Thiol oxidation inhibits nitric oxide-mediated pulmonary artery relaxation and guanylate cyclase stimulation

The mechanisms through which thiol oxidation and cellular redox influence the regulation of soluble guanylate cyclase (sGC) are poorly understood. This study investigated whether promoting thiol oxidation via inhibition of NADPH generation by the pentose phosphate pathway (PPP) with 1 mM 6-aminonicotinamide (6-AN) or the thiol oxidant diamide (1 mM) alters sGC activity and cGMP-associated relaxation to nitric oxide (NO) donors [S-nitroso-N-acetylpenicillamine (SNAP) and spermine-NONOate]. Diamide and 6-AN inhibited NO-elicited relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and stimulation of sGC activity in BPA homogenates. Treatment of BPA with the thiol reductant DTT (1 mM) reversed inhibition of NO-mediated relaxation and sGC stimulation by 6-AN. The increase in cGMP protein kinase-associated phosphorylation of vasodilator-stimulated phosphoprotein on Ser239 elicited by 10 microM SNAP was also inhibited by diamide. Activation of sGC by SNAP was attenuated by low micromolar concentrations of GSSG in concentrated, but not dilute, homogenates of BPA, suggesting that an enzymatic process contributes to the actions of GSSG. Relaxation to agents that function through cAMP (forskolin and isoproterenol) was not altered by inhibition of the pentose phosphate pathway or diamide. Thus a thiol oxidation mechanism controlled by the regulation of thiol redox by NADPH generated via the pentose phosphate pathway appears to inhibit sGC activation and cGMP-mediated relaxation by NO in a manner consistent with its function as an important physiological redox-mediated regulator of vascular function.     

Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase

Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P)+ oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide (NEM)/mol enzyme's SH = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of NEM/enzyme's SH = 300, two thiol residues were alkylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by NEM or by iodoacetamide. Biliverdin as well as the co-substrate NADPH protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group binds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxal phosphate the catalytic activity was irreversibly abolished. The location of a lysine residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thiol and epsilon-NH2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the NADPH binding site was inferred from the shifts in the UVmax of NADPH from 340 nm to 327 nm and of 3-acetyl NADPH from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by NADPH but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.     

Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment.

Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.     

Mechanisms of hydrogen peroxide formation in leukocytes: the NAD(P)H oxidase activity of myeloperoxidase.

The NADPH oxidase activity of polymorphonuclear leukocyte granules has not previously been attributed to myeloperoxidase because of its relative insensitivity to cyanide and its activation by aminotriazole. However it has been found that the NAD(P)H oxidase activity of myeloperoxidase or horseradish peroxidase was little affected by 2.0 mM cyanide although the peroxidase activity was nearly completely inhibited by 0.1 mM cyanide. Furthermore, the NAD(P)H oxidase activity of myeloperoxidase was considerably enhanced by aminotriazole although the peroxidase activity was inhibited.     

Cytosolic NADH redox and thiol oxidation regulate pulmonary arterial force through ERK MAP kinase

An ERK MAP kinase-mediated contractile mechanism previously reported to be activated by peroxide and stretch in bovine coronary arteries is shown in this study to be present in endothelium-denuded bovine pulmonary arteries and subject to regulation by modulation of cytosolic NAD(H) redox through the lactate dehydrogenase reaction. Although our previous work identified an acute PO2-dependent peroxide-mediated relaxation of bovine pulmonary arteries on exposure to lactate, a 30-min treatment with 10 mM lactate enhanced ERK phosphorylation and increased force generation to 30 mM KCl. Hypoxia inhibited these responses to lactate. Increases in ERK phosphorylation and the enhancement of force generation by lactate and stretch are attenuated in the presence of inhibitors of Nox oxidase (0.1 mM apocynin) or ERK activation (10 microM PD-98059) and by 0.1 mM ebselen. Additionally, incubation of pulmonary arteries with 10 mM pyruvate lowered basal levels of ERK phosphorylation, and it inhibited both the ERK phosphorylation and the enhancement in force generation to 30 mM KCl caused by stretch. Treatment of pulmonary arteries with the thiol oxidant diamide (1 microM) elicited what appears to be a peroxide-independent increase in force and ERK phosphorylation that were both attenuated by PD-98059. Thus pulmonary arteries possess a peroxide-elicited contractile mechanism involving ERK MAP kinase, which is stimulated by stretch and regulated through the control of Nox oxidase activity by the availability of cytosolic NADH.     

Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamide

In the present study, the effect of thiol redox and its possible role in the inhibitory effect of nicotinamide on renal brush-border membrane (BBM) phosphate uptake was examined. Addition of thiol reducing agent, dithiothreitol (DTT, 5 mM), caused an increase, while addition of thiol oxidant, diamide (DM, 5 mM) caused a reversible decrease in sodium-dependent BBM phosphate uptake. Kinetic analyses revealed an increase in both Vmax and Km by DTT, and a decrease in Vmax by DM. These results suggest that thiol redox influences BBM phosphate uptake with sulfhydryl (SH) groups relate to its capacity and disulfide (SS) groups to its affinity for phosphate. Since changes in cytosolic NAD levels may affect BBM thiol redox through changes in redox states of NADP and glutathione systems, we have examined such possibility by studying the effect of nicotinamide (NM). Incubation of proximal tubules with NM (10 mM) induced an oxidative effect on redox states of cytosolic NAD, NADP systems as inferred from decreased cellular lactate/pyruvate, malate/pyruvate, respectively. Measurements of cytosolic glutathiones and BBM thiols also revealed that NM pretreatment shifted the cytosolic glutathione redox (GSH/GSSG) and BBM thiol redox (SH/SS) toward more oxidized state. On the other hand, incubation of proximal tubules with NM suppressed phosphate uptake by the subsequently isolated BBM vesicles. The lower phosphate uptake by NM-pretreated BBM vesicles was reversed by DTT and was resistant to the inhibitory effect of DM. These results thus suggest that BBM thiol oxidation may be involved in the inhibitory effect of NM on BBM phosphate uptake.     

Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.     

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.     

Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases.

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status

Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.     

Activation of NADPH oxidase and extracellular superoxide production in seizure-induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.     

Glucose-6-phosphate dehydrogenase from a tetracycline producing strain of Streptomyces aureofaciens: some properties and regulatory aspects of the enzyme

Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.     

Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase

Heavy metal pollution can arise from many sources and damage many organisms. Exposure to the metal ions can leads to a reduction in cellular antioxidant enzyme activities and lowers cellular defense against oxidative stress. In this study we have tested effects of the some metal ions on the purified bovine kidney cortex glutathione reductase (GR). Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effect on the enzyme. The obtained IC?? values of Cd2+, Ni2+, and Zn2+ are 0.027, 0.8, and 1 mM, respectively. Kinetic characterization of the inhibition is also investigated. Cd2+ inhibition is noncompetitive with respect to both oxidized glutathione (GSSG) (Ki(GSSG) 0.060 ± 0.005 mM) and NADPH (Ki(NADPH) 0.025 ± 0.002 mM). Ni2+ inhibition is noncompetitive with respect to GSSG (Ki(GSSG) 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.712 ± 0.047 mM). The effect of Zn2+ on GR activity is consistent with noncompetitive inhibition pattern when the varied substrate is the GSSG (Ki(GSSG) 0.091 ± 0.005 mM) and the NADPH (Ki(NADPH) 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.     

Mechanism of action of the inhibition of pyridine-nucleotide-dependent flavine enzymes using the systemic fungicide Dexon]

