Glutathione transferases--structure and catalytic activity

The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Characterization, kinetics and comparative properties of thiol:protein disulfide oxidoreductase.

An enzyme capable of catalyzing thiol:protein disulfide exchange with glutathione and insulin has been purified to apparent homogeneity as judged by disc electrophoresis, electrophoresis with sodium dodecyl sulfate, equilibrium ultracentrifugation and amino acid sequence analysis of the glycoprotein. The isoelectric point of 4.10 and other physicochemical properties are somewhat different from those of similar enzymes described by other investigators, although there are similarities in substrate specificity and kinetic parameters. The solubilized enzyme catalyzes disulfide interchange with glutathione as well as with a variety of other thiol-containing compounds. Although insulin is the best protein substrate tested, vasopressin, oxytocin, and ribonuclease are also utilized. In the presence of glutathione, the enzyme catalyzes the restoration of the active conformation of “scrambled” ribonuclease. Catalysis depends on an active cysteine residue which can be alkylated/inactivated only in the presence of thiol-containing compounds.     

Glutathione peroxidase activities from rat liver

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.     

Human glutathione S-transferases. Characterization of the anionic forms from lung and placenta.

Anionic glutathione S-transferases were purified from human lung and placenta. Chemical and immunochemical characterization, including polyacrylamide-gel electrophoresis, gave strong evidence that the anionic lung and placental enzymes are chemically similar, if not identical, proteins. The electrophoretic mobilities of both proteins were identical in conventional alkaline gels as well as in gels containing sodium dodecyl sulphate. Gel filtration of the intact active enzyme established an Mr value of 45000; however, with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under dissociating conditions a subunit Mr of 22500 was obtained. Amino acid sequence analysis of the N-terminal region of the placental enzyme revealed a single polypeptide sequence identical with that of lung. Results obtained from immunoelectrophoresis, immunotitration, double immunodiffusion and rocket immunoelectrophoresis also indicated the anionic lung and placental enzymes to be closely similar. The chemical similarity of these two proteins was further supported by protein compositional analysis and fragment analysis after chemical hydrolysis. Immunochemical comparison of the anionic lung and placental enzymes with human liver glutathione S-transferases revealed cross-reactivity with the anionic omega enzyme, but no cross-reactivity was detectable with the cationic enzymes. Comparison of the N-terminal region of the human anionic enzyme with reported sequences of rat liver glutathione S-transferases gave strong evidence of chemical similarity, indicating that these enzymes are evolutionarily related. However, computer analysis of the 30-residue N-terminal sequence did not show any significant chemical similarity to any other reported protein sequence, pointing to the fact that the glutathione S-transferases represent a unique class of proteins.     

S-(2,3-dichlorotriazinyl)glutathione. A new affinity label for probing the structure and function of glutathione transferases.

S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I (GST I). Inactivation of GST I by SDTG at pH 6.5 followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase, leading to 42 +/- 3% residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration, consistent with the formation of a reversible complex with the enzyme (K(d) 107.9 +/- 2.1 micro m for the fast phase, and 224.5 +/- 4.2 micro m for the slow phase) before irreversible modification with maximum rate constants of 0.049 +/- 0.002 min(-1) and 0.0153 +/- 0.001 min(-1) for the fast and slow phases, respectively. Protection from inactivation was afforded by substrate analogues, demonstrating the specificity of the reaction. When the enzyme was inactivated (42% residual activity), approximately 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis, molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of alpha-helix H"'(3) and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits, which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus, modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites.     

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.     

