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A variant of red cell gluta thione reductase, characterized by greater electrophoretic mobility, in creased enzyme activity per unit of hemoglobin, and an autosomal mode of inheritance, has been found in Negro populations. There appears to be an association of this variant with primary gout.
Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.
A single introduction of vitamin E and D,L-methionine to rats kept on the vivarium ration or their addition to protein-free diet separately does not affect the glutathionperoxidase and glutathionreductase activities in the liver. A simultaneus introdyction of the vitamin and methionine causes an increase in the glutathionperoxidase activity, without changing the glutathionreductase activity. Vitamin E prevents from increasing the glutathionperoxidase and glutathionreductase activities due to selenium. Propyl gallate produces no such effect.
Enzymopenic haemolytic anaemias are rare diseases in which there is an excess iron turnover. The iron is mainly deposited in many tissues giving rise to fibrosis and tissue damage. There is always a specific arthropathy; by involving a number of joints at the same time the clinical picture may resemble active rheumatoid arthritis, and therefore an erroneous treatment with gold, chloroquine, D-penicillamine or corticosteroids is possible. The diagnostic procedure in a patient suffering from enzymopenic haemolytic anaemia and pathogenetic mechanisms are discussed.
Human platelets exposed in vitro to increasing amounts of BCNU rapidly develop a progressive, relatively selective, and almost complete deficiency of GSSG-R activity. Several other enzymes are not inhibited when intact platelets are exposed to the nitrosourea; lipoamide dehydrogenase was investigated because of the remarkable similarity of the structure of its active site with that of GSSG-R. BCNU inhibits lipoamide dehydrogenase and GSSG-R only when they are in the reduced state; in the intact platelet, lipoamide dehydrogenase (unlike GSSG-R) is oxidized and is therefore unaffected. This is the first documentation of lipoamide dehydrogenase activity in platelets. After BCNU exposure, there is a reduced release of 14C-serotonin in response to collagen; the cells become incapable of aggregating in response to even large doses of epinephrine, ADP, collagen, or arachidonic acid, with loss of both primary and secondary waves of aggregation. At higher doses of BCNU, there is also a diminished PF-3 activity of intact platelets; sonication of drug-treated platelets normalizes coagulant activity. The drug-induced functional abnormalities occur despite preservation of the number of platelets, their electron microscopic appearance, and their capacity to take up 14C-serotonin. BCNU induced GSSG-R deficiency precedes the development of the earliest evidence of platelet dysfunction, and almost all of the enzyme's activity must be abolished before any functional abnormality becomes detectable. A small fraction of GSSG-R activity is essential for platelet function, and BCNU provides a powerful new tool to investigate the role of the enzymatic reduction of glutathione in platelet physiology and pathology.
1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations. 2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective. 3. GSSG fully reactivated the inactive enzyme at 38 degrees C and neutral to acidic pH (5.5-7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.
Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity were measured in G6PD deficiency, heterozygous beta-thalassaemia and controls. It is confirmed that, in contrast to the high stimulation of GR by FAD commonly found in in thalassaemia indicating red-cell deficiency of FAD, and shown here to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD deficiency, leading to high in vitro activity. Unexpectedly, on the other hand, low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN, confirmed by response to oral riboflavin, was found in the majority of subjects with G6PD deficiency, similar to that found in heterozygous beta-thalassaemia. Whereas this is explained in thalassaemia by an inherited slow red-cell metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and high FAD. When G6PD deficiency occurs with heterozygous beta-thalassaemia, GR is usually saturated with FAD as in G6PD deficiency alone, unless there is an inherited, very slow red-cell metabolism of riboflavin to FMN. The part played by GR in haemolytic crises in G6PD deficiency is discussed.
In preparations of Wistar rat myocardium, Km values of GSH-Px to the substrates, H2O2 and GSH, as determined by the DTNB method, were 1.9 x 10(-4) mol/L and 2.0 x 10(-3) mol/L respectively. The Km value of GSSG-R to GSSG was 6.0 x 10(-5) mol/L by UV spectrophotometric assay. In vitro, the activity of GSH-Px was inhibited by 1.0, 6.0 and 16.0 mmol/L synthetic moniliformin (MF) with the corresponding activities of 89.4%, 65.2% and 47.9% of control values (n = 6). The activities of GSSG-R in the presence of 16.0 and 32.0 mmol/L MF were 74.4% and 61.9% of controls (n = 5) respectively. These results revealed that the inhibition of GSH-Px by MF was stronger than that of GSSG-R. As determined by Lineweaver-Burk and of Dixon plots, the inhibition of GSH-Px and GSSG-R by MF was competitive and noncompetitive, respectively. The affinity of GSH-Px to MF was higher than that of GSSG-R because the inhibition constant Ki of the former (6.0 mmol/L) was less than that of the latter (39 mmol/L). We suggest that the mechanism of toxic-damaging effects of MF on myocardium, may be closely related to the failure of remove of free radicals by some enzymes, as GSH-Px and GSSG-R, and that MF may be involved in keshan disease.
