Glutathione, oxidative stress and neurodegeneration.

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species and mitochondrial dysfunction. Here, we review the evidence for a disturbance of glutathione homeostasis that may either lead to or result from oxidative stress in neurodegenerative disorders. Glutathione is an important intracellular antioxidant that protects against a variety of different antioxidant species. An important role for glutathione was proposed for the pathogenesis of Parkinson's disease, because a decrease in total glutathione concentrations in the substantia nigra has been observed in preclinical stages, at a time at which other biochemical changes are not yet detectable. Because glutathione does not cross the blood-brain barrier other treatment options to increase brain concentrations of glutathione including glutathione analogs, mimetics or precursors are discussed.     

Effect of different dietary fat sources on production traits, lipid peroxide status and on the glutathione redox system in African catfish [Clarias gariepinus (Burchell)] fingerlings. Short communication

Lipids are used to provide the energy to cover the metabolic needs and to provide essential fatty acids, which are important for membrane function [12]. Fats may contain high level of long chain polyunsaturated fatty acids, which are prone to peroxidation [8] and will interact with the antioxidant defense system [1]. There is contradiction in the literature about whether the intake of fish oil enhance [7] or deplete [4] tissue antioxidant defenses and the glutathione redox system in different organisms. The aim of the present study was to examine the effects of different dietary oils on parameters of the lipid peroxide state and the glutathione redox system in C. gariepinus fingerlings.     

The glutathione system. I. Synthesis, transport, glutathione transferases, glutathione peroxidases

During the last 10–15 years significant progress has been achieved in all directions of studies of the glutathione system. A series of new enzymes involved into metabolism of glutathione has been discovered. Many of these enzymes are polyfunctional and their new activities have been recognized. The enzymes interact with hormones and signal transduction systems. Significant progress has been achieved in the studies of intracellular, intercellular and inter-organ transport. The important achievement is employment of not only selective compounds-analyzers but also gene engineering methods for identification of new functions.     

Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits.

The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury (time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect (P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase (P = 0.015) and glutathione reductase (P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione (P = 0.008) and total glutathione (P = 0.015). Morphological analysis (hematoxylin and eosin staining) showed significant polymorphonuclear cell infiltration of the damaged region at 24 h postinjury. We conclude that acute mechanical muscle stretch injury results in increased free radical production within 24 h after injury. Antioxidant enzyme and glutathione systems also appear to be affected during this early postinjury period.     

The effect of vitamin A deficiency on hepatic, renal and pulmonary glutathione S-transferase activities in the rat.

Feeding male weanling rats on a vitamin A-deficient diet for 6 weeks resulted in significant increases (44-57%) in glutathione S-aryl-, S-aralkyl- S-alkyl- and S-epoxidetransferase activities in the liver cytosol. Only the S-aralkyl- (27%) and S-alkyltransferase (14%) activities were significantly increased in the kidney as a result of deficiency. There was no effect on any of the pulmonary glutathione S-transferase activities. The increases in hepatic transferase activities were due primarily to increases (25-96%) in the apparent Vmax. There were no changes in the apparant Km of any of the four drug substrates employed. With 3,4-dichloronitrobenzene as the second substrate, the apparent Km for glutathione was increased by over 2-fold in vitamin A-deficient livers as compared with controls. The relationship between these results and enhanced susceptibility to chemical carcinogens in vitamin A deficiency is briefly discussed, and comparison is made between the effects of this nutritional state and pretreatment with drug inducers on the glutathione S-transferases.     

Lethal and mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine potentiated by oxidized glutathione, a seemingly harmless substance in the cellular environment.

Both the lethal and the mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on cells of Streptococcus pneumoniae were greatly potentiated by a component of yeast extract added to the cellular environment. This component was found to be an oxidation product of glutathione, glutathione disulfide (GSSG). At low concentrations in the medium, both GSSG and glutathione potentiated MNNG action, but at high concentrations, glutathione (and other sulfhydryl compounds) abolished the effect. Point mutations in a cellular gene conferred resistance to the potentiating effect, and they blocked uptake of either GSSG or glutathione into the cells as well. This gene apparently encodes a component of the system for glutathione transport in S. pneumoniae. The mechanism by which GSSG, an apparently innocuous substance in the environment, renders low levels of MNNG genotoxic and cytotoxic thus depends on its transport into the cell, where it is reduced by glutathione reductase and then activates intracellular MNNG. Also, it was observed that mutants of S. pneumoniae defective in DNA mismatch repair are more resistant to MNNG than are wild-type cells by a factor of 2.5.     

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

GAMMA-Glutamyl transpeptidase of sheep-kidney cortex. Isolation, catalytic properties and dissociation into two polypeptide chains.

