Effects of aging and cyclosporin treatment on the hepatobiliary efflux of glutathione

The aim of this study was to investigate the effects of cyclosporin (CyA) treatment on biliary glutathione efflux in rats of different ages (1, 2, 4, and 24 months). CyA treatment reduced the liver content of total glutathione in 1-, 2- and 24 month old rats (-30%, -43% and -30%, respectively). By contrast, oxidized glutathione (GSSG) concentration in liver tended to increase, although non significantly, in the rats aged 4 and 24 month (+36% and +28%, respectively). The oxidized-to-reduced glutathione ratio was significantly increased in 2-, 4- and 24 month old animals (+23%, +36% and >100%, respectively). Regarding biliary glutathione, our data indicate that efflux rates of total glutathione in control (untreated) rats increased to a maximum at 4 months, and decreased (-56%) in 24 month old rats, although values were still higher than those from young animals. CyA treatment significantly reduced biliary glutathione secretion except in 24 month old rats (-98%, -66% and -32%, at 1, 2 and 4 month, respectively). In addition, following inhibition of the intrabiliary catabolism of the tripeptide by acivicin, glutathione efflux rates into bile were significantly reduced by the drug only in 1- and 2 month old rats (-29% and -55%, respectively) and even tended to increase, although non significantly, in oldest animals. Our data indicate that inhibition of biliary glutathione efflux by CyA was greater in younger rats and support the view that increased intrabiliary catabolism of the tripeptide and inhibition of its canalicular transport could contribute to the decline in biliary glutathione secretion induced by the drug.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.     

Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.     

Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant

The ability of glutathione to scavenge the superoxide radical is a matter of serious contention in the literature: reported values for the second-order rate constant range from 10(2) to greater than 10(5) M(-1) s(-1). The physiological implications of this discrepancy will determine, for example, whether or not glutathione can compete with Mn-superoxide dismutase for reaction with the radical in the mitochondrial matrix, leading to formation of the potentially harmful glutathionyl radical. Several authors have investigated the kinetics of glutathione oxidation by superoxide using spectrophotometric assays, based on competition between either ferricytochrome c or epinephrine for reaction with the radical. However, these approaches have received criticism because the contributions of various secondary reactions to the overall kinetics have been largely overlooked (e.g., the reduction of ferricytochrome c by glutathione). In the present investigation, we have used electron paramagnetic resonance spectroscopy to monitor competition between GSH and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide. This method has been used previously and a rate constant of 1.8 x 10(5) M(-1) s(-1) obtained (Dikalov, S.; Khramtsov, V.; Zimmer, G. Arch. Biochem. Biophys. 326:207-218; 1996). However, we demonstrate that this value is a gross overestimation because the spectrum of the hydroxyl radical adduct of the spin trap was incorrectly assigned to the glutathionyl radical adduct. The relatively high yield of the DMPO hydroxyl radical adduct is shown to be due to the two-electron reduction of the corresponding superoxide radical adduct by glutathione. Taking these factors into consideration, we estimate the second order rate constant for the oxidation of glutathione by superoxide to be approximately 200 M(-1) s(-1).     

Mutagenicity of the glutathione and cysteine S-conjugates of the haloalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene and trichlorofluoroethene in the Ames test in comparison with the tetrachloroethene-analogues

The nephrotoxic and nephrocarcinogenic potential of the haloalkenes is associated with the conjugation of the chemicals to L-glutathione. Subsequent processing of the haloalkene glutathione S-conjugates via the cysteine conjugate beta-lyase pathway in the mammalian kidney yields nephrotoxic and mutagenic species. To investigate whether S-conjugates of the model chlorofluoroalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene (CAS # 431-52-7) and trichlorofluoroethene (CAS # 359-29-5) show comparable effects, we have synthesised the respective cysteine and glutathione S-conjugates and subjected them to the Ames test. The cysteine and glutathione S-conjugates of tetrachloroethene (CAS # 127-18-4), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) and S-(1,2,2-trichlorovinyl)glutathione (TCVG) were used as positive controls and reference substances. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)-L-cysteine (DCTFPC) and S-(2,2-dichloro-1-fluorovinyl)-L-cysteine (DCFVC) showed clear dose-dependent mutagenic effects with the Salmonella typhimurium tester strains TA100 and TA98. Using TCVC as a reference substance the following ranking in mutagenic response was established: TCVC>DCTFPC>DCFVC. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)glutathione (DCTFPG) and S-(2,2-dichloro-1-fluorovinyl)glutathione (DCFVG) showed potent dose-dependent mutagenic effects with the S. typhimurium tester strain TA100 in the presence of a rat kidney S9-protein fraction; tests carried out in the absence of the bioactivation system resulted only in background rates of revertants. Using TCVG as a reference substance the following ranking in mutagenic response was established: TCVG=DCTFPG>DCFVG.The data obtained provide a basis for further studies on the mutagenic and presumable carcinogenic potential of the substances.     

Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman's reagent

Evaluation of the kinetic parameters of the various reactions involved in the determination of glutathione provided the rationale for a modification of the frequently used assay (F. Tietze, 1969, Anal. Biochem. 27, 502-522) whereby the enzymatic reaction is no longer rate limiting. At pH 6.0, the nonenzymatic thiol interchange reaction of reduced glutathione (GSH) with Ellman's reagent becomes rate limiting, and inhibition of glutathione reductase up to 50% has no influence on the accuracy of the determination. The lower level of sensitivity is 10(-10) mol glutathione with a linear response up to 5 X 10(-9) mol. For determination of glutathione disulfide, GSH is alkylated by N-ethylmaleimide (NEM), and excess NEM is removed by extraction with ethyl acetate. Since the glutathione adduct is not stable, extracted samples are kept deep-frozen prior to analysis. Using this precaution, less than 0.05% of GSSG was determined in GSH-containing samples which had been previously freed from GSSG.     