Immunolocalization of antioxidant enzymes in adult hamster kidney

Summary Immunoperoxidase and immunogold techniques were used to localize the following antioxidant enzyme systems in the adult hamster kidney at the light and ultrastructural levels: superoxide dismutases, catalases, peroxidases and glutathione S-transferases. Each cell type in the kidney showed specific patterns of labelling of these enzymes. For example, proximal and distal tubular and transitional epithelial cells showed significant staining for all of these enzymes, while glomerular cells and cells of the thin loop of Henle did not show significant staining at the light microscope level. In addition, high levels of glutathione peroxidase were found in smooth muscle cells of renal arteries. At the ultrastructural level, each enzyme was found in a specific subcellular location. Manganese superoxide dismutase was found in mitochondria, catalase was localized in peroxisomes, while copper, zinc superoxide dismutase and glutathione S-transferase (liver and placental forms) were found in both the nucleus and cytoplasm. Glutathione peroxidase was found to have a broad intracellular distribution, with localization in mitochondria, peroxisomes, nucleus, and cytoplasm. Microvilli of tubular cells were labelled by antibodies to catalase, copper, zinc superoxide dismutase, glutathione peroxidase, and glutathione S-transferases. Cell types that were negative by light microscopy immunoperoxidase studies showed definite labelling with immunogold post-embedding ultrastructural techniques (glomerular cells and cells of the loop of Henle), demonstrating the greater sensitivity of the latter technique. These observations demonstrate that there are large variations in the levels of antioxidant enzymes in different cell types, and that even within a distinct cell type, the levels of these enzymes vary in different subcellular locations. Our results demonstrate for the first time the overall antioxidant enzyme status of individual kidney cell types, thereby explaining why different cell types have differing susceptibilities to oxidant stress. Possible physiological and pathological consequences of these findings are discussed.     

Emergence of novel enzyme quasi-species depends on the substrate matrix

Current research on enzyme evolution has shown that many enzymes are promiscuous and have activities with alternative substrates. Mutagenesis tends to relax substrate selectivity, and evolving enzymes can be regarded (summed over evolutionary time) as clusters of enzyme variants, or “quasi-species,” tested against a “substrate matrix” defined by all chemical substances to which the evolvants are exposed.In this investigation, the importance of the substrate matrix for identification of evolvable clusters of enzymes was evaluated by random sampling of variants from a library of glutathione transferase (GST) mutants. The variant GSTs were created by DNA shuffling of homologous Alpha class sequences. The substrate matrix was an array of alternative substrates used under defined experimental conditions. The measured enzyme activities produced a rectangular matrix, in which the rows can be projected as enzyme vectors in substrate-activity space and, reciprocally, the columns can be projected as alternative substrate vectors in enzyme-activity space. Multivariate analysis of the catalytic activities demonstrated that the enzyme vectors formed two primary clusters or functional “molecular quasi-species.” These quasi-species serve as the raw material from which more specialized enzymes eventually could evolve. The substrate vectors similarly formed two major groups. Identification of separate quasi-species of GSTs in a mutant library was critically dependent on the nature of the substrate matrix. When substrates from just one of the two groups were used, only one cluster of enzymes could be recognized. On the other hand, expansion of the substrate matrix to include additional substrates showed the presence of a third quasi-species among the GST variants already analyzed. Thus, the portrayal of the functional quasi-species is intimately linked to the effective substrate matrix. In natural evolution, the substrates actually encountered therefore play a pivotal role in determining whether latent catalytic abilities become manifest in novel enzymes.     

Two histone H1-specific protein-lysine N-methyltransferases from Euglena gracilis. Purification and characterization

Two forms of a histone H1-specific S-adenosylmethionine:protein-lysine N-methyltransferase (protein methylase III) have been purified from Euglena gracilis 48- and 214-fold, respectively, with yields of 3.4 and 4.6%. The enzymes were purified on DEAE-cellulose and histone-Sepharose affinity chromatography and found to be highly specific toward histone H1 as a substrate. However, one of the enzymes also methylates other histone subfractions to a limited extent. Of the proteins other than histones, only myosin showed measurable methyl-accepting capability. Both enzymes were found to be inhibited by S-adenosylhomocysteine (D and L forms), S-adenosyl-L-ethionine, and sinefungin. While the Ki values for S-adenosyl-L-ethionine were similar for both enzymes, the values for S-adenosyl-L-homocysteine and sinefungin were 10-fold lower for the second form. The Km values for histone H1 and S-adenosyl-L-methionine were found to be 3.1 X 10(-7) and 2.7 X 10(-5) M, respectively, for the first enzyme, and 4.4 X 10(-7) and 3.45 X 10(-5) M for the second. Peptide analysis of methyl-14C-labeled H1 revealed that the two enzymes methylate different sites within the histone H1 molecule. The two enzymes were found to have molecular weights of 55,000 and 34,000, respectively. Both enzymes have an optimum pH of 9.0, which is identical to that of other protein-lysine N-methyltransferases thus far identified.     

Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition

The procedure developed for purification of the N-ethylmaleimide-activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition. In addition the microsomal glutathione transferase purified in unactivated form could be activated 15-fold with N-ethylmaleimide to give the same specific activity with 1-chloro-2,4-dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N-ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'-dithiopyridyl and binding of radioactive N-ethylmaleimide. Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1. Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N-ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.     

Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat.

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation

Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.     

Changes in the post-translational modification of lysosomal enzymes during development of Dictyostelium

The form of post-translational modification present on two lysosomal enzymes--acid phosphatase and alpha-mannosidase--changes as part of the developmental program of Dictyostelium discoideum. Prior to 8 h of development, all enzyme molecules are of a single modification type (early form enzyme). Starting at 8 h of development, enzyme molecules with a second type of modification (late-form enzymes) begin to appear in the cell. We separated the early and late forms of these enzymes from each other by chromatography on DEAE-cellulose. We found that the change in protein modification affects the enzymes' in vitro properties. The early and late forms of both of these enzymes differ in thermostability and susceptibility to proteolytic inactivation. We also found that the late form of alpha-mannosidase is preferentially secreted. We suggest that by synthesizing molecules with a second form of modification, the cell confers new characteristics to its lysosomal enzymes.     

Isolation of mutants of Schizosaccharomyces pombe unable to synthesize cadystin, small cadmium-binding peptides

Schizosaccharomyces pombe synthesize small cadmium-binding peptides cadystin, structure of which is (gamma-Glu-Cys)n-Gly, in response to cadmium. Mutants unable to synthesize cadystin were found in the mutants hypersensitive to cadmium. Some of them lack activity of either gamma-glutamylcysteine synthetase (EC 6.3.2.2) or glutathione synthetase (EC 6.3.2.3), enzyme involved in glutathione biosynthesis. Some mutants have the same activity levels of these enzymes as wild type has. These results indicate that some steps of cadystin biosynthesis are catalyzed by the enzymes catalyzing glutathione biosynthesis.     

Species-related variations in antioxidant components of gastric and duodenal mucosa

Enzymatic and non-enzymatic antioxidant profiles of the gastric and duodenal mucosa of rat, rabbit, cat and pig were investigated and found to exhibit significant variations. Rat gastric and duodenal mucosa exhibited the highest levels of basal glutathione of the various tissues examined. The highest activity of glutathione reductase was found in the gastric and duodenal mucosa of rat as compared with that in these tissues from the other species. The gastric mucosa of cat and pig showed similar activities of glutathione peroxidase, which was significantly lower than those in rat or rabbit gastric mucosa. The activity of this antioxidant enzyme was similar in rat, rabbit and pig duodenal mucosa and lower than that in cat duodenal mucosa. Strong correlations were found between activities of the functionally coupled antioxidant enzymes glutathione peroxidase and glutathione reductase in gastric but not in duodenal mucosa. The activity of superoxide dismutase showed negligible regional or species-related variations in activity.     

Antioxidant Defenses in Wild Growing Halophyte Crithmum maritimum from Inland and Coastline Populations

Traditional Mediterranean diet includes the halophyte Crithmum maritimum L. (Apiaceae) which can be found in the coastline of the Balearic Islands but also inland. Both areas differed in the environmental conditions, mainly in salinity which can affect the oxidative status of this species. The aim was to evaluate the antioxidant enzyme activities, polyphenols and the lipid peroxidation in leaves of wild C. maritimum growing in a natural coastal area influenced by marine salinity and an inland area without marine influence. The activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase as well as polyphenol and reduced glutathione content were significantly higher in the samples from coastline population, whereas no significant differences were found in glutathione reductase activity and in malondialdehyde levels. The production of H₂O₂ was also significantly higher in the population from coastline. In conclusion, C. maritimum adapt their antioxidant defense machinery to the different salinity conditions, avoiding the instauration of oxidative stress.