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PubMedDate not supplied

Glutathione and N-acetylcysteine conjugates of 2-chloroethyl isocyanate. Identification as metabolites of N,N'-bis(2-chloroethyl)-N-nitrosourea in the rat and inhibitory properties toward glutathione reductase in vitro.

The antitumor agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) is known to be unstable in aqueous solution, and to degrade spontaneously to reactive alkylating and carbamoylating intermediates. Whereas the alkylating component is believed to be responsible for the antitumor effects of this drug, it has been speculated that the carbamoylating species 2-chloroethyl isocyanate (CEIC) may mediate some of the serious adverse effects of BCNU therapy. In order to determine whether CEIC is released from BCNU in vivo, rats were administered an ip injection of the drug and a targeted search was made by ionspray LC-MS/MS techniques for the glutathione (GSH) conjugate of CEIC in bile and for the corresponding N-acetylcysteine (NAC) adduct in urine. Both of these S-linked conjugates were identified on the basis of their HPLC and MS/MS characteristics, which were identical to those of the respective reference compounds prepared by synthesis. Quantitative studies indicated that, following an ip dose of BCNU (24 mg kg-1), excretion of the GSH conjugate in bile over 4 h accounted for 3.90 +/- 0.64% of the administered dose, while excretion of the mercapturic acid derivative in urine over 24 h accounted for a further 18.1 +/- 3.3% (n = 4). Experiments conducted in vitro demonstrated that the S-linked conjugates of CEIC were of limited stability under simulated physiological conditions, decomposing to generate free GSH and NAC. In addition, both adducts inhibited rat liver glutathione reductase in vitro, when they were essentially equipotent to BCNU.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMedDate not supplied

Investigations of glutathione conjugation in vitro by 1H NMR spectroscopy. Uncatalyzed and glutathione transferase-catalyzed reactions.

Conjugation reactions of glutathione (GSH) and related thiols with diethyl maleate (DEM) and other alpha, beta-unsaturated carbonyl compounds have been investigated by 1H NMR spectroscopy. The products from the reaction with DEM and diethyl fumarate (DEF) are shown to be the diastereomers of S-(alpha,beta-diethoxycarbonylethyl)glutathione. During the course of the reaction, DEM isomerized to DEF, and the rate of isomerization was dependent upon whether the solvent was 1H2O or 2H2O. The observed rate data exhibit apparent second order kinetic behavior. The reaction of maleate with GSH was considerably slower, and solvent-dependent isomerization was observed, while little reaction of fumarate with GSH was observed at pH 6.5. Reaction of DEM with N-acetyl-L-cysteine followed a similar course to that of GSH, and although L-cysteine reacted rapidly with DEM, it did not promote the isomerization of DEM. Reactions involving penicillamine and N-acetylpenicillamine were considerably slower. Conjugation reactions catalyzed by commercial GSH transferases and selected rat and human purified isoenzymes were also investigated. Of those isoenzymes studied, rat GSH transferase 4-4 was found to exert the greatest degree of stereo control in conjugation reactions with DEF.

Animals↗
PubMed2002

The cyclopentenone product of lipid peroxidation, 15-A(2t)-isoprostane (8-isoprostaglandin A(2)), is efficiently conjugated with glutathione by human and rat glutathione transferase A4-4.

Glutathione transferases (GSTs) are a large family of enzymes that can be divided into different classes based on structure. There has been considerable interest in the ability of GSTs to conjugate and inactivate endogenously derived reactive lipid peroxidation products that contain alpha,beta-unsaturated carbonyl moieties such as 4-hydroxyalkenals. One enzyme with prominent activity toward these substrates is human GST A4-4. Recently, we described a novel series of compounds termed A(2)/J(2)-isoprostanes (IsoPs) that are formed endogenously in humans from the free radical-initiated peroxidation of arachidonic acid. These compounds contain alpha,beta-unsaturated carbonyl groups and have structures similar to cyclooxygenase-derived PGA(2) and PGJ(2). Because of their chemical reactivity, these compounds may mediate tissue injury associated with oxidant stress. Herein, we report that the A-ring IsoP 15-A(2t)-IsoP (8-iso-PGA(2)) is efficiently conjugated to glutathione (GSH) by human GST A4-4 with a k(cat)/K(m) value of >200 s(-)(1) mM(-)(1). The k(cat)/K(m) value for conjugation of 15-A(2t)-IsoP by the homologous rat GST A4-4 is >2000 s(-)(1) mM(-)(1). Similar high enzyme activities were observed when PGA(2) was used as a substrate. In contrast, the human GSTs A1-1, M1-1, M2-2, P1-1, and T1-1 and rat GST T2-2 did not significantly metabolize 15-A(2t)-IsoP. These studies have therefore defined a potentially important route by which cyclopentenone IsoPs are metabolized that may serve as a mechanism for the inactivation of these highly reactive compounds.

