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 binding of the retro-analogue of glutathione disulfide to glutathione reductase

The retro-analogue of glutathione disulfide was bound to the GSSG binding site of crystalline glutathione reductase. The binding mode revealed why the analogue is a very poor substrate in enzyme catalysis. The observed binding mode difference between natural substrate and retro-analogue is explained.     

Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde

Because of the importance of glutathione (GSH) and glutathione disulfide (GSSG) in cellular signal transduction, gene regulation, redox regulation, and biochemical homeostasis, accurate determination of cellular glutathione levels is critical. Several procedures have been developed, but many suffer from overestimating GSSG or from cellular substances interfering or competing with GSH determination. Assays based on HPLC, with enzymatic reduction of GSSG by glutathione reductase and NADPH, appear to be valid but are limited in sample throughput and availability of equipment. The fluorescence probe o-phthalaldehyde (OPA, phthalic dicarboxaldehyde) reacts with GSH and has a high quantum yield, yet its use has been limited due to unidentified interfering and fluorescence-quenching substances in liver. This paper describes assay conditions under which these limitations are avoided. By using a phosphate-buffered assay at lower pH, interference with nonspecific reactants is minimal. Since enzymatic reduction is not possible due to the reaction of OPA with NAD(P)H and other stronger reducing agents, leading to an overestimation of GSSG levels, dithionite was used to reduce GSSG. High sample throughput combined with sensitive (20-pmol limit of detection) and accurate determination of GSH and GSSG using OPA is achievable with any monochromatographic spectrofluorometer. Sample preparation and storage conditions are described that return the same levels of GSH and GSSG for at least 4 weeks.     

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.     

Energy-linked cardiac transport system for glutathione disulfide

The relationship between the rate of glutathione disulfide (GSSG) export and the energy state was studied in isolated perfused rat heart. The intracellular GSSG level was maintained at saturation for transport (7.5 nmol GSSG X min-1 X g heart-1) by continuous perfusion with 20 microM t-butyl hydroperoxide. GSSG release was substantially restricted upon the addition of inhibitors of mitochondrial respiration such as KCN, antimycin A or rotenone. In contrast, no effect was observed on GSSG release during potassium-induced cardiac arrest, although changes in oxygen consumption and coronary flow were similar to those observed with KCN. The dependence of the GSSG transport rate on the cytosolic free ATP/ADP ratio reveals that GSSG transport is half-maximal at (ATP/ADP)free approximately equal to 10. The capacity of GSSG transport was unchanged by infusion of epinephrine, norepinephrine or dibutyryl cyclic AMP.     

Determination of glutathione and glutathione disulfide in biological samples: An in-depth review

Glutathione (GSH) is a thiol-containing tripeptide, which plays central roles in the defence against oxidative damage and in signaling pathways. Upon oxidation, GSH is transformed to glutathione disulfide (GSSG). The concentrations of GSH and GSSG and their molar ratio are indicators of cell functionality and oxidative stress. Assessment of redox homeostasis in various clinical states and medical applications for restoration of the glutathione status are of growing importance. This review is intended to provide a state-of-the-art overview of issues relating to sample pretreatment and choices for the separation and detection of GSH and GSSG. High-performance liquid chromatography, capillary electrophoresis and gas chromatography (as techniques with a separation step) with photometric, fluorimetric, electrochemical and mass spectrometric detection are discussed, stress being laid on novel approaches.     

Conversion of glutathione to glutathione disulfide, a catalytic function of gamma-glutamyl transpeptidase.

A purification procedure, based on that previously used for rat kidney gamma-glutamyl transpeptidase, was used for the purification of glutathione oxidase (which converts glutathione to gluthathione disulfide). The two activities co-purified, the ratio of the activities remaining constant through all steps of the isolation procedure. The purified enzyme was separable into 12 isozymic species by isoelectric focusing. All 12 isozymes exhibited a constant ratio of transpeptidase to glutathione oxidase activities, strongly supporting the conclusion that conversion of glutathione to glutathione disulfide is a catalytic function of gamma-glutamyl transpeptidase. Modulation of oxidase activity by inhibitors and acceptor substrates of transpeptidase is discussed in relation to the possible glutathione binding sites involved in gamma-glutamyl transfer and oxidase activities of the enzyme.     

Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method

The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.     

