Redox regulation of the signaling pathways leading to eNOS phosphorylation

Oxidative stress mediates positive and negative effects on physiological processes. Recent reports show that H(2)O(2) induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-phosphorylation-dependent pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H(2)O(2) (100 microM) activated the Akt/eNOS pathway, higher levels (500 microM) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfhydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorylation events induced by 100 microM H(2)O(2). GSH was not a target thiol in this system because buthionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane surface and intracellular free thiols was associated with the inhibition of phosphorylation, suggesting that oxidation of non-GSH thiols inhibits the H(2)O(2)-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols, suggesting a more prominent role for the former. Similarly, DEM and CDNB inhibited TNF-alpha-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS.     

Contrasting effects of thiol-modulating agents on endothelial NO bioactivity

The bioactivity of endothelium-derived nitric oxide(NO) is an important component of vascular homeostasis that issensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define itsrole in modulating endothelial NO bioactivity. In porcine aorticendothelial cells (PAECs), we depleted intracellular GSH (>70%) using1) buthionine-(S,R)-sulfoximine (BSO), whichinhibits GSH synthesis; 2) diamide, which oxidizes thiols;or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putativelydepletes GSH through glutathione S-transferase activity.Cellular GSH depletion with BSO had no effect on endothelial NObioactivity measured as A-23187-induced cGMP accumulation. In contrast,oxidation of intracellular thiols with diamide inhibited bothA-23187-induced cGMP accumulation and the cGMP response to exogenousNO. Diamide treatment of either PAECs, PAEC membrane fractions, orpurified endothelial nitric oxide synthase (eNOS) resulted insignificant inhibition (~75%) of eNOS catalytic activity measured asL-[³H]arginine-to-L-[³H]citrullineconversion. This effect appeared related to oxidation of eNOS thiols asit was completely reversed by dithiothreitol. Glutathione depletionwith CDNB inhibited A-23187-stimulated cGMP accumulation but not thecGMP response to exogenous NO. Rather, CDNB treatment impaired eNOScatalytic activity in intact PAECs, and this effect was reversed byexcess NADPH in isolated purified eNOS assays. Consistent with theseresults, we found spectral evidence that CDNB reacts with NADPH andrenders it inactive as a cofactor for either eNOS or glutathionereductase. Thus thiol-modulating agents exert pleiotropic effects onendothelial NO bioactivity, and these data may help to resolve a numberof conflicting previous studies linking GSH status with endothelialcell NO bioactivity.

     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2,4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30-40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G1----S). Since the latter type of effect most probably is Ca2+ dependent and since the spindle is sensitive to Ca2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase. All compounds were found to cause an increase of ATP with some of the applied concentrations. The results with diethyl maleate suggest that an increase of ATP is favored by an attack on mitochondrial reduced glutathione. The possible analogy between this effect and an increase of ATP and Ap4A in bacteria during oxidative stress is considered.     

Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: differential effects of vitamin E and disulfiram

Exposure of isolated rat hepatocytes to allyl alcohol (AA), diethyl maleate (DEM) and bromoisovalerylurea (BIU) induced lipid peroxidation, depletion of free protein thiols to about 50% of the control value and cell death. Vitamin E completely prevented lipid peroxidation, protein thiol depletion and cell death. A low concentration (0.1 mM) of the lipophylic disulfide, disulfiram (DSF), also prevented the induction of lipid peroxidation by the hepatotoxins; however, in the presence of DSF, protein thiol depletion and cell death occurred more rapidly. Incubation of cells with a high concentration (10 mM) of DSF alone led to 100% depletion of protein thiols and cell death, which could not be prevented by vitamin E. The level of free protein thiols in cells, decreased to 50% by exposure to AA, DEM and BIU, could be reversed to 75% of the initial level by dithiothreitol (DTT) treatment, indicating that the protein thiols were partially modified into disulfides and partially into other, stable thiol adducts. The 100% depletion of protein thiols by DSF was completely reversed by DTT treatment. The involvement of lipid peroxidation in protein thiol depletion was studied by measuring the effect of a lipid peroxidation product, 4-hydroxynonenal (4-HNE), on protein thiols in a cell free liver fraction. 4-HNE did not induce lipid peroxidation in this system, but protein thiols were depleted to 30% of the initial value, irrespective of the presence of vitamin E. DTT treatment could reverse this for only 25%. Similar, DSF-induced protein thiol depletion could be reversed completely by DTT. We conclude that (at least) two types of protein thiol modifications can occur after exposure of hepatocytes to toxic compounds: one due to interaction of endogeneously generated lipid peroxidation products with protein thiols, which is not reversible by the action of DTT, and one due to a disulfide interchange between disulfides like DSF and protein thiols, which can be reversed by the action of DTT.     