Effects of riboflavin repletion of rats at various stages of development were evaluated by biochemical and behavioral parameters. One group of dams received diets containing a suboptimal level of riboflavin, approximately 15 mug, and another group, control, received approximately 40 mug of the vitamin daily 2 weeks before mating. Rats fed the control diet received approximately 120 mug riboflavin daily during pregnancy and lactation; suboptimals received approximately 15 mug daily. Some rats fed the control diet were pair-fed to rats fed the suboptimal ration. A group of dams fed the suboptimal diet was switched to control after parturition. At weaning, male offspring were fed the same riboflavin levels their respective dams received before mating except one group, whose dams were fed the suboptimal diet, received the control diet. Male progeny of dams pair-fed the control diet to suboptimal rats were either pair-fed to offspring of suboptimal dams or to offspring riboflavin-repleted at weaning. Rats that always received the suboptimal diet had significantly higher general activity scores at 60 days of age than the scores of other animals. Brains from rats always fed the suboptimal diet and those receiving riboflavin repletion at weaning had lower, sometimes significantly, DNA, RNA, and protein contents than those from other animals. Riboflavin restriction during gestation and lactation, but not gestation alone, appeared to produce permanent alterations in general activity scores and brain nucleic acid and protein contents of male rat progeny.
Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.
Candida albicans is a dimorphic yeast capable of producing alternate morphological forms (yeast or mycelium) in response to environmental changes. The intracellular level of glutathione, which helps to maintain the redox potential of the cell, is decreased significantly during the thermal induction of yeast-to-mycelium conversion. The reason for the decline of glutathione in the mycelial form is not understood. We have, therefore, investigated the levels of glutathione reductase, glutathione S-transferase, gamma-glutamyltranspeptidase, and glutathione peroxidase, four key enzymes involved in glutathione metabolism, in the yeast and mycelial forms. Yeast cells of C. albicans 3153A were induced in Lee's medium (pH 6.5) at 37 degrees C for 3 h to produce germ tubes. Cell lysates were prepared from yeast and mycelial cells, and glutathione reductase, glutathione S-transferase, gamma-glutamyltranspeptidase, and glutathione peroxidase were assayed spectrophotometrically. There was a 640% increase of the level of gamma-glutamyltranspeptidase in the germ tubes as compared with the yeast cells. No other significant alteration of the levels of enzymes was noted. This increased activity of gamma-glutamyltranspeptidase, which cleaves the glutamic acid residue of glutathione (Glu-Cys-Gly) appears to be, at least in part, responsible for the rapid decrease of the intracellular glutathione in C. albicans during the yeast-to-mycelium conversion.
Cellular lysis in freshly isolated hepatocytes incubated with varying concentrations of selenite could be related to the reductive metabolism of selenite. A decrease in intracellular GSH levels was observed concomitant with an increased rate of accumulation of oxidized glutathione in the incubation medium. Pretreatment of hepatocytes with an inhibitor of GSSG-reductase (1,3-bis(2-chloroethyl)-1-nitrosourea), prior to the addition of 50 microM selenite, resulted in substantially lower GSH-levels. The rate of GSSG reductase-catalyzed metabolism of selenite (50 microM) could be estimated to approximately 7 nmoles reduced/h per 10(6) cells. The results indicate that this was the major metabolic pathway for toxic concentrations of selenite in isolated hepatocytes. Furthermore, selenite considerably decreased cellular NADPH levels. In hepatocytes isolated from starved rats, the presence of alanine and glucose in the incubation medium protected against selenite-mediated cellular lysis. These observations suggest that an insufficient NADPH generation could be critical for selenite reduction and toxicity in isolated hepatocytes.