Interference of plasmatic reduced glutathione and hemolysis on glutathione disulfide levels in human blood

The blood reduced glutathione (GSH)/GSH disulfide (GSSG) ratio is an index of the oxidant/antioxidant balance of the whole body. Nevertheless, data indicating GSH and GSSG physiological levels are still widely divergent, especially those on GSSG, probably due to its low concentration. Standardization in methodological protocols and sample manipulation could help to minimize these discrepancies. Therefore, we have investigated how plasma reduced GSH, which is rapidly oxidized after blood withdrawal, could alter the blood GSSG measurement if the sample is not suitably processed. We have observed that an increase in plasma GSH concentration, due to red blood cell hemolysis, is responsible for a significant overestimation of blood GSSG level. Our results show that, before performing blood GSSG determination, thiols have to be rapidly blocked, to avoid possible pitfalls in GSSG measurement, in particular when hemolysis is present.     

Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.     

Increase in glutathione disulfide level regulates the activity of microsomal glutathione S-transferase in rat liver

Glutathione disulfide stimulates the activity of rat liver microsomal glutathione S-transferase 2-fold after incubation at 25 degrees C for 10 min. When the microsomes were incubated with the disulfide for over 20 min, the transferase activity increased to the same extent as in the case of N-ethylmaleimide (6-fold). Even in the presence of reduced glutathione, some enhancement of the transferase activity was observed. The data presented here are evidence that increase in glutathione disulfide level, e.g. by lipid peroxidation, on endoplasmic reticulum causes the upregulation of microsomal glutathione S-transferase activity.     

Intracellular fate of ferritin in HeLa cells following microinjection

It is known that following iron overload newly synthesized ferritin molecules accumulate in lysosomes. However, the way in which these molecules enter the lysosomes has not been clarified. In order to assess if these molecules can be taken up by lysosomes from the cell sap, i.e., by way of autophagy, ferritin was introduced into HeLa cells through microinjection with a glass capillary. The fate of the ferritin was studied after varying intervals with the electron microscope. Shortly after microinjection ferritin molecules could be observed in the cell sap. After both 1 and 2 h, they were found in clusters and still mainly in the cell sap. After 4 h, ferritin molecules were present not only in the cell sap and in autophagic vacuoles but also in occasional secondary lysosomes. After 12 h, they were seen mainly in lysosomes, undergoing degradation. In no instance were ferritin molecules translocated into other organelles such as mitochondria, Golgi apparatus, or endoplasmic reticulum. The present study demonstrates that ferritin can be introduced into cells by glass capillary microinjection without cell damage. From its initial location in the cell sap ferritin is taken up into the lysosomal vacuome. Autophagy is considered to be the principal mechanism for the transfer of the ferritin molecules into lysosomes.     

Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry

A sensitive method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human saliva was developed and validated. GSH was captured and stabilized by the addition of N-ethylmaleimide (NEM). Solid-phase extraction (SPE) using an Oasis MAX extraction cartridge was employed for sample preparation and analysis was performed on a Shimadzu LCMS-2010 A that was operated in the single ion monitoring mode using positive ion electrospray ionization (ESI) as the interface. The monitored ion for GSH-NEM was m/z 433 and that for GSSG was m/z 613. Chromatography was carried out on an Atlantis HILIC silica column (150 mm x 2.1 mm, 5 microm) with acetonitrile and formate buffer as the mobile phase at the flow rate of 0.2 ml/min. The calibration curve was linear over the range of 0.1-100 microM for GSH-NEM. The extraction recoveries of GSH-NEM spiked at concentrations of 25 and 50 microM were 97.1 and 104.4%, respectively. Similar results were obtained for GSSG. The newly developed hydrophilic interaction chromatography with mass spectrometry (HILIC/MS) method showed superior sensitivity for the determination of GSH and GSSG in human saliva samples.     

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.     

Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples

By combining the least complicated and expedient methods of sample handling with the sensitivity and specificity of the GSH assay by enzymatic recycling and the small volumes and software capabilities of microtiter plate technology we have devised a rapid, sensitive, and easy assay for GSH and GSSG in biological samples. The assay is sensitive to 5 pmol in sample volumes of 50 microliters, although other volumes could be used. The use of a computer-driven microplate with software capable of linear kinetic data storage and analysis on each well, Maxline series microplate readers and Softmax software, enables the user not only to assay large numbers of samples per day but also to have immediate calculated results. We suggest by examples that measurements of total GSH as well as changes in GSH:GSSG in vitro and in vivo are feasible with this technology.     

Refolding of the mixed disulfide of bovine trypsinogen and glutathione.

The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.     

Hepatoprotection by L-cysteine-glutathione mixed disulfide,a sulfhydryl-modified prodrug of glutathione

L-Cysteine-glutathione disulfide, a ubiquitous substance present in mammalian cells, was shown to be highly effective in protecting mice against acetaminophen-induced hepatotoxicity. Since the corresponding D-cysteine-glutathione disulfide was totally ineffective in this regard, an enzymatic mechanism that provides glutathione directly to cells is postulated.