Animals↗
PubMed2004

Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl)glutathione and by glutathione S-transferase-mediated activation of dihalomethanes.

The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A >> C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.

Animals↗
PubMed2005

Interactions of sodium selenite, glutathione, arsenic species, and omega class human glutathione transferase.

Human monomethylarsenate reductase [MMA(V) reductase] and human glutathione S-transferase omega 1-1 (hGSTO1-1) [because MMA(V) reductase and hGSTO1-1 are identical proteins, the authors will utilize the designation "hGSTO1-1"] are identical proteins that catalyze the reduction of arsenate, monomethylarsenate [MMA(V)], and dimethylarsenate [DMA(V)]. Sodium selenite (selenite) inhibited the reduction of each of these substrates by the enzyme in a concentration-dependent manner. The kinetics indicated a noncompetitive inhibition of the MMA(V), DMA(V), or arsenate reducing activity of hGSTO1-1. The inhibition of the MMA(V) reducting activity of hGSTO1-1 by selenite was reversed by 1 mM DL-dithiothreitol (DTT) but not by reduced glutathione (GSH), which is a required substrate for the enzyme. Neither superoxide anion nor hydrogen peroxide was involved in the selenite inhibition of hGSTO1-1. MALDI-TOF and MS/MS analysis demonstrated that five molecules of GSH were bound to one monomer of hGSTO1-1. Four of the five cysteines of the monomer were glutathionylated. Cys-32 in the active center, however, exists mostly in the sulfhydryl form since it was alkylated consistently by iodoacetamide. MALDI-TOF mass spectra analysis of hGSTO1-1 after reaction with GSH and sodium selenite indicated that selenium was integrated into hGSTO1-1 molecules. Three selenium were found to be covalently bonded to the monomer of hGSTO1-1 with three molecules of GSH. It is proposed that the reaction products of the reduction of selenite inhibited the activity of hGSTO1-1 by reacting with disulfides of glutathionylated cysteines to form bis (S-cysteinyl)selenide and S-selanylcysteine and had little or no interaction with the sulfhydryl of Cys-32 in the active site of the enzyme.

Amino Acid Sequence↗
PubMed2007

Kinetics and mechanism of the oxidation of the glutathione dimer by hypochlorous Acid and catalytic reduction of the chloroamine product by glutathione reductase.

Oxidized glutathione (GSSG) reacts with two molar equivalents of HOCl/OCl- (a neutrophil-derived oxidant and a common biocide) to form the dichloro (bis-N-chloro-gamma-l-glutamyl) derivative (NDG). The reaction of less than two molar equivalents of HOCl with GSSG does not yield the unsymmetrical monochloro derivative (NCG) but rather a stoichiometric amount of NDG and GSSG. This result is explained by a faster reaction of the second equivalent of HOCl with NCG than that of the first equivalent of HOCl with GSSG. The rates of reaction of GSSG2-, GSSG3-, and GSSG4- (successive deprotonation of the ammonium groups) have been investigated, and it is clear that GSSG2- is unreactive, whereas GSSG4- is about twice as reactive as GSSG3-. Accordingly, the following mechanism is proposed (constants for 5 degrees C): H+ + OCl- = HOCl, pK1 = -7.47; GSSG2- = GSSG3- + H+, pK2 = 8.5; GSSG3- = GSSG4- + H+, pK3 = 9.5; GSSG3- + HOCl --> NCG3- + H2O, k4 = 2.7(2) x 106 M-1 s-1; GSSG4- + HOCl --> NCG4- + H2O, k5 = 3.5(3) x 107 M-1 s-1; NCG3- --> NDG4- + H+, k6 = fast; and NCG4- + HOCl --> NDG4- + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. NDG decomposes at pH 7.4 in a first-order process with kdec = 4.22(1) x 10-4 s-1 (t1/2 = 27 min). Glutathione reductase (EC 1.6.4.2) is capable of catalyzing the reduction of NDG by NADPH. The only NDG-derived product that is observed (by NMR) after the reduction by NADPH is GSH. Thus, in the presence of the GOR/NADPH system, GSH is capable of redox buffering a 3/2 mol equiv of HOCl rather than a 1/2 mol equiv as previously assumed.

