Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The crystal structure of class Pi glutathione S-transferase from porcine lung (pGST P1-1) in complex with glutathione sulphonate has been refined at 2.11 A resolution, to a crystallographic R-factor of 16.5% for 21, 165 unique reflections. The refined structure includes 3314 protein atoms, 46 inhibitor (glutathione sulphonate) atoms and 254 water molecules. The model shows good stereochemistry, with root-mean-square deviations from ideal bond lengths and bond angles of 0.011 A and 2.8 degrees, respectively. The estimated root-mean-square co-ordinate error is 0.2 A. The protein is a dimer assembled from identical subunits of 207 amino acid residues. The tertiary structure of the pGST P1 subunit is organized as two domains, the N-terminal domain (domain I, residues 1 to 74) and the larger C-terminal domain (domain II, residues 81 to 207). Glutathione sulphonate, a competitive inhibitor, binds to the G-site region (i.e. the glutathione-binding region) of the active site located on each subunit. Each G-site is, however, structurally dependent of the neighbouring subunit as structural elements forming a fully functional G-site are provided by both subunits, with domain I as the major supporting framework. A number of direct and water-mediated polar interactions are involved in sequestering the glutathione analogue at the G-site. The extended conformation assumed by the enzyme-bound inhibitor as well as the strategic interactions between inhibitor and protein, closely resemble those observed for the physiological substrate, reduced glutathione bound at the active site of class Mu glutathione S-transferase 3-3. Hydrogen bonding between the sulphonyl moiety of the inhibitor and the hydroxyl group of an evolutionary conserved tyrosine residue, Tyr7, provides the first direct structural evidence for a catalytic protein group in glutathione S-transferases that is involved in the activation of the substrate glutathione. The catalytic role for Tyr7 has subsequently been confirmed by mutagenesis and kinetic studies. Comparison of the known crystal structures for class Pi, class Mu and class Alpha isoenzymes, indicates that the cytosolic glutathione S-transferases share a common fold and that the structural features for catalysis are similar.
J Mol Biol 1994 Oct 14
PMID:Refined crystal structure of porcine class Pi glutathione S-transferase (pGST P1-1) at 2.1 A resolution. 793 43

Glutathione S-transferases (GSTs) constitute a major detoxification mechanism in helminth organisms and are regarded vaccine candidates against helminth infections. Onchocerca volvulus glutathione-binding proteins were purified from the aqueous soluble fraction of homogenised adult females by affinity chromatography on glutathione-agarose. The eluted proteins had a specific GST activity of 1.6 mumol min-1 mg-1. Immunohistochemical studies localised these antigens in the hypodermis, the wall of the seminal receptacle and spermatozoa of adult worms. A lambda gt11 clone was isolated from an expression library of O. volvulus by immunoscreening. Sequence analysis revealed that it encoded a pi-class GST with 60% identity with Caenorhabditis elegans and up to 45% identity with mammalian pi-class GSTs. Antibodies affinity selected with recombinant GST demonstrated cross-reactivity between Litomosoides sigmodontis and O. volvulus GSTs.
Mol Biochem Parasitol 1994 Jul
PMID:Molecular characterisation and localisation of an Onchocerca volvulus pi-class glutathione S-transferase. 798 70

Dichloroacetylene causes trigeminal neuropathy in humans and animals. Glutathione conjugation of dichloroacetylene affords S-(1,2-dichlorovinyl)glutathione (DCVG), which is hydrolyzed to S-(1,2-dichlorovinyl)-L-cysteine (DCVC). This study was undertaken to test the hypothesis that the neurotoxicity of dichloroacetylene may be associated with glutathione S-conjugate formation and brain uptake and bioactivation of the dichloroacetylene-derived S-conjugates. With the Oldendorf technique, the Brain Uptake Index for [35S]DCVC and [35S]DCVG was determined and compared with the uptake of [35S]methionine and [14C]sucrose. Brain uptake of DCVC exceeded uptake of methionine and DCVG uptake was comparable to methionine uptake. Both [35S]DCVC and [35S]DCVG were recovered intact in brain tissue. The uptake of the 35S-labeled S-conjugates was inhibited by unlabeled DCVC and DCVG in a concentration-dependent manner. The data indicated that DCVC, but not DCVG, was transported by the sodium-independent system-L transporter for neutral amino acids. In vitro studies revealed that DCVG can be hydrolyzed to DCVC by brain tissue in a concentration-dependent manner.
Brain Res Mol Brain Res 1993 Jan
PMID:Brain uptake of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the glutathione and cysteine S-conjugates of the neurotoxin dichloroacetylene. 838 9