Plasma glutathione turnover in the rat: effect of fasting and buthionine sulfoximine

Hepatic glutathione (GSH) plays an important role in the detoxification of reactive molecular intermediates. Because of evidence that the intrahepatic turnover of glutathione in the rat may be largely accounted for by efflux from hepatocytes into the general circulation, the quantitation of plasma GSH turnover in vivo could provide a noninvasive index of hepatic glutathione metabolism. We developed a method to estimate plasma glutathione turnover and clearance in the intact, anesthetized rat using a 30-min unprimed, continuous infusion of 35S-labelled GSH. A steady state of free plasma glutathione specific radioactivity was achieved within 10 min, as determined by high-pressure liquid chromatography with fluorometric detection after precolumn derivatization of the plasma samples with monobromobimane. The method was tested after two treatments known to alter hepatic GSH metabolism: 90 min after intraperitoneal injection of 4 mmol/kg buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and after a 48-h fast. Liver glutathione concentration (mean +/- SEM) was 5.00 +/- 0.53 mumol/g wet weight in control rats. It decreased to 3.10 +/- 0.35 mumol/g wet weight after BSO injection and to 3.36 +/- 0.14 mumol/g wet weight after fasting (both p less than 0.05). Plasma glutathione turnover was 63.0 +/- 7.46 nmol.min-1.100 g-1 body weight in control rats, 35.0 +/- 2.92 nmol.min-1.g-1 body weight in BSO-treated rats, and 41.7 +/- 2.28 nmol.min-1.g-1 body weight after fasting (both p less than 0.05), thus reflecting the hepatic alterations. This approach might prove useful in the noninvasive assessment of liver glutathione status.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

Direct measurement of the rate of glutathione synthesis in 1-chloro-2,4-dinitrobenzene treated human erythrocytes

Cell glutathione scavenges free radicals, degrades peroxides, removes damaging electrophiles and maintains the redox state. The aim of this study was to develop an effective and efficient method to measure the rate of glutathione synthesis from its constituent amino acids in whole erythrocytes (RBCs). RBCs (10% haematocrit) were exposed to 0.3 mM 1-chloro-2,4-dinitrobenzene (CDNB) to lower their total glutathione content by 70% and then incubated with glucose, and N-acetylcysteine as a cysteine source. Over 3 h, glutathione levels increased at a constant rate of 1.2 micromol (L RBC)(-1)min(-1), almost 5 times faster than the rate of glutathione synthesis in RBCs with normal glutathione levels. Glutathione at concentrations normally found in RBCs is known to inhibit glutamate cysteine ligase (the major rate controlling enzyme for glutathione synthesis). The rate of glutathione recovery was substantially reduced in RBCs treated with buthionine sulfoximine, a specific inhibitor of glutamate cysteine ligase. Our results indicate that the measurement of glutathione recovery rate after CDNB treatment can be used to estimate de novo synthesis of glutathione. Application of this direct method for measuring glutathione synthesis will increase understanding of the interactions of effectors that determine glutathione levels in RBCs under various physiological and pathological conditions.     

Glutathione metabolism under the influence of hydroperoxides in the lactating mammary gland of the rat. Effect of glucose and extracellular ATP

Tert-butyl hydroperoxide decreases GSH and total free glutathione (GSH+2GSSG) contents of acini from lactating mammary glands. The decrease in total free glutathione can be explained by an increase in mixed disulfide formation and by excretion of GSS G to the extracellular medium, and subsequent degradation catalyzed by gamma-glutamyl transpeptidase. Low concentrations of glucose prevented the changes in glutathione levels induced by the peroxide. In the presence of extracellular ATP, glucose did not prevent these changes. However, incubations with the peroxide, did not alter the rate of other metabolic pathways by acini.Abbreviations used GSH Reduced glutathione - GSSG Glutathione disulfide - GSSR Glutathione mixed disulfide - GGT Gamma-glutamyl transpaptidase - tbOOH Tert-butyl hydroperoxide     

Virus-induced changes in the subcellular distribution of glutathione precursors in Cucurbita pepo (L.)

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene.     

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.     

Investigating the antimicrobial,antioxidant and cytotoxic activities of the biological synthesized glutathione selenium nano-incorporation

BioMetals - Aqueous glutathione selenium nano-incorporation (GSH-SeN-Inco) was prepared by gamma radiation in presence of microbial glutathione (GSH) and selenium dioxide. The novel prepared...     

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.     

State of the liver antioxidant system and content of matrix metalloproteinase-2 of the large intestine under the effect of maleimide derivative in experimental colorectal carcinogenesis in rats

The maleimide derivative--1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2.5-dione (MI-1) with cytostatic activity did not cause substantial changes of liver antioxidant system and level of matrix metalloproteinase-2 in intestinal mucosa after chronic treatment (for 20 weeks). MI-1 did not cause significant changes in the content of thiobarbituric-active products and plasma membrane protein carbonyl groups in the rat liver. However activities of superoxide dismutase, glutathione peroxidase, and content of reduced glutathione were decreased in both doses--0.027 and 2.7 mg/kg. The level of matrix metalloproteinase-2 in intestinal mucosa was decreased just in maximum dose--2.7 mg/kg. The contents of thiobarbituric-active products, protein carbonyl groups, reduced glutathione, matrix metalloproteinase-2, activities of glutathione peroxidase and glutathione-S-transferase in the liver cells have increased in 1.2-dimethylhydrazine-induced colon cancer in rats. The activities of enzymes of the first line of antioxidant defense--superoxide dismutase and catalase were decreased to 40%. The maleimide derivative prevents development of oxidation stress and partially reduce them to control level.