Gamma-Glutamyl transpeptidase was isolated from sheep kidney cortex as an apparently homogeneous, highly active protein. At optimal pH and in the absence of acceptors, the enzyme catalyzes the release of about 510 mumol of p-nitroaniline per mg protein per min from the model substrate L-gamma-glutamyl-p-nitroanilide. Polyacrylamide gel electrophoresis in a sodium dodecylsulfate buffer system showed the presence of a large (Mr approximately 65000) and a small (Mr approximately 27000) polypeptide chain. Dissociation into two polypeptide chains was also achieved in 8 M urea. Amidination with dimethylsuberimidate produced a crosslinked protein of molecular weight approximately 90000. In the course of this work a convenient procedure was developed for the determination of gamma-glutamyl transpeptidase activity using L[glycine-2-3H]glutathione as the substrate. In this procedure the release of cysteinyl-[2-3H]glycine from glutathione is followed, after separation of the radioactive di-peptide from unreacted glutathione on a small Dowex-1 acetate column. The reactions with gamma-glutamyl-p-nitroanilide and glutathione are both strongly activated by several metal ions (Ca2+, Mg2+, Na+ and K+) and by a number of amino acids and peptide acceptors. The products of the reaction with glutathione were identified as cysteinylglycine, gamma-glutamylglutathione and glutamate. The formation of these products is consistent with the function of gamma-glutamyl transpeptidase in both the gamma-glutamyl transfer reaction and in the hydrolysis of the gamma-glutamyl bond. The activating effect of metal ions in the reaction with glutathione was shown to be dependent on the acceleration of the transfer reaction; the rate of hydrolysis of the gamma-glutamyl bond remaining unchanged.     

Complete glutathione system in probiotic Lactobacillus fermentum ME-3

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.     

Distribution of the cystine/glutamate antiporter system xc- in the brain, kidney, and duodenum.

System x(c)(-), one of the main transporters responsible for central nervous system cystine transport, is comprised of two subunits, xCT and 4F2hc. The transport of cystine into cells is rate limiting for glutathione synthesis, the major antioxidant and redox cofactor in the brain. Alterations in glutathione status are prevalent in numerous neurodegenerative diseases, emphasizing the importance of proper cystine homeostasis. However, the distribution of xCT and 4F2hc within the brain and other areas has not been described. Using specific antibodies, both xCT and 4F2hc were localized predominantly to neurons in the mouse and human brain, but some glial cells were labeled as well. Border areas between the brain proper and periphery including the vascular endothelial cells, ependymal cells, choroid plexus, and leptomeninges were also highly positive for the system x(c)(-) components. xCT and 4F2hc are also present at the brush border membranes in the kidney and duodenum. These results indicate that system x(c)(-) is likely to play a role in cellular health throughout many areas of the brain as well as other organs by maintaining intracellular cystine levels, thereby resulting in low levels of oxidative stress.     

Acid-volatile selenium formation catalyzed by glutathione reductase.

The production of acid-volatile selenide (apparently H2Se) was catalyzed by glutathione reductase in an anaerobic system containing 20 mM glutathione, 0.05 mM sodium selenite, a TPNH-generating system, and microgram quantities of highly purified yeast glutathione reductase. H2Se production in this system was proportional to glutathione reductase concentration and was maximal at pH 7. Significant nonenzymic H2Se production occurred in the system lacking glutathione reductase and TNPH. A concentration of arsenite (0.1 mM) which does not inhibit glutathione reductase inhibited selenide volatilization, as did bovine serum albumin (1.67 mg/ml). Both appear to inhibit Se volatilization by reacting with the selenide product(s). The selenotrisulfide derivative of glutathione (GSSeSG) was readily converted to H2Se by glutathione reductase and TPNH without the addition of glutathione. These results suggest that GSSeSG formed nonenzymically from glutathione and selenic undergoes stepwise reduction by glutathione reductase (or excess GSH) to GSSeH and finally to H2Se. The same pathway operates when glutathione is used as the reducing agent but to a lesser extent.     

Diet, DDT, and the toxicity of drugs and chemicals.

The toxic and carcinogenic effects of many compounds depend on their activation to reactive molecules in the cytochrome P-450 system of the endoplasmic reticulum of cells. Changes in dietary input alter P-450 levels in different tissues and so alter toxicity. In this way, low protein diets protect against carbon tetrachloride poisoning. Fats, proteins, and nonnutrients such as flavones, antioxidants, and contaminants like DDT all affect P-450 levels. The activated molecules may be diverted from their target sites by inactivation processes, such as epoxide hydratase or glutathione trappin. This is also under nutritional control. Low protein diets render animals sensitive to acetaminophen by reducing glutathione levels. In the sequence of events leading from initial contact of toxin with organism to eventual cell injury or neoplasm, nutritional factors are of import at every stage. Assessments of the toxicity of chemicals that do not take into account the nutritional variable in man are likely to be incorrect.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.     