The current studies were designed to test two hypotheses: (1) differences in steady-state reduced glutathione levels are responsible for subcompartment differences in susceptibility to acute ozone injury, and (2) elevation of reduced glutathione concentrations accounts for the tolerance to further injury produced by repeated ozone exposure. Glutathione was measured in well-defined subcompartments of the lung of both rats and monkeys to compare alterations occurring in both target (distal trachea and terminal bronchiole) and nontarget areas (lobar bronchus, major daughter, minor daughter bronchus, and parenchyma) of the lung in species that differ in sensitivity to ozone exposure (rat is less susceptible than monkey). Glutathione concentrations were decreased in trachea of rats exposed to 0.4 ppm ozone for 2 h and increased in lobar bronchus and distal bronchiole after 2 h exposure at 1 ppm. In monkey, glutathione levels in most subcompartments were not altered by either 0.4 or 1.0 ppm ozone exposure for 2 h. The exceptions were the major daughter subcompartment (200% of control at 0.4 ppm exposure) and the distal bronchiole (55% of control at 1 ppm exposure). Ninety day ozone exposures (6 h/day x 5 days/week) in rats produced an elevation in glutathione (164% of control value) only in distal bronchiole at the 1 ppm exposure level. In a similar manner, glutathione levels in the distal bronchiole of monkeys exposed for 90 days to 1 ppm O3 were 165% of the corresponding control values. These results suggest the following: glutathione levels in target and nontarget areas of the lung and in susceptible versus less susceptible species are not the primary determinant in the differences observed in ozone toxicity; the response of lung subcompartments to short-term ozone exposure varied depending on airway subcompartment and species; increased glutathione levels may be one reason for adaptation of some airway epithelial cells from rats and monkeys exposed to O3 for long periods; and use of well-defined segments of the lung provides a means of assessing changes in target areas of the lung without dilution from nontarget areas.
Am J Respir Cell Mol Biol 1996 Jan
PMID:Ozone-induced alterations in glutathione in lung subcompartments of rats and monkeys. 853 88

Glutathione S-transferases (GST) are a family of multifunctional enzymes involved in the metabolization of a broad variety of xenobiotics and reactive endogenous compounds. The interest in plant glutathione S-transferases may be attributed to their agronomic value, since it has been demonstrated that glutathione conjugation for a variety of herbicides is the major resistance and selectivity factor in plants. The three-dimensional structure of glutathione S-transferase from the plant Arabidopsis thaliana has been solved by multiple isomorphous replacement and multiwavelength anomalous dispersion techniques at 3 A resolution and refined to a final crystallographic R-factor of 17.5% using data from 8 to 2.2 A resolution. The enzyme forms a dimer of two identical subunits each consisting of 211 residues. Each subunit is characterized by the GST-typical modular structure with two spatially distinct domains. Domain I consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side by an irregular segment containing three short 3(10)-helices, while domain II is entirely helical. The dimeric molecule is globular with a prominent large cavity formed between the two subunits. The active site is located in a cleft situated between domains I and II and each subunit binds two molecules of a competitive inhibitor S-hexylglutathione. Both hexyl moieties are oriented parallel and fill the H-subsite of the enzyme's active site. The glutathione peptide of one inhibitor, termed productive binding, occupies the G-subsite with multiple interactions similar to those observed for other glutathione S-transferases, while the glutathione backbone of the second inhibitor, termed unproductive binding, exhibits only weak interactions mediated by two polar contacts. A most striking difference from the mammalian glutathione S-transferases, which share a conserved catalytic tyrosine residue, is the lack of this tyrosine in the active site of the plant glutathione S-transferase.
J Mol Biol 1996 Jan 19
PMID:Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. 855 21

