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.     

Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange

Rabbit muscle phosphofructokinase is rapidly inactivated at pH 8.0 by incubation with low concentrations of oxidized glutathione, Coenzyme A glutathione mixed disulfide, and oxidized Coenzyme A. The inactivation is first order in disulfide concentration over the concentration ranges examined (50-200 microM), and is approximately 8-fold slower at pH 7.0 than at pH 8.0. The substrates ATP and fructose 6-phosphate protect against inactivation while effector molecules such as AMP, cAMP, and citrate do not. The oxidation of the enzyme by disulfides is fully reversible. The equilibrium constant for the reaction Ered + GSSG in equilibrium Eox + GSH at pH 8.0 is 7.1 in the absence of substrates and 2.5 in the presence of 0.1 mM ATP. For comparison, the equilibrium constant for the reaction CoASH + GSSG in equilibrium CoASSG + GSH was found to be 3.1 at pH 8.0. These equilibrium constants for thiol/disulfide exchange are such that modulation of phosphofructokinase activity by thiol/disulfide exchange in vivo is feasible. The ability of the thiol/disulfide ratio in vivo to modulate the activity of the fructose 6-phosphate/fructose 1,6-diphosphate futile cycle is discussed. The possibility is considered that modulation of the thiol/disulfide ratio in vivo may serve as a "third messenger" in response to cAMP levels, and that the activity of key enzymes of glycolysis/gluconeogenesis may be regulated in response to changing thiol/disulfide ratios.     

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.     

Thiol/disulfide exchange between rabbit muscle phosphofructokinase and glutathione. Kinetics and thermodynamics of enzyme oxidation

Reversible thiol/disulfide exchange equilibria between rabbit muscle phosphofructokinase and glutathione redox buffers results in a dependence of the activity of the enzyme on the thiol to disulfide ratio of the redox buffer (Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086-12091). The transition between fully reduced (active) and fully oxidized (inactive) enzyme is half complete at a [GSH]/[GSSG] ratio of 6.5 +/- 1 at pH 8.0 and 5.6 +/- 0.9 at pH 7.2. In the presence of excess GSSG approximately 40-50% of the activity is lost in a rapid process (k = 110 M-1 min-1), while the remaining activity is lost more slowly (k = 1.9 M-1 min-1). Two equivalents of radiolabeled glutathione are incorporated covalently, one coincident with each phase of inactivation. The most rapidly oxidized sulfhydryl group is also the most rapidly reduced by GSH in the reverse reaction (k = 150 M-1 min-1). Reduction of a more slowly reacting protein-glutathione mixed disulfide is required to regenerate the original activity (k = 0.33 M-1 min-1). The thiol/disulfide oxidation equilibrium constant (Kox) for the most rapidly oxidized sulfhydryl group is estimated to be 0.7 while that for the more slowly oxidized group is 6.1. The sulfhydryl group which is more easily oxidized kinetically is the more thermodynamically resistant to oxidation. The magnitude of the equilibrium constants for these reversible oxidations would suggest that the oxidation state (and activity) of phosphofructokinase would not be significantly affected by typical metabolic changes in the glutathione oxidation state in vivo.     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.     

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles,Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.     

Influence of glutathione on the reactivation of enzymes containing cysteine or cystine

Refolding of dimeric porcine cytosolic or mitochondrial malate dehydrogenases and of tetrameric pig heart and skeletal muscle lactate dehydrogenases (containing 5-7 cysteine residues), as well as reformation of the four cystine cross-bridges of bovine pancreatic ribonuclease, were studied in the presence of reduced and oxidized glutathione (GSH and GSSG). At the intracellular GSH level (5 mM) reduced ribonuclease can be reoxidized by 0.01-0.5 mM GSSG (pH 7.4) both at 20 degrees C and 37 degrees C. In this physiological range of GSSG concentrations and pH, the dehydrogenases show at least partial reactivation. With GSSG concentrations greater than 5 mM, reactivation is found to be completely inhibited for all the enzymes given. The results show that at the intracellular level of GSH and GSSG, thiol groups in reduced, unfolded ribonuclease are oxidized to form intramolecular cystine cross-bridges, while thiol groups of typical cysteine enzymes, such as lactate and malate dehydrogenase, remain in their reduced state during refolding. The rate of reactivation of lactate dehydrogenase (porcine muscle) is not affected by GSSG. In the case of ribonuclease, increasing concentrations of GSSG increase the rate of reactivation: At 20 degrees C, the halftime of the correct disulfide bond formation varies from approximately equal to 80 h in the presence of 0.01 mM GSSG to approximately equal to 10 h in the presence of 0.25 mM GSSG. A further increase in the rate of reactivation at higher GSSG concentrations is accompanied by a decrease in yield. Reactivation of ribonuclease is also observed at the low glutathione level found in blood plasma (5-25 microM GSH).     

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.     

Reactions of nitrosobenzene with reduced glutathione.

Nitrosobenzene (NOB) formed acid labile conjugates with reduced glutathione (GSH) and hemoglobin within red cells. In vitro, NOB rapidly reacted with GSH with formation of phenylhydroxylamine (PH), oxidized glutathione (GSSG), and a water-soluble compound identified as glutathionesulfinanilide (GSO-AN). Free aniline (AN), aminophenols and azoxybenzene were not detected. The proportion of PH formed increased with increasing GSH concentration and at higher pH values. Spectroscopic analysis revealed the formation of a labile adduct following a second order reaction (K = 5 x 10(3) M-1 . sec-1 at pH 7.4 and 37 degrees). This reaction was reversible because nearly all NOB could be extracted with ether from the labile intermediate. On the other hand, the labile intermediate was transformed into GSO-AN (with increasing rate at lower pH values) or it was cleaved by GSH with formation of GSSG and PH. Intermediate formation of NOB and thiol radicals was ruled out by analysis of the equilibrium data. A tentative scheme is presented for the proposed reaction mechanism.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Nonenzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxidoreduction buffers.