The actions of Dexon on the NADH-ferricyanide oxidoreductase and the NADPH oxidase system of electron transfer particles (ETP) from beef heart as well as on the NADPH-cytochrome c oxidoreductase from brewer's yeast (Saccharomyces carlsbergensis Hansen) were investigated. The inhibition of the NADH dehydrogenase activity of ETP and that of the yeast enzyme correspond with respect to the following characteristics: 1) increase in the inhibition, 2) enhancement of the Dexon sensitivity by one order of magnitude after preincubation in the presence of NAD(P)H, 3) irreversibility of the inhibition, 4) no detectable changes in the spectral properties and in coenzyme activity of FMN after acid extraction from Dexon-treated enzyme. The inhibition of the NADH dehydrogenase activity of ETP is diminished by both NAD+ and FMN. However, no interaction of Dexon with NAD(P)H or FMN could be detected in the absence of enzyme or apoenzyme. The concentration of half-inhibition by Dexon for the yeast enzyme corresponds with its FMN concentration. It is proposed that both apoenzyme, NAD(P)H and FMN are involved in the interaction with Dexon. Possible mechanisms of binding are both complanar complexations of the ring systems and a triazene formation between FMNH2 and Dexon. The NADPH oxidase activity of the ETP is partly inhibited; the share inhibited by Dexon may represent the pathway via the transhydrogenase reaction.     

Short-term exercise preserves myocardial glutathione and decreases arrhythmias after thiol oxidation and ischemia in isolated rat hearts

The purpose of this study was to determine if exercise (Ex) protects hearts from arrhythmias induced by glutathione oxidation or ischemia-reperfusion (I/R). Female Sprague-Dawley rats were divided into two experimental groups: sedentary controls (Sed) or short-term Ex (10 days of treadmill running). Twenty-four hours after the last session, hearts were excised and exposed to either perfusion with the thiol oxidant diamide (200 μM) or global I/R. Ex significantly delayed the time to the onset of ventricular arrhythmia after irreversible diamide perfusion. During a shorter diamide perfusion protocol with washout, Ex significantly decreased the incidence of arrhythmia, as evidenced by a delayed time to the first observed arrhythmia, lower arrhythmia scores, and lower incidence of ventricular fibrillation. Ex hearts exposed to I/R (30-min ischemia/30-min reperfusion) also showed lower arrhythmia scores and incidence of ventricular fibrillation compared with Sed counterparts. Our finding that Ex protected intact hearts from thiol oxidation was corroborated in isolated ventricular myocytes. In myocytes from Ex animals, both the increase in H(2)O(2) fluorescence and incidence of cell death were delayed after diamide. Although there were no baseline differences in reduced-to-oxidized glutathione ratios (GSH/GSSG) between the Sed and Ex groups, GSH/GSSG was better preserved in Ex groups after diamide perfusion and I/R. Myocardial glutathione reductase activity was significantly enhanced after Ex, and this was preserved in the Ex group after diamide perfusion. Our results show that Ex protects the heart from arrhythmias after two different oxidative stressors and support the hypothesis that sustaining the GSH/GSSG pool stabilizes cardiac electrical function during conditions of oxidative stress.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn2+ (up to 2 mM) and activated above this concentration. Ca2+ inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Wheat Chloroplastic Glutathione Reductase Activity is Regulated by the Combined Effect of pH, NADPH and GSSG

To study the mechanism of regulation of chloroplastic glutathionereductase (GR) under photooxidative conditions, GR activity,and the levels of NADPH, GSH and GSSG were measured in wheatchloroplasts under photooxidative light. GR was extremely labile,and the concentrations of GSH and GSSG progressively diminishedin chloroplasts prepared without ascorbate. The NADPH leveldid not significantly change during photooxidative treatment.The addition of 10 mM ascorbate to the incubation medium preventedthe decrease of GSH and GSSG and strongly protected GR activity.However, ascorbate had no effect on NADPH-dependent inhibitionof the chloroplastic GR purified from wheat leaves. We studiedthe effect of NADPH, temperature, pH and GSSG on the purifiedenzyme. The inhibition by NADPH was greatly dependent on temperatureand pH. NADPH inhibited GR by around 93% up to pH 7.5, but withina range of 8.0 to 9.5 the inhibition was only marginal. ThepH dependence of the NADPH inhibitory effect could be due, atleast in part, to different rates in the generation of NADPH-X,a derivative of NADPH which inactivates several pyridin nucleotidedehydrogenases. Furthermore, the NADPH-dependent inhibitionwas almost completely prevented by GSSG, but not by GSH. (Received July 9, 1998; Accepted April 30, 1999)