Steady-state kinetic studies of glutathione reductase

The steady-state kinetic studies of yeast glutathione reductase, performed when [GSSG] = 10[NADPH] in the assay mixture, show that at concentrations of GSSG under 450 microM the enzymatic mechanism pathway is ping-pong. Furthermore, in the case of higher values, the enzymatic kinetics follows a sequential pathway. However when the glutathione reductase reaction passes to the ping-pong mechanism, the inhibition effect by excess of NADPH is stronger than when the reaction takes place over the sequential mechanism.     

Glutathione reductase: solvent equilibrium and kinetic isotope effects

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456.     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.     

A 'branched' mechanism of the reverse reaction of yeast glutathione reductase. An estimation of the enzyme standard potential values from the steady-state kinetics data

The reduced glutathione-linked NADP+ reduction, catalyzed by yeast glutathione reductase, follows a 'sequential' or 'ping-pong' mechanism at high or low NADP+ concentrations, respectively. The pattern of the NADPH and NADP+ cross-inhibition reflects not only the competition for the binding site, but the shift of the reaction equilibrium as well. A 'branched' scheme of the glutathione reductase reaction is presented. The enzyme standard potential (-255 mV, pH 7.0) was estimated from the ratio of the NADPH and NADP+ rate constants corresponding to the ping-pong mechanism.     

Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human jejunal glutathione reductase: purification and evaluation of the NADPH- and glutathione-induced changes in redox state

Human proximal jejunal glutathione reductase (EC 1.6.4.2) was purified to homogeneity by affinity chromatography on 2', 5'-ADP-Sepharose 4B. In most of its molecular and kinetic properties, the enzyme resembled glutathione reductase from other sources: The subunit mass was 56 kDa; the isoelectric point and pH optimum were 6.75 and 7.25, respectively; Michaelis constants, determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [Km(NADPH) = 20 microM, Km(GSSG) = 80 microM]. The response of the enzyme to reducing conditions, on the other hand, had unique features: Preincubation with 1 mM NADPH resulted in 90% loss of activity which could be partially reversed by 2 mM GSSG, but not GSH. (Treatment with GSSG regenerated 68% of the original activity.) Reduction by GSH also caused inactivation which potentially amounted to greater than 80%. This inactivation could not be reversed by GSSG. The protective effect of GSSG against inactivation by GSH was studied. Except where [GSSG] far exceeded [GSH], the presence of GSSG in the preincubation medium decreased the extent of inhibition without affecting the rate constant for approach to equilibrium activity. At [GSSG] greater than [GSH] a decrease in the rate constant for inactivation was also observed. The results were interpreted in terms of a three-step mechanism: (1) preequilibrium reduction of Eox to Ered; (2) rate-limiting change in conformation from Ered to E'red, and (3) irreversible conversion to catalytically inferior products.     

Kinetic properties of sheep brain glutathione reductase.

The kinetic properties of sheep brain glutathione reductase (GSSGR) were investigated. The enzyme showed Ping-Pong kinetics with double substrate inhibition in the forward direction. Km values for NADPH and GSSG were found to be 60.9 and 116.9 mumol/l, and Ki values were found to be 42.1 and 347.3 mumol/l, respectively. NADP+ inhibition at low fixed concentration of NADPH was mixed-type with a Ki of 281.5 mumol/l and alpha of 0.048. It is concluded that the enzyme shows a hybrid Ping-Pong-ordered branched mechanism.     

Hysteretic behaviour and GSSG substrate inhibition shown by glutathione reductase from Phycomyces blakesleeanus

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.     

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed‐type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox‐active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.     

Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.     

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     

Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates

Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction. For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes. Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates. Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate. The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex. At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex. Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex. As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random. By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli

A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex. This step, H4F dissociation from the E X NADPH X H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM. The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, khyd = 950 s-1, essentially irreversible, Keq = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pKa = 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4. This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.     

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

The kinetic mechanism of human placental aldose reductase and aldehyde reductase II

The kinetic mechanism of NADPH-dependent aldehyde reductase II and aldose reductase, purified from human placenta, has been studied using L-glucuronate and DL-glyceraldehyde as their respective substrates. For aldehyde reductase II, the initial velocity and product inhibition studies (using NADP and gulonate) indicate that the enzyme reaction sequence is ordered with NADPH binding to the free enzyme and NADP being the last product to be released. Inhibition patterns using menadione (an analog of the aldehydic substrate) and ATP-ribose (an analog of NADPH) are also consistent with a compulsory ordered reaction sequence. Isotope effects of deuterium-substituted NADPH (NADPD) also corroborate the above reaction scheme and indicate that hydride transfer is not the sole rate-limiting step in the reaction sequence. For aldose reductase, initial velocity patterns, product, and dead-end inhibition studies indicate a random binding pattern of the substrates and an ordered release of product; the coenzyme is released last. A steady-state random mechanism is also consistent with deuterium isotope effects of NADPD on the reaction sequence catalyzed by this enzyme. However, the hydride transfer step seems to be more rate determining for aldose reductase than for aldehyde reductase II.     

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.     

Glutathione metabolism of the erythrocyte. The enzymic cleavage of glutathione–haemoglobin preparations by glutathione reductase

A complex of haemoglobin and GSH was prepared by incubating haemoglobin with GSH and acetylphenylhydrazine. GSH could be released from the crude preparation by incubation with NADPH. However, when the haemoglobin preparation was separated from glutathione reductase by DEAE-Sephadex chromatography, NADPH no longer released GSH. Rather, the addition of a combination of either partially purified human erythrocyte or crystalline glutathione reductase and NADPH was required to release GSH from the haemoglobin-GSH complex. This complex is commonly believed to represent a mixed disulphide of GSH and the cysteine-beta-93 thiol group. This interpretation was supported by the finding that prior alkylation of available haemoglobin thiol groups prevented the formation of the complex. By using haemoglobin-[(35)S]GSH complex as a substrate, it was shown that GSH itself released the radioactivity from the complex only very slowly. In contrast, the release of [(35)S]GSH was very rapid in the presence of NADPH and glutathione reductase. This suggests that the cleavage of the haemoglobin-GSH complex is not mediated by GSH with cyclic reduction of GSSG formed, but rather proceeds enzymically through glutathione reductase.     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.