Catalysis↗
PubMed2002

Glutathione synthetase deficiency: is gamma-glutamylcysteine accumulation a way to cope with oxidative stress in cells with insufficient levels of glutathione?

Glutathione (GSH) plays a major role in the cellular defence against oxidative stress and other vital cellular functions. It therefore seems inevitable that patients with severe depletion of GSH will not survive. However, at least some with glutathione synthetase (GS) deficiency do. This study was done to determine whether these patients have a mechanism to compensate for their GSH deficiency. Cell-free extracts of cultured fibroblasts from 9 patients with GS deficiency and 9 control subjects were analysed by HPLC for low-molecular-weight thiol compounds. The patients' cells contained 7.4 nmol of GSH per mg of protein (median; range 2.8-25.2) compared to 33.0 nmol in control fibroblasts (range 26.7-51.4) (p < 0.01). On the other hand, the patients' cells accumulated 18.1 nmol of gamma-glutamylcysteine (gamma-GC) per mg of protein (median; range 6.9-71.7), whereas the control cells contained 0.1 nmol (range 0.05-0.16) (p < 0.01). The cysteine concentrations in the patients' cells were 20.7 nmol/mg protein (median; range 9.4-52.9) compared to 8.9 nmol in control cells (range 3.0-12.4) (p < 0.01). Cultured fibroblasts from patients with GS deficiency have low levels of GSH, but instead accumulate gamma-GC. We suggest that gamma-GC, which contains both reactive groups of GSH (i.e. the sulphydryl and gamma-glutamyl groups), can compensate for GSH in the cellular defence against oxidative stress. Thus, gamma-GC may alleviate, but only partly prevent, serious consequences of insufficient GSH levels in affected patients. Since the sum of the levels of GSH and gamma-GC in GS-deficient cells (median 31.5 nmol/mg protein, range 16.2-79.0) was similar to the level of GSH alone in control cells (33.0 nmol/mg protein, range 26.7-51.4), we propose that the cultured fibroblasts may have a mechanism to regulate in a coordinated way the levels of GSH and gamma-GC; for instance, by both compounds acting as feedback inhibitors of gamma-GC synthetase.

Adolescent↗
PubMed1991

The relationships between glutathione, glutathione-S-transferase and cytotoxicity of platinum drugs and melphalan in eight human ovarian carcinoma cell lines.

The role of glutathione (GSH) and GSH-S-transferase (GST) activity in modulating the cytotoxicity of four platinum drugs and melphalan was evaluated in eight human ovarian carcinoma cell lines. The cell lines were established from solid and ascitic tumours from pretreated and untreated patients, and showed a wide spectrum of sensitivity to several platinum II and platinum IV drugs; cisplatin, carboplatin, CHIP and tetraplatin. Intracellular glutathione concentration measured in the cell lines showed a significant (P = 0.05) correlation with IC50 values for cisplatin (r = 0.91), carboplatin (r = 0.87) and CHIP (r = 0.88). The correlation between GSH levels and IC50 values for melphalan (r = 0.76) or tetraplatin (r = 0.60) was not as significant. GST activity showed no correlation with IC50 values, for the four platinum drugs. To determine the significance of the elevated GSH concentration in the refractory cell lines, the effect of D,L-buthionine-S, R-sulfoximine (BSO) mediated GSH depletion on platinum drug cytotoxicity was examined in one of the most sensitive (CH1) and two of the least sensitive (relatively resistant; SKOV-3, HX/62) cell lines. Comparison was made with the effect of GSH depletion on melphalan cytotoxicity in these three lines. These lines were differentially sensitive to BSO, with the two most platinum drug resistant lines being more tolerant to BSO than the sensitive CH1 line. Depletion of cellular GSH, ranging between 61 and 88%, had a differential effect on the sensitivity to PtII vs PtIV drugs in the three cell lines: cytotoxicity of the PtIV drugs, tetraplatin and CHIP, was substantially enhanced in both the resistant and sensitive cell lines; in contrast, the cytotoxicity of the PtII drugs, cisplatin and carboplatin, was only significantly increased in one of the two relatively resistant lines (SKOV-3) and in the sensitive (CH1) line after GSH depletion. Moreover the dose modification factor (DMF) for the PtII agents were lower than those for PtIV agents in the three cell lines. The dose modification factor for tetraplatin after BSO treatment was similar to that observed for melphalan in all three cell lines. In the SKOV-3 cell line extending the BSO pretreatment period to 48 h from 24 h marginally reduced the cytotoxicity of cisplatin, whereas the cytotoxicity of the other three drugs remained similar to that observed after 24 h BSO pretreatment. In contrast, extending the BSO treatment to 24 h after drug exposure potentiated the cytotoxicity of cisplatin, CHIP and tetraplatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Carboplatin↗
PubMed1996

The relationship between tumour glutathione concentration, glutathione S-transferase isoenzyme expression and response to single agent carboplatin in epithelial ovarian cancer patients.

There is evidence to suggest that glutathione (GSH) and glutathione-S-transferases (GST) are important factors in determining sensitivity to cytotoxic drugs in vitro and in preclinical in vivo model systems. To define the relationship between tumour GSH concentration, GST isoenzyme expression and response to carboplatin in epithelial ovarian cancer (EOC), tumour samples from 39 patients with assessable disease after primary surgery were analyzed for GSH content and GST expression. Response was assessed after completing six courses of single agent carboplatin therapy. GSH was measured by high performance liquid chromatography (HPLC) in fresh tumour samples taken at primary laparatomy. GST isoenzyme expression was assessed by immunohistochemistry of fixed tumour material using antibodies specific for pi, alpha and mu classes. GST isoenzyme expression was defined as positive if the staining intensity was strong and more than 10% of tumour cells were involved. The mean GSH concentrations were: 8351 +/- 4496, 7211 +/- 5026, 6559 +/- 4573 and 3758 +/- 1885 (nmol g-1 tissue dry weight mean +/- s.d.) for tumours from patients who subsequently achieved a complete response (CR, n = 18), partial response (PR, n = 10) or who had static disease (SD, n = 7) or progressive disease (PD, n = 4) respectively. There was no relationship between GSH concentration and response (ANOVA, P = 0.32). There were also no relationship between GST isoenzyme expression and response (P Fisher's exact test 0.51-0.55 and chi-squared test 0.98-0.99). In conclusion, there was no association between the concentration of GSH or expression of GST isoenzymes and response to single agent carboplatin in primary previously untreated EOC.

Antineoplastic Agents↗
PubMed1997

Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells.

The cytotoxic mechanism of a conjugate of doxorubicin (DXR) and glutathione (GSH) via glutaraldehyde (GSH-DXR) was investigated using DXR-sensitive (AH66P) and -resistant (AH66DR) rat hepatoma cells. GSH-DXR accumulated in AH66DR cells as well as in AH66P cells without efflux by P-gp and exhibited the potent cytocidal activity against both cells compared with DXR. To examine whether thiol from GSH-DXR affected the expression of cytotoxicity, two conjugates of DXR, with modified peptides containing alanine or serine substituted for cysteine in GSH were prepared and their cytotoxicities determined. Substitution of these amino acids for cysteine resulted in an approximately two- to fourfold reduction in cytotoxic activity against both cell lines compared with the effect of GSH-DXR. Depletion of intracellular GSH by treatment of both cells with buthionine sulphoximine did not change the cytotoxic activity of DXR, BSA-DXR or GSH-DXR. By co-treating the cells with tributyltin acetate, an inhibitor of glutathione S-transferase (GST), and either DXR, BSA-DXR or GSH-DXR, the cytotoxicity was markedly increased. Interestingly, GSH-DXR showed non-competitive inhibition of GST activity and its IC50 value was 1.3 microM. These results suggested that the inhibition of GST activity by GSH-DXR must be an important contribution to the expression of potent cytotoxicity of the drug.

Animals↗
PubMed1998

Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines.

Resistance to chemotherapy is a major problem in the treatment of patients with head and neck squamous cell carcinoma (HNSCC). Important factors involved are drug detoxification by glutathione (GSH) and reduced drug accumulation due to active transport out of the cell by so-called 'multidrug resistance-related proteins'. We have studied a panel of eight HNSCC cell lines showing differences in sensitivity to the anti-cancer drug cisplatin. Our previous studies indicated that the IC50 values were inversely correlated with the intracellular accumulation of platinum (Pt). In the present study, cellular GSH levels were found not to be related to the IC50 values. The expression levels of the enzymes glutathione S-transferase (GST) alpha, mu, and pi, the multidrug resistance-related proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) were determined semiquantitatively by means of immunocytochemistry. The levels of the GSTs, P-gp and LRP were not found to be correlated with the IC50 values of the HNSCC cell lines. Surprisingly, however, an inverse correlation was found between MRP levels and IC50 values. The MRP expression levels were in agreement with the results of the MRP functional assay, based on the transport of calcein across the cell membrane as performed for two of the cell lines. Further studies should prove whether other pump mechanisms or DNA repair are involved in the cisplatin accumulation and the subsequent HNSCC cell growth inhibition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Serum selenium, plasma glutathione (GSH) and erythrocyte glutathione peroxidase (GSH-Px)-levels in asymptomatic versus symptomatic human immunodeficiency virus-1 (HIV-1)-infection.

OBJECTIVES: Antioxidant defense status was investigated in HIV-infected patients by measuring serum selenium, erythrocyte glutathione peroxidase (GSH-Px) activity, plasma thiol (-SH) and glutathione (GSH) concentrations along with the assessment of the clinical stage and surrogate markers of HIV-disease. DESIGN, SETTING AND SUBJECTS: Serum selenium levels were determined cross-sectionally in 104 sequentially selected HIV-infected patients (83 outpatients and 21 patients with ongoing AIDS defining events). The patients were classified into three stages of the disease, I, II and III according to the 1993 Centers For Disease Control (CDC) classification system for HIV-infection. GSH-Px activities, plasma SH and plasma GSH concentrations were determined in a subset of 24 patients at stage I and 12 patients at stage III with an active AIDS-defining disease. RESULTS: Mean serum selenium levels were lower in CDC stage II (68.7 +/- 20.9 micrograms/l; P < 0.01; n = 34) and stage III (51.4 +/- 14.7 micrograms/l; P < 0.01; n = 37) HIV-infected patients than in healthy subjects (89.2 +/- 20.9 micrograms/l; n = 72) and stage I patients (82.3 +/- 20.5; microgram/l; n = 33). Serum selenium levels were positively correlated with CD4-count (r = 0.42; P < 0.001; n = 104) and inversely with levels of soluble tumor necrosis factor receptors type II (r = -0.58; P < 0.01; n = 35), neopterin (r = -0.5; P < 0.001; n = 80) and beta 2-microglobulin (r = -0.4; P < 0.001; n = 94). Hepatitis C virus (HCV) and HIV-coinfected patients at CDC stages I and II showed markedly lower selenium concentrations compared to HIV-infected patients without concomitant HCV-infection. Serum selenium and GSH-Px activity in hospitalized AIDS patients was significantly lower as compared to asymptomatic patients and healthy subjects, whereas plasma SH and GSH concentrations were lower in both, asymptomatic -and AIDS-patients, than in the controls. CONCLUSION: The results show that stages I-III of HIV-disease are characterized by significant impairments of antioxidative defenses provided by selenium, GSH-Px, SH-groups and GSH.