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells

Thiol reagents activateK-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independentouabain-resistant K flux, in red blood cells (RBCs) of several species,upon depletion of cellular glutathione (GSH). K-Cl COT isphysiologically active in high potassium (HK), high GSH (HG) dog RBCs.In this unique model, we studied whether the same inverse relationshipexists between GSH levels and K-Cl COT activity found in other species.The effects of GSH depletion by three different chemical reactions[nitrite (NO₂)-mediated oxidation, diazene dicarboxylicacid bis-N,N-dimethylamide (diamide)-induceddithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD).After 85% GSH depletion, all three interventions stimulated K-Cl COThalf-maximally with the following order of potency: diamide > NO₂ > CDNB. Repletion of GSH reversed K-Cl COTstimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activationby NO₂ and diamide. K-Cl COT activation at concentrationratios of oxidant/GSH greater than unity was irreversible, suggestingeither nitrosothiolation, heterodithiol formation, or GST-mediateddinitrophenylation of protein thiols. The data support the hypothesisthat an intact redox system, rather than the absolute GSH levels,protects K-Cl COT activity and cell volume regulation from thiol modification.

     

Relationship between 1-chloro2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthiomine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 M 1-chloro-2,4-dinitrobenzene and 250 M buthiomine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthiomine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 M 2-cyclohexene-l-one, which depletes free glutathione by conjugation, resulted in a comAbbreviation BSO DL-buthiomine-S-R-sulfoximine - CDNB 1-chloro-2,4-dinitrobenzene - CHX 2-cyclohexene-l-one - GSH glutathione - GST glutathione-S-transferase - MAPS microtubule-associated proteins - MF microfilaments - MT microtubules.     

Spindle disturbances in mammalian cells. III. Toxicity, c-mitosis and aneuploidy with 22 different compounds. Specific and unspecific mechanisms

Early investigations have shown that many chemically different compounds can cause disturbances of the spindle function (c-mitosis) in eukaryotic cells and that there is an unspecific (physical) mechanism based on the partitioning of the compound into cellular hydrophobic compartments. This suggests that the approach should be quantitative when testing compounds for this type of activity in vitro; effect/no effect is not the most pertinent question. The present study demonstrates how a set of reference compounds can be used in attempts to identify compounds that act by a more specific (chemical) mechanism to disturb the spindle function. All experiments were performed with an established cell line (V79 Chinese hamster). The results suggest that there is a good qualitative coupling in these cells between c-mitosis and aneuploidy with chemical treatment. Among compounds that are particularly active in relation to their lipophilic character are some chlorophenols, caffeine, diamide, diethyl maleate, 1-chloro-2,4-dinitrobenzene and tertiary butylhydroperoxide. This points to Ca2+-sequestering by mitochondria and/or cellular pH regulation (chlorophenols), Ca2+ release and sequestering by the endoplasmic reticulum (caffeine), enzymatic conjugation to glutathione (diethyl maleate, chlorodinitrobenzene) and hydroperoxide metabolism (t-butylhydroperoxide) as important target functions for specific activity.     