The influence of altered levels of endogenous catecholamines following adrenalectomy or 6-hydroxydopamine (6-OH) treatment (alone or in combination) on enzymatic (glutathione reductase, catalase, glutathione peroxidase and Cu, Zn superoxide dismutase) and non-enzymatic (glutathione) antioxidant components of heart, liver, kidney, lung and erythrocytes in male Wistar rats was investigated. Functional antioxidant status was assessed in terms of susceptibility to t-butylhydroperoxide-induced sulfhydryl group oxidation (an indirect measure of glutathione depletion) and lipid peroxidation, as measured by thiobarbituric acid-reactive substance (TBARS) formation. Reduced levels of adrenaline and noradrenaline resulted from adrenalectomy and 6-OH treatment, respectively, while a combination of these treatments led to a reduction in the levels of both catecholamines. Adrenalectomy was associated with alterations in glutathione reductase activity in the heart and liver (increased). 6-OH treatment alone produced an elevation in glutathione reductase activity only in the heart. In adrenalectomized animals, 6-OH treatment produced no further increases in glutathione reductase activities of heart or liver. In lung, however, the combination of adrenalectomy and 6-OH treatment caused an elevation in both glutathione peroxidase and glutathione reductase activities. Glutathione levels of liver alone were elevated following adrenalectomy, while those of erythrocytes and liver (but not other tissues investigated) were increased by the combination of adrenalectomy and 6-OH treatment. The kidney was relatively resistant to the effects of sympathectomy and showed no changes in any of the antioxidant components measured. Adrenalectomy alone or in combination with 6-OH produced an increased in susceptibility to peroxide-induced sulfhydryl group oxidation only in the heart. 6-OH treatment caused a reduction in peroxide-induced TBARS formation only in the kidney. Both adrenalectomy and the combination of adrenalectomy and 6-OH treatment were associated with reduced TBARS formation in the liver, lung and kidney, but not heart. Results from this study demonstrate that the effects of sympathectomy on antioxidant status vary among tissues. Differences between adrenalectomy and 6-OH treatment on antioxidant components are suggestive of differential actions of adrenaline and noradrenaline on tissue antioxidant status which may have important implications under conditions associated with elevations in levels of these catecholamines including chronic stress and myocardial infarction.
Mol Cell Biochem 1995 Nov 08
PMID:Alteration of antioxidant status following sympathectomy: differential effects of modified plasma levels of adrenaline and noradrenaline. 860 10

Glutathione (GSH) depletion by buthioninine sulfoximine (BSO) is being explored clinically as a means of enhancing the efficacy of cancer chemotherapy. We investigated the kinetics of GSH depletion and altered gamma-L-glutamyl-L-cysteine synthetase (gamma-GC-S) gene expression in two human malignant glioma cell lines, HBT5 and HBT28, and examined how these relate to GSH resynthesis and changes in DNA interstrand cross-link induction and cytotoxicity of 1,3-bis(2-chloroethyl)-nitrosourea (BCNU). GSH content was 54 and 126 nmol/mg/protein in HBT 5 and HBT 28, respectively, and after a 24-hr exposure to 100 microM BSO was decreased by 95% in HBT 5 and 91% in HBT 28. Basal gamma-GC-S enzyme activity in HBT 28 was twice that in HBT 5, and steady state gamma-GC-S gene transcripts were 2.6-fold higher in HBT 28 than in HBT 5, with no apparent amplification or rearrangement of the gene in either cell line. BSO exposure (100 microM) for 24 hr increased gamma-GC-S gene transcripts by 1.7-fold in HBT 5 and 2.8-fold in HBT 28. After BSO removal, the rate of GSH resynthesis in HBT 28 was twice that in HBT 5. Continuous BSO exposure increased the level of BCNU-induced DNA interstrand cross-links, and cytotoxicity was significantly higher in cells exposed continuously to BSO than in cells with only a 24-hr BSO preexposure. This increase was, however, greater in HBT 28 than in HBT 5. These findings indicate significant heterogeneity in the effects of BSO on gamma-GC-S gene expression and in the ability of BSO to sensitize tumors and cell lines to BCNU. The data also suggest that by preventing GSH resynthesis, a greater level of cytotoxicity is achieved with continuous BSO exposure than with BSO preexposure alone.
Mol Pharmacol 1996 Jun
PMID:Buthionine sulfoximine induction of gamma-L-glutamyl-L-cysteine synthetase gene expression, kinetics of glutathione depletion and resynthesis, and modulation of carmustine-induced DNA-DNA cross-linking and cytotoxicity in human glioma cells. 864 39