With the glutathione system that leads to rapid regeneration of reduced lysozyme (Saxena, V. P., and Wetlaufer, D. B. (1971) Biochemistry 9, 5015), reduced pancreatic ribonuclease (RNase) regenerated activity in high yield (greater than 90%) but at a considerably lower rate (t1/2 approximately 75 min). Systematic examination of the effects upon regeneration of the concentrations and ratios of reduced and oxidized glutathione (GSH and GSSG) showed the same broad optima for RNase as were earlier found for lysozyme: [GSSG] = 5 X 10(-4) M, [GSH] = 5 X 10(-3) M. Regeneration of reduced RNase by air oxidation was shown to be inhibitable by 10(-4) M EDTA, whereas the glutathione regeneration was unaffected by EDTA. In addition the air-oxidative regeneration showed a strong temperature dependence, in contrast with the glutathione system. The mechanisms of these two kinds of regenerations are therefore different. Six potentially catalytic metal ions were tested in the air-oxidative regeneration of RNase: Cu2+, Co2+, Mn2+, Fe3+, Zn2+, and Ni2+. Of these, only Cu2+ enhanced the rate of regeneration of RNase activity, although both Cu2+ and Co2+ catalyzed thioloxidation of reduced RNase. The rates and yields of RNase regenerations were independent of protein concentration from 3 X 10(-7) M to 1.2 X 10(-5) M in the glutathione system. Preincubation of freshly dissolved reduced RNase under nonoxidizing conditions before adding glutathione did not change the rate or extent of regeneration. Studies of its pH dependence showed that the glutathione regeneration depends on the deprotonation of prototropic groups with 7.5 less than pK less than 8.0. The major ion exchange chromatographic peaks from glutathione and air-oxidative regenerations appeared to be identical with native RNase, by the criteria of specific activity, chromatographic mobility, and circular dichroic spectra. The glutathione system permits regeneration at much higher RNase concentration than the air regeneration, with rates and yields comparable to the greatest reported for air regeneration.     

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.     

Thiol/disulfide redox equilibrium between glutathione and glycogen debranching enzyme (amylo-1,6-glucosidase/4-alpha-glucanotransferase) from rabbit muscle

Rabbit skeletal muscle glycogen debranching enzyme is inactivated in a kinetically biphasic manner by GSSG at pH 8.0. The rapid phase results in the loss of 30% activity, while the slower phase leads to total enzyme inactivation. Both the glucosidase and the transferase activities of the enzyme are inhibited by GSSG. The inactivation by disulfides is fully and rapidly reversed in a biphasic manner by reduction with excess reduced dithiothreitol or GSH. After a fast initial recovery of 70% of the initial activity, the remaining 30% of the activity is recovered more slowly. Equilibration of the enzyme with a redox buffer of GSH and GSSG shows a monophasic equilibration of the activity. The ratio of GSH/GSSG where the enzyme is 50% active (R0.5) is 0.06 +/- 0.03. The R0.5 does not vary significantly with the total concentration of glutathione species suggesting formation of protein-SSG mixed disulfides. The ratios of the observed second-order rate constants for GSSG inactivation and GSH reactivation do not lead to a correct value of the observed thiol/disulfide oxidation equilibrium constant. Although the enzyme has sulfhydryl groups, the oxidation of which leads to activity changes, the kinetic and thermodynamic resistance to oxidation suggests that the enzyme is not likely to be subject to regulation by thiol/disulfide exchange in vivo.     

Effect of cadmium on antioxidative enzymes,glutathione content,and glutathionylation in tall fescue

The aim of this work was to assess the effect of different Cd2+concentrations on some antioxidant enzymes in Festuca arundinacea. Increased activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione S-transferase, and glutathione reductase were ascertained in response to low Cd²⁺ concentrations (0–20 μM), whereas the enzyme activities were less increased or decreased at a higher Cd²⁺ dosage (50 μM) and a longer exposure. The content of reduced glutathione (GSH) decreased significantly with increasing Cd²⁺ concentrations, whereas the content of oxidized glutathione (GSSG) increased proportionally to the amount of Cd²⁺ applied. Further experiments, performed by incubating the enzyme extracts with oxidized glutathione, evidenced that the addition of GSSG to the incubation mixtures caused significant decreases of some enzymatic activities. Finally, the effect of glutathione S-transferase, FaGST I, extracted from fescue seedlings and purified till homogeneity, on these enzyme activities was investigated. It was found that FaGST I enhanced the decreased enzymatic activities caused by GSSG.     

Protective effect of propionyl-L-carnitine against ischaemia and reperfusion-damage

Summary Reperfusion of isolated rabbit heart after 60 min of ischaemia resulted in poor recovery of mechanical function, release of reduced (GSH) and oxidized glutathione (GSSG), reduction of tissue GSH/GSSG ratio and shift of cellular thiol redox state toward oxidation, suggesting the occurrence of oxidative stress. Pretreatment of the isolated heart with propionyl-L-carnitine (10^–7M) improved the functional recovery of the myocardium, reduced GSH and GSSG release and attenuated the accumulation of tissue GSSG. This effect was specific for propionyl-L-carnitine as L-carnitine and propionyl acid did not modify myocardial damage.     

Variation in glutathione status associated with induction and initiation of diapause in eggs of the bivoltine strain of the silkworm Bombyx mori

For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.