Plasmoredoxin, a novel redox-active protein unique for malarial parasites.

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development. Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m-1.s-1 at pH 6.9 and 25 degrees C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites. Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.     

Role of the N-terminus of glutathione in the action of yeast glyoxalase I.

A number of S-substituted glutathiones and the corresponding N-substituted S-substituted analogues have been found to be linear competitive inhibitors of yeast glyoxalase I at 26 degrees C over the pH range 4.6-8.5. N-Acetylation of S-(p-bromobenzyl)glutathione weakens binding by 13.7-fold. N-benzoylation by 25.6-fold, N-trimethylacetylation by 53.3-fold and N-carbobenzoxylation by 7.8-fold, indicating a minor steric component in the binding at the N-site. The Ki-weakening effect of N-substitution of glutathione depends on the chemical nature of the S-substituent, indicating flexibility in the glutathione and/or glyoxalase I contributions to the binding site for glutathione derivatives. The effect of N-acylation on Ki is in accord with a charge interaction of the free enzyme with S-blocked glutathione in a region of reasonably high dielectric constant. There is a slight pH effect on Ki for S-(m-trifluoromethylbenzyl)glutathione but not for S-(p-bromobenzyl)glutathione.     

Revisiting the interaction of the radical anion metabolite of nitrofurantoin with glutathione.

There have been several conflicting reports as to the scavenging nature of glutathione toward the nitro radical anion of the drug nitrofurantoin. We produced the radical anion enzymatically using the xanthine oxidase/hypoxanthine system at pH 7.4 and pH 9.0 in the presence of various levels of glutathione from 10 to 100 mM and monitored any changes in the radical concentration via electron spin resonance spectroscopy. Independent of glutathione concentration, there was no decrease in the steady-state concentration of the radical. In fact, there was an average 30% increase in the concentration of the radical anion, which suggests enhanced enzyme activity in the presence of glutathione (GSH). These results, together with observations of the effects of glutathione on the stability of the radical anion generated by radiolysis or dithionite, rule out any detectable reaction between the nitrofurantoin radical anion and GSH under physiologically relevant conditions.     

Epigenetic formation of lactate dehydrogenase isozymes in the house mouse, Mus musculus.

The origin of mouse lactate dehydrogenase (LDH) sub-bands was investigated by using our miniaturized polyacrylamide gel electrophoretic apparatus. Mouse LDH isozymes are generated by combinations of three types of A subunit, the primary type and two epigenetically modified forms. These are designated A1, A2, and A3 in the order of their electrophoretic mobilities towards the anode. The A1 subunit arises from the covalent binding of molecules of glutathione through disulfide bonds to the original subunit, A3. The A2 subunit arises from the covalent binding of molecules of cysteine through disulfide bonds to the A3 subunit. All isozymes can be explained as tetramers composed of the three kinds of A subunit (A1, A2, or A3) in combination with B subunits to yield a total of 35 isozymes. The kinetic properties of these sub-bands were also examined. There was no difference between A24 and A34 in the Km for pyruvate and for lactate. Thermostability at 56 degrees C was greater for A34 than for A24. The activities of tetramers at the electrophoretic position of A3B1 and A4 in extracts containing all five isozymes were increased by treatment of the extracts with high concentrations of reduced glutathione or cysteine with the concomitant disappearance or decrease in activity of tetramers at the position of B4 and A3B1. These results suggest that, in the presence of reduced glutathione or cysteine, LDH isozymes containing the B subunit are first dissociated and then the A subunits are preferentaially recombined.     

The Activities of Antioxidant Enzymes and the Glutathione Content of the Digestive Organs in Marine Invertebrates from Possiet Bay,Sea of Japan

The main components of the antioxidant (AO) system, that is, the activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, as well as the glutathione content of cells of the digestive organs, have been measured in 26 species of marine invertebrates that belong to four taxonomic groups from the Possiet Bay, Sea of Japan. It has been shown that the activities of antioxidant enzymes and glutathione content are species specific. In the digestive organs of echinoderms, the activities of antioxidant enzymes and the glutathione content are generally higher compared with those in mollusks. All the studied species exhibit the greatest variability in the activities of catalase and glutathione peroxidase; the lowest variability occurred in activities of superoxide dismutase and glutathione content. The possible causes of the differences in the levels of the investigated components of the AO system are discussed.     

Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.     

Complete glutathione system in probiotic <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> ME-3

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.