The role of thiols in cellular response to radiation and drugs

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.     

Thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels

Cellular redox change regulates pulmonary vascular tone by affecting function of membrane and cytoplasmic proteins, enzymes, and second messengers. This study was designed to test the hypothesis that functional modulation of ion channels by thiol oxidation contributes to regulation of excitation-contraction coupling in isolated pulmonary artery (PA) rings. Acute treatment with the thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC50 was 335 and 58 microM for 40 mM K+ - and 2 microM phenylephrine-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was affected by neither functional removal of endothelium nor 8-bromoguanosine-3'-5'-cyclic monophosphate (50 microM) and HA-1004 (30 microM). A rise in extracellular K+ concentration (from 20 to 80 mM) attenuated the thiol oxidant-induced PA relaxation. Passive store depletion by cyclopiazonic acid (50 microM) and active store depletion by phenylephrine (in the absence of external Ca2+ both induced PA contraction due to capacitative Ca2+ entry. Thiol oxidation by diamide significantly attenuated capacitative Ca2+ entry-induced PA contraction due to active and passive store depletion. The PA rings isolated from left and right PA branches appeared to respond differently to store depletion. Although the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5-fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels, which subsequently attenuate Ca2+ influx and decrease cytosolic free Ca2+ concentration in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K+ channels and inhibition of store-operated Ca2+ channels appear to be independent of functional endothelium and of the cGMP-dependent protein kinase pathway.     

Inhibition of vascular NADH/NADPH oxidase activity by thiol reagents: lack of correlation with cellular glutathione redox status

Vascular NAD(P)H oxidase activity contributes to oxidative stress. Thiol oxidants inhibit leukocyte NADPH oxidase. To assess the role of reactive thiols on vascular oxidase, rabbit iliac/carotid artery homogenates were incubated with distinct thiol reagents. NAD(P)H-driven enzyme activity, assessed by lucigenin (5 or 250 microM) luminescence, was nearly completely (> 97%) inhibited by the oxidant diamide (1mM) or the alkylator p-chloromercuryphenylsulfonate (pCMPS, 0.5mM). Analogous inhibition was also shown with EPR spectroscopy using DMPO as a spin trap. The oxidant dithionitrobenzoic acid (0.5mM) inhibited NADPH-driven signals by 92% but had no effect on NADH-driven signals. In contrast, the vicinal dithiol ligand phenylarsine oxide (PAO, 1 microM) induced minor nonsignificant inhibition of NADPH-driven activity, but significant stimulation of NADH-triggered signals. The alkylator N-ethyl maleimide (NEM, 0.5mM) or glutathione disulfide (GSSG, 3mM) had no effect with each substrate. Coincubation of N-acetylcysteine (NAC, 3mM) with diamide or pCMPS reversed their inhibitory effects by 30-60%, whereas NAC alone inhibited the oxidase by 52%. Incubation of intact arterial rings with the above reagents disclosed similar results, except that PAO became inhibitor and NAC stimulator of NADH-driven signals. Notably, the cell-impermeant reagent pCMPS was also inhibitory in whole rings, suggesting that reactive thiol(s) affecting oxidase activity are highly accessible. Since lack of oxidase inhibition by NEM or GSSG occurred despite significant cellular glutathione depletion, change in intracellular redox status is not sufficient to account for oxidase inhibition. Moreover, the observed differences between NADPH and NADH-driven oxidase activity point to complex or multiple enzyme forms.     

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd⁺⁺-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd⁺⁺ cytotoxic effect and deccreased the LD_50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd⁺⁺. Thus the results demostrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd⁺⁺ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd⁺⁺ cytotoxic response. Comparison of reduced glutathione levels and of Cd⁺⁺ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd⁺⁺ resistance.Abbreviations BSO DL-buthionine-(R,S)-sulfoximine - DEM diethyl maleate - DMSO dimethyl sulfoxide - GSH reduced glutathione - MT metallothionein     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.     

Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes

The significance of glutathione S-conjugate in the regulation of glutathione synthesis was studied using human erythrocyte gamma-glutamylcysteine synthetase. Feedback inhibition of the enzyme by reduced glutathione was released by the addition of the glutathione S-conjugate (S-2,4-dinitrophenyl glutathione). A half-maximal effect of glutathione S-conjugate on gamma-glutamylcysteine synthetase activity was obtained at approximately 1 microM; 50 microM glutathione S-conjugate in the presence of 10 mM glutathione actually increased the enzyme activity twofold above uninhibited levels. Glutathione S-conjugate had no effect on the enzyme activity in the absence of glutathione. When erythrocytes were exposed to the electrophile 1-chloro-2,4-dinitrobenzene, which forms a glutathione S-conjugate by the catalytic reaction of glutathione S-transferase, the level of glutathione synthesis increased. These data suggest that glutathione S-conjugate plays a role in stimulating the synthesis of glutathione.     

Partial purification and some biochemical properties of neonatal rat cutaneous glutathione S-transferases

1. Previous studies have demonstrated the presence of glutathione S-transferases in the skin of rodents and humans. This study represents the first attempt to purify cytosolic glutathione S-transferases from skin of 3-day-old rats. 2. A partial purification of the enzyme was achieved by a two-step procedure: affinity chromatography followed by HPLC. Two peaks, one major (P-1) and one minor (P-2), were resolved by HPLC containing about 82% and 10% of the recovered activity, respectively. 3. The major form exhibited an overall purification of about 2270-fold with a specific activity of about 73 mumoles/min/mg protein towards 1-chloro-2,4-dinitrobenzene. 4. The kinetic data for P-1 yielded mean Km values of 2.39 mM for 1-chloro-2,4-dinitrobenzene and 0.72 mM for reduced glutathione, while the respective average Vmax values were found to be 212 and 101 mumoles/min/mg protein. 5. Significantly inhibition of enzyme activity was noted in the presence of 0.2 mM HgCl2, 0.63 microM 1.2-naphthoquinone, 1.0 microM triphenyltin chloride, and 12.5 microM 17 beta-estradiol-3-sulfate.     

The glutathione S-transferase activity in the pyloric caeca of rainbow trout, Salmo gairdneri

Pyloric caeca of trout contain 1.9 mmol GSH/kg tissue. Cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.06 mmol/min/g protein. Cholate (3.3 mM) inhibits cytosolic transferase activity by 55% at pH 6.6 and by 4% at pH 7.4. The transferases do not bind 8-anilino-1-naphthalene sulphonate at pH 7.4. The cytosolic transferases are inactivated progressively by 1-chloro-2,4-dinitrobenzene, 50% of their activity being lost in 5.0 min. A minority of the activity does not bind to a glutathione-affinity matrix. At pH 6.6 its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.88 and 9.1 mM respectively. The rest of the activity is eluted from the affinity matrix as a single peak. Its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.33 and 2.9 mM respectively. Its subunit Mr is 22.4 kDa.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

The glutathione S-transferase activity in the kidney of rainbow trout (Salmo gairdneri)

Trout kidney contains 2.3 mmol GSH/kg. The cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.35 mumol/min/mg protein. There is no detectable activity with 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, p-nitrobenzyl chloride or p-nitrophenyl acetate. A variable proportion of the activity does not bind to a glutathione-affinity matrix. Its Km values for GSH and 1-chloro-2,4-dinitrobenzene are 3.0 and 5.1 mM, respectively. The rest of the activity is eluted from the affinity matrix as one main and two minor peaks. The main peak has Km values for GSH and 1-chloro-2,4-dinitrobenzene of 0.4 and 4.5 mM, respectively. Its subunit Mr is 22,900. The activity in the main peak is inhibited progressively by 1-chloro-2,4-dinitrobenzene with a rate constant of 0.11/min.