In the present studies we have described a glutathione-dependent system in sheep liver microsomes that protects against membrane lipid peroxidation initiated by either Fe+2/NADPH or Fe+2/ascorbate. Glutathione protected against lipid peroxidation in microsomes containing a wide range of alpha-tocopherol levels (0.02-0.11 microgram/mg protein). The addition of glutathione disulfide alone had no effect on microsomal lipid peroxidation, however, it prolonged the protection afforded by glutathione, particularly in assays containing Fe+2/NADPH. Whereas the glutathione-dependent protection was very labile, with loss of activity demonstrated in microsomes stored at 4 degrees C for 24 hours, the combined effect of glutathione and glutathione disulfide was not affected by storage. The glutathione S-transferase inhibitors, bromosulphothalein and S-hexylglutathione, reversed the protection observed with glutathione, indicating a possible role for microsomal glutathione S-transferases in this protection, but not that observed by the combination of glutathione and glutathione disulfide. In general, our findings support previous results observed with rat liver microsomes and suggest that microsomal glutathione S-transferases may be involved in the glutathione-dependent protection in sheep liver microsomes.
Biochem Mol Biol Int 1996 Mar
PMID:Glutathione-dependent protection against lipid peroxidation in sheep liver microsomes. 882 16

Glutathione (GSH) is an abundant cellular thiol which has been implicated in many cellular processes including protection against xenobiotics, carcinogens and free radicals. Utilization of GSH in both enzymic and non-enzymic defence mechanisms results in its conversion to the oxidized form (GSSG), and it must be recycled to GSH to maintain the high intracellular ratio of GSH to GSSG. Glutathione reductase (GLR) is a flavoenzyme, which catalyses reduction of GSSG to GSH using the reducing power of NADPH. We show that yeast mutants deleted for GLR1, encoding glutathione reductase, lack GLR activity and accumulate increased levels of GSSG. In addition, the glr1 mutant strain was unaffected in the inducible adaptive response to hydrogen peroxide, but showed increased sensitivity to oxidants including both peroxides and superoxide, indicating a requirement for GLR in protection against oxidative stress. Furthermore, GLR1 expression was elevated two to threefold in the presence of oxidants, and regulation was dependent upon the yAP-1 transcriptional activator protein. Thus, GLR1 is one of a growing number of genes involved in the protection of yeast cells against oxidative stress and regulated by yAP-1.
Mol Microbiol 1996 Jul
PMID:Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation. 884 43

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated from Arabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast, Schizosaccharomyces pombe, and shared only a small region of similarity with the Escherichia coli protein. A 4.3 kb SstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons. When the Arabidopsis cDNA cloned in a special shuttle vector was expressed in a S. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in the gsh2- mutant, and restored to substantial levels by the expression of the Arabidopsis cDNA. The S. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols, 109Cd2+ binding activity, and cadmium resistance. Since the Arabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.
Plant Mol Biol 1996 Sep
PMID:Cloning of the cDNA and genomic clones for glutathione synthetase from Arabidopsis thaliana and complementation of a gsh2 mutant in fission yeast. 891 26


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