In vitro inhibition of topoisomerase IIα by reduced glutathione

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 μM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity.     

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.     

Selenite negatively regulates caspase-3 through a redox mechanism

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.     

Mass spectrometric analysis of nitric oxide-modified caspase-3.

Caspases are a family of cysteine proteases activated during apoptosis. Modification of caspases by nitric oxide and its relevance during apoptosis is currently a controversial subject. In this study we analyzed the S-nitrosated form of caspase-3 at a molecular level. By using electrospray ionization-mass spectrometry, we detected poly-S-nitrosation of caspase-3 with an average of about 2 molecules of NO bound per enzyme. Although NO treatment completely inhibited enzyme activity, S-nitrosation was not restricted to the active site cysteine. Rather, we detected multiple relative mass increases of 30 +/- 1 Da in both the p12 and p17 subunits of caspase-3, corresponding to single to triple S-nitrosation. The stability of these S-nitrosations differed in physiologically relevant concentrations of 5 mM glutathione. Whereas all S-nitroso bonds in the p12 subunit were cleaved with release of NO and partial formation of protein-mixed disulfides with glutathione, a single S-nitrosation in the p17 subunit remained stable. Since this S-nitrosation was not observed in a mutant form of caspase-3 lacking the active site cysteine, we conclude that NO nitrosates the active site cysteine of caspase-3 and that this modification is notably inert to fast trans-nitrosation with glutathione. Furthermore, we provide evidence that treatment of caspase-3 with NO can lead to mixed disulfide formation with glutathione, demonstrating the oxidative character of NO.     

Redox control of caspase-3 activity by thioredoxin and other reduced proteins

Caspases are cysteine proteinases that play a critical role in the execution phase of apoptosis. The active site cysteine residue must be reduced for caspase activity. Thioredoxins are redox proteins that catalyze the reduction of cysteine residues. We have examined the ability of various recombinant human thioredoxins to activate caspase-3. The EC(50) for caspase-3 activation by reduced thioredoxin-1 was 2.5 microM, by reduced glutathione 1.0 mM and by dithiothreitol 3.5 mM. A catalytic site redox-inactive mutant thioredoxin-1 was almost as active as thioredoxin-1 in activating caspase-3. Caspase activation was shown to correlate with the number of reduced cysteine residues in the thioredoxins. Reduced insulin and serum albumin were as effective on a molar basis as thioredoxin-1 in activating caspase-3. Thus, caspase-3 activation is not a specific effect of thioredoxins but is a property shared by other reduced proteins.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl)glutathione and N-ethylmaleimide showed intermediate inhibitory effects.     

Dihydroheptaprenyl and dihydrodecaprenyl monophosphates induce apoptosis mediated by activation of caspase-3-like protease

Dolichyl phosphate, an essential carrier lipid in the biosynthesis of N-linked glycoprotein, has been found to induce apoptosis in rat glioma C6 cells and human monoblastic leukemia U937 cells. In the present study, dolichyl phosphate and structurally related compounds were examined regarding their apoptosis-inducing activities in U937 cells. Dihydroheptaprenyl and dihydrodecaprenyl phosphates, of which isoprene units are shorter than that of dolichyl phosphate, induced apoptosis in U937 cells. This phenomenon occurred in a dose- and time-dependent manner, as seen with dolichyl phosphate-induced apoptosis. Derivatives of the same isoprene units of dolichyl phosphate, such as dolichol, dolichal or dolichoic acid, did not induce DNA fragmentation. Farnesyl phosphate and geranylgeranyl phosphate also failed to induce apoptosis. During apoptosis, the caspase family of cysteine proteases play important roles. We observed that apoptosis induced by dihydroprenyl phosphate was mediated by caspase-3-like (CPP32-like) activation but not by caspase-1-like (ICE-like) activation. This caspase-3-like activation was inhibited by a specific inhibitor of caspase-3, DEVD-CHO, but not by an caspase-1 inhibitor YVAD-CHO. We interpret these results to mean that dihydroprenyl phosphates with more than seven isoprene units have apoptosis-inducing activity and that their signal is mediated by caspase-3-like activation.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Modulation of rat liver S-adenosylmethionine synthetase activity by glutathione.

Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated Ki values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a KOX of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance.     

Purification and properties of the glutathione reductase of Chromatium vinosum.

The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.     

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.     

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.     

Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress

The aim of this study was to determine whether exposure to heat stress would lead to oxidative stress and whether this effect varied with different exposure periods. We kept 1-, 6- and 12-month-old male Wistar rats at an ambient temperature of either 22 degrees C or 40 degrees C for 3 and 7 days and measured glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST) activities and levels of thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH) and oxidized glutathione (GSSG) in erythrocytes and determined GSH/GSSG ratio, total glutathione and the redox index. G-6-PD and CAT activities were found to be significantly increased in 1- and 6-month-old rats after 3 and 7 days of heat stress, but G-6-PD activities decreased in 12-month-old rats. Cu, Zn-SOD activity decreased in 1-month-old rats after heat stress, whereas it increased in 6- and 12-month-old rats. GST activity increased in all groups. GSH and total GSH levels and GSH/GSSG ratios decreased in 1- and 6-month-old rats but they increased in 12-month-old rats after heat stress. GSSG levels increased in 1- and 6-month-old rats but decreased in 12-month-old rats after heat stress. TBARS levels increased in all groups. Seven days of stress is more effective in altering enzyme activities and levels of GSH, GSSG and TBARS. When the effects of both heat stress and aging were examined together, it was interesting to note that they mostly influenced G-6-PD activity.     

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

alpha-fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase-3-like proteases.

alpha-Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1-10 microM AFP produces significant suppression of tumor cell growth by inducing dose-dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase-3-like activity resulting in cleavage of poly(ADP-ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP-mediated cell death. Using fluorogenic caspase substrates, we found that caspase-3-like proteases were activated as early as 4 h after treatment of Raji cells with 15 microM AFP, whereas caspase-1, caspase-8, and caspase-9-like activity was not detected during the time interval 0.5-17 h. AFP treatment of Raji cells increased Bcl-2 protein, showing that AFP-induced apoptosis is not explained by downregulation of the Bcl-2 gene. This also suggests that AFP operates downstream of the Bcl-2-sensitive step. AFP notably decreased basal levels of soluble and membrane-bound Fas ligand. Incubation of AFP-sensitive tumor cells (HepG2, Raji) with neutralizing anti-Fas, anti-tumor necrosis factor receptor (TNFR)1 or anti-TNFR2 mAb did not prevent AFP-induced apoptosis, demonstrating its independence of Fas-dependent and TNFR-dependent signaling. In addition, it was found that cells resistant to TNF-induced (Raji) or Fas-induced (MCF-7) apoptosis are, nevertheless, sensitive to AFP-mediated cell death. In contrast, cells sensitive to Fas-mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP-mediated cell death involves activation of the effector caspase-3-like proteases, but is independent of upstream activation of the initiator caspase-1, caspase-8, and caspase-9-like proteases.     

Disulfide reduction and sulfhydryl uptake by Streptococcus mutans

Incubation of Streptococcus mutans cells with certain disulfide compounds resulted in accumulation of reduced sulfhydryl compounds in the extracellular medium or in both the medium and the cells. Oxidized lipoic acid and lipoamide competed for reduction. At high concentrations, these compounds were reduced at rates comparable to that of glucose metabolism, and all of the increase in sulfhydryls was in the medium. Cystamine did not compete with these compounds for reduction but was also reduced at high rates and low apparent affinity, and all of the cysteamine produced from cystamine accumulated in the medium. In contrast, glutathione disulfide (GSSG) and L-cystine were reduced slowly but with high apparent affinity, and 60 to 80% of the increase in sulfhydryls was intracellular. NADH-dependent lipoic acid or lipoamide reductase activity was present in the particulate (wall-plus-membrane) fraction, whereas NADPH-dependent GSSG reductase activity was present in the soluble (cytoplasmic) fraction. Two transport systems for disulfide and sulfhydryl compounds were distinguished. GSSG, L-cystine, and reduced glutathione competed for uptake. L-Cysteine was taken up by a separate system that also accepted L-penicillamine and D-cysteine as substrates. Uptake of glutathione or L-cysteine, or the uptake and reduction of GSSG or L-cystine, resulted in up to a 10-fold increase in cell sulfhydryl content that raised intracellular concentrations to between 30 and 40 mM. These reductase and transport systems enable S. mutans cells to create a reducing environment in both the extracellular medium and the cytoplasm.     

Purification by affinity chromatography of yeast glutathione reductase, the enzyme responsible for the NADPH-dependent reduction of the mixed disulfide of coenzyme A and glutathione.

Glutathione reductase (NAD(P)H : oxidised-glutathione oxidoreductase, EC 1.6.4.2) was purified from baker's yeast by a new procedure involving affinity chromatography on 2',5'-ADP-Sepharose 4B. The yield was 65% of essentially homogeneous enzyme. The activity was assayed with both glutathione disulfide (GSSG) and the mixed disulfide of coenzyme A and glutathione (CoAssg). The two disulfide substrates gave coinciding activity profiles and a constant ratio of the activities in different chromatographic and electrophoretic systems. No evidence was obtained for the existence of a reductase specific for CoASSG distinct from glutathione reductase. It is concluded that normal baker's yeast contains a single reductase active with both GSSG and CoASSG.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.