Adult↗
PubMed2001

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Amino Acid Sequence↗
PubMed1973

Interrelationship of glutathione-cystine transhydrogenase and glutathione reductase in developing rat intestine.

1. Glutathione reductase and glutathione-cystine transhydrogenase activity in supernatant fractions of whole homogenates and homogenates of mucosal and muscular layers were determined in developing rat intestine after determination of the optimum conditions for assay of the two enzymes. In jejunum from adult rat, the K(m) values for GSSG reductase and GSH-cystine transhydrogenase activities were 0.25mm-GSSG and 0.23mm-cystine respectively. 2. The two activities could be differentiated by stability studies since GSSG reductase was stable at 60 degrees C for 10min and could be stored at 4 degrees C for 24h without loss of activity. GSH-cystine transhydrogenase, on the other hand, was denatured at 60 degrees C and completely inactive after 24h storage at 4 degrees C. 3. Based on calculations of total activities, both enzymes increased from the eighteenth day until the animals were young adults. 4. Total GSSG reductase activity increased at a greater rate with age than total GSH-cystine transhydrogenase activity as evidenced by activity ratios for GSH-cystine transhydrogenase/GSSG reductase of 0.44 and 0.12 in ileum from suckling and adult rats respectively, and 0.31 and 0.24 in jejunum from suckling and adult rats respectively. 5. In mucosa from adult rats GSSG reductase was more active in the ileum than in the jejunum, whereas GSH-cystine transhydrogenase activity was higher in the jejunum. 6. GSH-cystine transhydrogenase was active only in the muscle cells of the ileum of 7-day-old rats but became localized primarily in the mucosal layer in the adult rat. However, GSSG reductase activity was distributed evenly between the two layers throughout the intestine.

Aging↗
PubMed1991

Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site.

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Amino Acid Sequence↗
PubMed1992

Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1.

Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis. The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn. The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity. Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx. 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger. In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes. Asp99 primarily contributes to catalysis rather than to binding. The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism. The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

Amino Acids↗
PubMed2002

The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis.

Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage.

Animals↗
PubMed2002

Effects of palm oil on lipid peroxidation, reduced glutathione, glutathione peroxidase, and vitamin A levels in the corpus uteri, cornu uteri and corpus luteum of young and adult female sheep.

The aim of the current study was to determine whether a rumen protected palm oil based diet affect malondialdehyde (MDA), glutathione peroxidase (GSH-Px), reduced glutathione (rGSH) and vitamin A levels in the tissues of cornu uteri, corpus uteri and corpus luteum over the barley based isoenergetic and isonitrogenous diet, and whether the response is different between ewes and ewe-lambs. During the breeding season, half of Morkaraman ewes (2-4-year-old, n = 10) and ewe-lambs (7-8-months-old, n = 10) was offered a barley based diet and the other half was offered a protected palm oil based diet for 42 +/- 0.7 days. At the end of the experiment all animals were slaughtered and measurements carried out in the tissues collected. In all animals tested, cornu uteri had the highest MDA levels followed by corpus uteri and corpus luteum (P < 0.01) but no differences were between the tissues observed in GSH-Px and rGSH levels (P > 0.05). Vitamin A levels were, however, higher in corpus luteum than in cornu uteri and corpus uteri (P < 0.05). Corpus uteri MDA levels were not different (P > 0.05) but rGSH levels were higher for the palm oil fed groups (P < 0.05). GSH-Px and rGSH levels were higher for ewe-lambs than ewes (P < 0.05). In conclusion, it appears that MDA, rGSH, GSH-Px, and vitamin A work in a different fashion for corpus uteri, cornu uteri and corpus luteum, and for ewes and ewe-lambs. Dietary palm oil did not significantly affect the parameters studied except higher rGSH levels in corpus uteri. Levels of antioxidatively active substances, such as rGSH and GSH-Px were lower in ewes compared with those in ewe-lambs.

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