diets containing the different vegetable oils during the 4-week feeding period. Highest adduct levels of etheno-dC (430 +/- 181 adducts/10(9) parent bases), HNE-dGp (617 +/- 96 adducts/10(9) parent bases) and 8-Oxo-dG (37,400 +/- 12,200 adducts/10(9) parent bases) were seen in rats on sunflower oil diet (highest linoleic acid content). Highest adducts levels of etheno-dA (133 +/- 113 adducts/10(9) parent bases) were found in coconut oil diet (lowest content of linoleic acid). Weakly positive correlations between linoleic acid content in the four diet groups were only observed for levels of HNE-dGp and 8-Oxo-dG. Neither the diet based on olive oil (which contains mainly oleic acid) nor the diet based on rapeseed oil (containing alpha-linolenic acid) exerted any significant protective effect against oxidative DNA damage. Our results indicate that a high linoleic acid diet may contribute to oxidative stress in the liver of female rats leading to a marginal increase in oxidative DNA-damage.     

Crystallization of GST2, a human class alpha glutathione transferase

Single crystals of human GST2, a class alpha glutathione transferase have been grown in polyethylene glycol 2000 by the hanging-drop vapour diffusion method. The crystals belong to space group C2 and have cell dimensions a = 100.8 A, b = 95.4 A, c = 105.2 A and beta = 92.4 degrees. The X-ray diffraction pattern extends to better than 3 A resolution.     

Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.     

Selenium metabolism and glutathione peroxidase activity in cultured human lymphoblasts

The metabolism of selenite, selenocysteine (SeCys), and selenomethionine (SeMet) was studied in three human lymphoblast cell lines with defects in the transsulfuration pathway and in control cells without this defect. There were very little differences in the induction of glutathione peroxidase (GPX) activity by selenite and SeCys among these cells. However, markedly higher levels of SeMet were required to induce GPX activity in transsulfuration defective cells than in control cells. Surprisingly, the addition of pyridoxal phosphate (PLP) to the media resulted in elevated GPX activity in all cells regardless of the chemical form of Se used. There is no explanation for this effect of PLP, but it is not through direct reaction with GPX or on the alteration of sulfhydryl groups.     

Reinvestigation of a selenopeptide with purportedly high glutathione peroxidase activity

A 15-amino acid long selenopeptide (15SeP) was recently reported to possess nearly the same catalytic activity as glutathione peroxidase (Gpx) for the reduction of hydrogen peroxide by glutathione (Sun, Y., Li, T. Y., Chen, H., Zhang, K., Zheng, K. Y., Mu, Y., Yan, G. L., Li, W., Shen, J. C., and Luo, G. M. (2004) J. Biol. Chem. 279, 37235-37240). Such a finding is startling considering the high efficiency of the natural enzyme and the modest catalytic properties of most short peptides. As 15SeP had been subjected only to limited chemical characterization, we prepared it by a new route involving selenocysteine-mediated native chemical ligation. High resolution matrix-assisted laser desorption ionization mass spectrometry confirmed the identity of the reaction product, whereas circular dichroism spectroscopy showed that 15SeP assumes a random coil conformation in solution. Although low levels of peroxidase activity were detectable under standard assay conditions, the peptide is >5 orders of magnitude less active than native Gpx. Our observations are incompatible with claims ascribing remarkable catalytic properties to 15SeP and suggest that the efficiency of Gpx derives from its well defined three-dimensional structure.