Hepatic levels of GSH and Phase II detoxication enzymes were compared to biochemical and histological indices of hepatic damage in 4- to 76-week-old nontransgenic mice and their transgenic littermates that overexpress the hepatitis B virus large envelope protein. The mice were fed a low-sucrose AIN-76A diet ad libitum. Hepatic-specific activities of quinone reductase (QR) and glutathione S-transferase (GST) were increased 2- to 10-fold beginning at 12 weeks of age in transgenic mice and correlated with increases in serum alanine aminotransferase (ALT) (r = 0.84 and 0.59, respectively). Quantitative histological analysis demonstrated that apoptosis was the predominant feature in 4- to 12-week-old transgenic mice, whereas necrosis and inflammation predominated at later time points. Surprisingly, 3-fold elevations in ALT were observed beginning at 52 weeks of age in nontransgenic mice, and hepatic-specific activities of QR and GST were also modestly increased in elderly nontransgenic animals. In contrast to transgenic mice, apoptosis was not a prominent feature. The strongest histological correlates to ALT in 4- to 76-week-old nontransgenic mice were necrosis and inflammation (r > 0.96), which in turn may have been evoked by hepatic fat accumulation. Profiles of specific GST isoforms were quantitated chromatographically and identified by sequencing tryptic digests. The Ya1 subunit of alpha-class GST was markedly increased from undetectable levels in transgenic mice, while more modest increases were observed in nontransgenic mice more than 1 year old. Fivefold elevations of the Yb1 subunit, a constitutively expressed mu-class GST, were found in transgenic mice older than 4 weeks of age, while 2-fold increases were observed in nontransgenic animals that were more than 1 year old. These studies demonstrate that selected increases in Phase II detoxication enzymes are a stereotyped response to chronic hepatitis that is strikingly reminiscent of the treatment of mice with anticarcinogenic enzyme inducers.
Styrene is both hepatotoxic and pneumotoxic in mice. Its mode of action is not clear, but it may be related to oxidative stress including a very large decrease in reduced glutathione (GSH). The current studies evaluated if: (1) the more toxic R-styrene oxide had a greater effect on reduced GSH levels than the less toxic S-styrene oxide, (2) the ratio of reduced to oxidized forms of glutathione was altered by styrene or styrene oxide, (3) other enzymes involved in the oxidant status of the cell, namely glutathione reductase, glutathione peroxidase and gamma-glutamylcysteine synthetase were altered, and (4) lipid peroxidation, as measured by the determination of malondialdehyde, increased. R-Styrene oxide (300mg/kg, ip) caused greater decreases in mouse liver and lung GSH than did S-styrene oxide (300mg/kg, ip). Styrene (600mg/kg, ip) caused decreases in both GSH and GSSG in both liver and lung. Styrene and styrene oxide did not cause significant increases in lipid peroxidation in either liver or lung. Styrene and styrene oxide had minimal effects on glutathione reductase and glutathione peroxidase in liver and lung. Styrene increased gamma-glutamylcysteine synthetase activity. The results suggest that while styrene and its metabolite styrene oxide cause significant decreases in GSH levels, they have little effect on the enzymes glutathione reductase and glutathione peroxidase and that in response to decreased glutathione levels there is an increase in its synthesis via induction of gamma-glutamylcysteine synthetase activity.
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Two cytochromes P450 (PB1 and PB2) have been isolated from the livers of rats treated with phenobarbital. PB2 (mol. wt. 53 500) is novel and is the first example of a phenobarbital-inducible enzyme with a Soret peak at 447 nm. Using an enzyme-linked immunosorbent assay, some immunochemical and structural similarities were observed between these cytochromes. PB1 and PB2 were induced by phenobarbital, Aroclor 1254, trans-stilbene oxide and to a lesser extent by isosafrole. Immunohistochemical localization of these proteins in the liver of untreated rats showed PB1 to be localized in a large area and PB2 in a narrow range of cells around the central vein. This demonstrates the heterogeneity of hepatocytes even within the centrilobular area and indicates that the synthesis of these two proteins is regulated differently although both are induced by the same agent, phenobarbital. Two 3-methylcholanthrene inducible cytochromes MC1 (mol. wt. 54 500) and MC2 (mol. wt. 57 000) were present at very low levels, MC2 mostly in the periportal region but also diffusely distributed throughout the lobule including some centrilobular cells, MC1 concentrated in the centrilobular region. The localization of two major groups of glutathione transferases (GST's) was also different. 'C' type proteins (Yb Yb') and microsomal epoxide hydrolase (EH), were concentrated around the central vein, whereas the 'B' type proteins (Ya Yc) and cytochrome P450 reductase were distributed in a larger area of this region. Thus, the localization was different for some members of the same enzyme family, whilst similarities in the localization existed across the border of the families: (i) PB2, MC1, EH and GST 'C' type proteins were concentrated in a narrow area around the central vein; (ii) PB1 and GST 'B' type proteins occupied a large centrilobular area; (iii) MC2 levels were very low, predominantly periportal but also diffusely distributed throughout the lobule. Treatment of the animals with inducers increased the staining intensity and in several cases extended the areas of cells containing these proteins over the adjacent zone without fundamentally altering their distributions. However, treatment with beta-naphthoflavone led to a shift of MC1 to the periportal area. This suggests that the expression of these proteins in certain cells is not an irreversible quality of differentiation but depends on the degree of suppression and derepression of regulatory components.(ABSTRACT TRUNCATED AT 400 WORDS)