Glutathione VII. Differentiation among substrates by the thiol-oxidizing agent, diamide (original) (raw)
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Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures
The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 M concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 M concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.
The FASEB Journal, 2002
Fluorescence microscopy of A549 cells stained with a glutathione (L-γ-glutamyl-Lcysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the perinuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol-reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)-dependent inhibition of GSH synthesis. However, confocal visualization of antibody-stained A549 cells in the z-plane revealed the majority of the perinuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z-plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z-section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near-complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z-plane. The antibody also provided initial visualization of the compartmentalization of protein-GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti-GSH antibody-stained Jurkat T lymhocytes, we also demonstrated population variations in the cellular compliment of GSH and protein-GSH mixed disulfides, formed in response to diamide. In addition, we showed cell-cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.
Biochemistry, 1978
An enzyme catalyzing thiol-disulfide interchange of glutathione and disulfides and the reaction between glutathione and thiosulfate esters has been purified 40 000-fold from rat liver cytosol. The enzyme, named thioltransferase (Askelöf, P., Axelsson, K., Eriksson, S., & Mannervik, B. (1974) FEBS Lett. 38, 263--267), was homogeneous in several electrophoretic systems, had an isoelectric point at pH 9.6, and contained 8.6% (w/w) carbohydrate. The catalytic activity had a distinct optimum at pH 7.5. A series of substrates was tested at a constant glutathione level; the kcat values (at 4mM glutathione) were all in the range of about 10(4) min-1. The substrates included mixed disulfides of glutathione, other low-molecular-weight disulfides, S-sulfocysteine and S-sulfoglutathione, and peptide disulfides such as insulin, oxytocin, ribonuclease, and the mixed disulfide of glutathione and egg-white lysozyme. The enzymatic reaction was inhibited by an excess of glutathione (greater than 4mM).
Analytical …, 2000
A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or ␣-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or ␣-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce ␣-lipoate, while Q 7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce ␣-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of ␣-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit ␣-lipoate reduction in Q 7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.
Biomedical Chromatography, 2009
This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Effects of N-acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster ovary cells
Glutathione peroxidase mimicry of diphenyl diselenide: Plausible contribution of proteins’ thiols
Toxicology Advances
Organoseleniums are a class of compounds attracting attention across the globe owing to their Glutathione peroxidase (GPx) mimicry, which confers on them a strong antioxidant activity. Diphenyl diselenide (DPDS) is an Organoselenium whose GPx mimetic property has been suggested to rely on the oxidation of non-protein or protein thiols critical to the activities of some sulfhydryl enzymes. This study, therefore investigated the GPx mimic/antioxidant property of DPDS as well as the role of thiols of two key sulfhydryl enzymes, cerebral Na + /K +-ATPase (sodium pump) and hepatic delta-aminolevulinic acid dehydratase (δ-ALAD) in the GPx mimicry of DPDS. Albino Wistar rats were euthanized, and the liver and brain were removed and used to assay for the effect of DPDS on lipid peroxidation induced by two prooxidants [Fe 2+ (10 µM) and H 2 O 2 , (1 mM)] as well as the activities of the sulfhydryl enzymes. The results revealed that DPDS profoundly (P < 0.05) counteracted Fe 2+ and H 2 O 2-induced lipid peroxidation in the rats' hepatic and cerebral tissues. Furthermore, the results of assay systems for lipid peroxidation and sodium pump revealed that DPDS inhibited Na + /K +-ATPase and lipid peroxidation in the brain tissue homogenates in the same reaction system. A similar result was obtained in the assay system for lipid peroxidation and hepatic δ-ALAD as DPDS simultaneously inhibited the enzyme's activity and lipid peroxidation. This suggests that the GPx mimetic property of DPDS may be linked to the enzymes' loss of activity, which further validates the suggestions that the enzymes' inhibition, as well as the antioxidant action of DPDS, rely on the oxidation of critical thiols of the enzymes. However, the GPx mimicry of DPDS should be investigated in the presence of thiol-blocking or oxidizing agents in biological systems in order to further ascertain the role of protein thiols.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1995
A novel method for glutathione-protein mixed disulphide (GSSP) determination, based on the use of protein sulphydryl groups as endogenous reductant and on the spectrophotometric determination of reduced glutathione, is described. The procedure is based on the observation that acid-precipitated proteins from different rat tissues rapidly release GSH from GSSP when brought to neutral pH. The basal GSSP content determined in rat liver, heart, lung, testis, spleen and brain corresponded to that reported in the literature and determined by more complex sample preparation or labor-intensive analytical procedures.
Formation and reduction of glutathione-protein mixed disulfides during oxidative stress
Biochemical Pharmacology, 1987
Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose-and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione.