protein-GSH Mixed Disulfides Research Articles

Does oxidative stress play any role in diabetic cataract formation? ----Re-evaluation using a thioltransferase gene knockout mouse model

Oxidative stress is a known risk factor in senile cataract formation. In recent years, it has been suggested that oxidation may also be associated with cataract induced by hyperglycemia, but this concept has not been well examined or validated. Since thioltransferase (TTase) is one of the key enzymes that regulates redox homeostasis and protects against oxidative stress in the lens, we have used TTase gene knockout (KO) mice as a model to examine this new concept. Lenses from 4 months old TTase KO and wild-type (WT) mice were incubated in TC199 culture medium containing 30 mM glucose for 48 h. Each lens was assessed for opacity, graded by LOCSII system, and the wet weight was recorded after which it was homogenized in lysis buffer and analyzed for water-soluble protein and free glutathione (GSH). In vivo studies were carried out using 4 months old TTase KO and WT mouse groups. Each mouse received two consecutive days of intraperitoneal streptozotozin (STZ) injections to induce diabetes. The lenses were examined weekly for 4 weeks using a slit-lamp biomicroscope, and then extracted and analyzed for levels of GSH, water-soluble protein, ATP and protein-GSH mixed disulfide (PSSG). TTase KO lenses cultured in high glucose developed a mild cortical opacity but slightly more than that of the WT lenses. Both groups had similar contents of soluble proteins and GSH. Exposure to high glucose did not change the soluble protein level but did suppress GSH by 20% in lenses with or without TTase. STZ-induced diabetic KO mice also developed a higher degree of mild cortical lens opacity compared to that of the diabetic WT controls. Similar 15–20% losses in lens GSH and ATP were found after one-month induced diabetes in WT and KO mice. There was a 20% greater amount of PSSG in the lenses of TTase KO than the WT control. Under diabetic condition, both groups displayed more glutathionylated proteins in the beta-actin (42 kDa) and lens crystallin proteins (18–22 kDa) regions, and some additional modified proteins at 15–17 kDa and 60–70 kDa, with a total 20–30% PSSG increment in both groups. In conclusion, we have found that hyperglycemia induced some oxidative stress-associated biochemical changes with mild lens opacity in both WT and KO mice. However, these changes were only marginally higher in the TTase KO mouse than that of the WT control, suggesting that TTase deletion may only play a minor role in the early stage of hyperglycemia-induced cataract formation in the mice.

Redox Modulation Matters: Emerging Functions for Glutaredoxins in Plant Development and Stress Responses.

Glutaredoxins (GRXs) are small ubiquitous glutathione (GSH)-dependent oxidoreductases that catalyze the reversible reduction of protein disulfide bridges or protein-GSH mixed disulfide bonds via a dithiol or monothiol mechanism, respectively. Three major classes of GRXs, with the CPYC-type, the CGFS-type or the CC-type active site, have been identified in many plant species. In spite of the well-characterized roles for GRXs in Escherichia coli, yeast and humans, the biological functions of plant GRXs have been largely enigmatic. The CPYC-type and CGFS-type GRXs exist in all organisms, from prokaryotes to eukaryotes, whereas the CC-type class has thus far been solely identified in land plants. Only the number of the CC-type GRXs has enlarged dramatically during the evolution of land plants, suggesting their participation in the formation of more complex plants adapted to life on land. A growing body of evidence indicates that plant GRXs are involved in numerous cellular pathways. In this review, emphasis is placed on the recently emerging functions for GRXs in floral organ development and disease resistance. Notably, CC-type GRXs have been recruited to participate in these two seemingly unrelated processes. Besides, the current knowledge of plant GRXs in the assembly and delivery of iron-sulfur clusters, oxidative stress responses and arsenic resistance is also presented. As GRXs require GSH as an electron donor to reduce their target proteins, GSH-related developmental processes, including the control of flowering time and the development of postembryonic roots and shoots, are further discussed. Profiling the thiol redox proteome using high-throughput proteomic approaches and measuring cellular redox changes with fluorescent redox biosensors will help to further unravel the redox-regulated physiological processes in plants.

Oxidative Responses Induced by Pharmacologic Vitreolysis and/or Long-term Hyperoxia Treatment in Rat Lenses

ABSTRACTPurpose: The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo.Methods: Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors.Results: No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis.Conclusions: Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.

Redox Mechanisms Involved in the Selective Activation of Nrf2-mediated Resistance Versus p53-dependent Apoptosis in Adenocarcinoma Cells

We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.

Influence of Dietary Quercetin on Glutathione Redox Status in Mice

Flavonoids such as quercetin have been shown to serve as a protective defense against oxidative damage in vivo. However, the bioavailability of quercetin depends on the food source and type of glycosidic moiety linked to the molecule. In this study, mice were fed 1 mg/day quercetin in the form of quercetin aglycone, rutin, apple, or onion, and reduced glutathione (GSH), oxidized glutathione (GSSG), and protein-GSH mixed disulfides were determined to investigate the influence of dietary quercetin on the GSH redox status in metabolically active tissues, mitochondria, and plasma of mice. All quercetin treatment groups produced increases in the GSH:GSSG ratio and decreases in mixed disulfide levels in hepatic tissue. Cardiac tissue did not change in response to dietary quercetin; however, cardiac mitochondria demonstrated a reduction in the GSH:GSSG ratio and an increase in protein mixed disulfide levels. No significant changes were observed in the plasma GSH:GSSG ratio, but mixed disulfide levels were decreased for all of the diets. The changes in plasma redox status did not parallel the changes in the tissues. Onion fed mice demonstrated the greatest increases in GSH:GSSG ratios and the greatest decreases in protein mixed disulfide levels of all diets compared. For all treatment groups, increases in the GSH:GSSG ratios corresponded with decreases in protein mixed disulfide levels. The results of this study indicate that quercetin influences GSH:GSSG ratios and protein thiolation in a tissue-specific manner and that these effects are dependent on food source and bioavailability.

S-glutathionylation in human platelets by a thiol–disulfide exchange-independent mechanism

Protein–glutathione mixed disulfide formation was investigated in vitro by exposure of human platelets to the thiol-specific oxidant azodicarboxylic acid-bis-dimethylamide (diamide). We found that diamide causes a decrease in the reduced form of glutathione (GSH), paralleled by an increase in protein–GSH mixed disulfides (S-glutathionylated proteins), which was not accompanied by any significant increase in the basal level of glutathione disulfide (GSSG). The increase in the appearance of S-glutathionylated proteins was inversely correlated with ADP-induced platelet aggregation. Platelet cytoskeleton was analyzed by SDS–PAGE followed by Western immunoblotting with anti-GSH antibody. The main S-glutathionylated cytoskeletal protein proved to be actin, which accounts for 35% of the platelet total protein content. Our results suggest that neither GSSG formation nor a consequent thiol–disulfide exchange mechanism is involved in actin S-glutathionylation of human platelets exposed to diamide. Instead, a mechanism involving the initial oxidative activation of actin thiol groups, which then react with GSH to the protein–GSH mixed disulfides, makes it likely that platelet actin is S-glutathionylated without any significant increase in the GSSG content.

Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells.

Fluorescence microscopy of A549 cells stained with a glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the peri-nuclear 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 peri-nuclear 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 lymphocytes, 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.

Physical Exercise Induces Oxidation of Plasma Protein Thiols to Cysteine Mixed Disulfides in Humans.

We have reported that strenuous physical exercise causes a decrease in protein-bound sulfhydryl groups (p-SHs), such as albumin cysteine residues, in human plasma (Inayama et al., 1996, Life Sci., 59, 573-578). We further investigated the fate of plasma protein thiols after moderate exercise. Six untrained healthy female volunteers ran for 30-min at the individual ventilatory threshold. We observed an increase in protein cysteine mixed disulfides (p-S-Cys) after running, as evidenced by reducing plasma proteins with dithiothreitol to detect the increase of cysteine, along with the concomitant decrease in p-SHs in plasma. However, plasma protein-bound glutathione (GSH) and S-nitroso-protein were undetectable before and after exercise. Test tube experiments suggest that p-S-Cys are probably formed by the hydrolysis of protein GSH mixed disulfides by γ-glutamyltranspeptidase and peptidase, and/or by the oxidative addition of p-SHs to cystine.

Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures.

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 microM buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mM 3-amino-1,2,4-triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH-depleted cells with malonate (40 mM) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3-amino-1,2,4-triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre-treatment of cultures with 400 microM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein-GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mM) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein-GSH mixed disulfides three-fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein-GSH mixed disulfide formation under these circumstances may play a protective role.

Glutathione homeostasis in alveolar epithelial cells in vitro and lung in vivo under oxidative stress

We studied the acute effects of cigarette smoke condensate (CSC), H2O2, and tumor necrosis factor (TNF)-alpha on the glutathione (GSH) redox system in a human type II epithelial cell line (A549) in vitro. CSC, in vitro and in vivo after intratracheal instillation of CSC in the rat, produced a depletion of intracellular soluble GSH, concomitant with GSH-conjugate formation, without significant elevation of oxidized GSH (GSSG), protein-GSH mixed disulfides (PrSSG), nor any GSH efflux from the cells. By contrast, H2O2 (500 microM) after 5-min exposure to A549 cells caused significant depletion of intracellular GSH associated with an efflux of GSSG and a significant increase in the formation of PrSSG. TNF-alpha, in concentrations of 100 U/ml and 1,000 U/ml, produced a significant depletion of GSH in A549 cells after 4- and 24-h exposure, with an associated elevation of GSSG. The activities of glutathione peroxidase, gamma-glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase were significantly decreased in epithelial cells and in rat lungs after CSC exposure, without change in glutathione S-transferase and glutathione reductase activities. By contrast, H2O2 and TNF-alpha did not alter these enzyme activities in epithelial cells. Thus GSH depletion and alteration in enzyme activities in alveolar epithelial cells by CSC, H2O2, and TNF-alpha occur by different mechanisms.

Cysteines of chloroplast NADP-malate dehydrogenase form mixed disulfides

Chloroplast NADP-malate dehydrogenase (NADP-MDH) from pea and from spinach was N-terminally truncated by limited proteolysis with Staphylococcus aureus protease V8. The resulting monomeric enzymes lacking, respectively, the 37 and 38 N-terminal amino acids were inactive. Reduction and addition of low concentrations of guanidine-HCl (50–100 mM) resulted in a highly active enzyme of 850 units per mg protein. Equilibration of the truncated enzyme with various glutathione (GSH) redox buffers and assaying its activity in the presence of guanidine-HCl was used to establish the existence of protein—GSH mixed disulfides. This finding was further confirmed using incorporation of radioactively labelled thiol. The possible function of such cysteine modifications under oxidative stress and their regeneration by the thioredoxin system in the light is discussed.

The Effect and Recovery of Long-term H 2O 2 Exposure on Lens Morphology and Biochemistry

Oxidative stress has long been speculated to play an important role in cataractogenesis. In the H 2O 2-induced cataract model, rat lens showed extensive biochemical damage but very mild morphological changes after being exposed to H 2O 2 (0·5 mM) for 24 hr in culture. This damage included reduced glutathione (GSH) depletion, protein-GSH mixed disulfide (PSSG) elevation but not protein-protein disulfide (PSSP) formation. In order to understand the role of protein-thiol mixed disulfide formation in relation to the sequence of events during cataract induction, we conducted a long term H 2O 2 exposure study for up to 96 hr to monitor the dynamic changes in GSH and PSSG levels, the formation of PSSP aggregate, protein solubility, and the progression in lens opacity. Rat lenses were cultured in 0·5 mM H 2O 2 and harvested at intervals of 24, 48, 72 and 96 hr for the examination of morphological and biochemical changes. Contralateral lenses cultured in H 2O 2-free media were used as controls. It was found that the lenses had only patchy opacity at the equator after 24 hr, but became hydrated suddenly at 48 hr (31% heavier than the control), with an opacity which involved the entire outer cortical region. By 72 hr incubation, the nucleus was opacified. Lens GSH progressively decreased with time of H 2O 2 exposure, 40% was lost by 24 hr and over 95% by 48 hr. There was a concomitant elevation of PSSG, 16-fold over the controls by 24 hr and 45-fold by 48 hr followed by a decline to 34-fold after 72 hr. In addition, the level of protein-cysteine mixed disulfide (PSSC) was elevated after 48 hr incubation in H 2O 2. At this time point, PSSP aggregates began to appear both in water soluble (WS) and urea soluble (US) fractions along with a drastic reduction in protein solubility. Western blot analysis of the protein fractions identified β and γ, but not α-crystallin in the disulfide-containing aggregates. The lens clarity and biochemical changes partially recovered if the oxidant was removed within 24 hr, indicating a potential therapeutic role for antioxidants. The complete normalization of PSSG level under this recovery condition signifies that cells may have a natural defense system for controlling PSSG elevation.

The possible mechanism of naphthalene cataract in rat and its prevention by an aldose reductase inhibitor (ALØ1576)

The naphthalene-induced cataract in rats has been studied for many years as a possible model of human aging-related cataract. While the molecular mechanism of this cataract is unclear, it has recently been demonstrated that the aldose reductase inhibitor ALØ1576 can prevent lens opacification in this system. The present study was undertaken to investigate the molecular basis for the effects of naphthalene on the lens and the role of pigmentation in the cataractogenic mechanism. Cataracts were induced in five strains of rats (two pigmented, three albino) by oral administration of naphthalene. Initial lens changes were observed after 1 week by slit-lamp; by 3 weeks a distinct shell-like opacity was present in the deep cortex. Little difference in the course of opacification was found between the pigmented and albino strains. Major biochemical effects were a decrease of 20–30% in glutathione (GSH) by 1 week of feeding, disulfide cross-linking of lens proteins present by 3 weeks, and a nearly 20-fold increase in the content of protein-GSH mixed disulfide. No effect was seen in the ability of the affected lenses to accumulate actively [ 3H]choline or 86Rb from the medium in organ culture nor in the activity of the Na + K + -ATPase . ALØ1576 (10 mg kg −1 day −1) completely prevented all morphological and biochemical changes in the lenses of the naphthalene-fed rats in both pigmented and non-pigmented strains. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats. Naphthalene dihydrodiol was found in the aqueous humor and lens of naphthalene-fed rats. It is proposed that naphthalene dihydrodiol produced in the liver reaches the aqueous humor and penetrates the lens where it is further metabolized ultimately to form the toxic species, naphthoquinone.

Oxidative changes in hypoxic rat heart tissue

Reactive O2 species appear to be generated both during hypoxia and at reoxygenation, but it has not been established whether these species interact with heart tissue and cause injury. Oxidative changes were evaluated in isolated rat heart perfused with Krebs-Henseleit medium containing 10 mM glucose and 2.5 mM calcium. After 5-10 min hypoxia, tissue glutathione (GSH) decreased while glutathione disulfide (GSSG), protein carbonyls, and thiobarbituric acid reactive substances (TBARS) increased compared with controls. Similarly, sarcolemmal and sarcoplasmic reticular Ca-ATPase activity (an enzyme susceptible to oxidative inactivation) decreased in response to 10 min hypoxia. These changes were more pronounced after 60 min of hypoxia when protein-GSH mixed disulfides were also increased. There were no further oxidative changes after 4 min reoxygenation when the release of lactate dehydrogenase (LDH) was maximal. Myocardial protein thiol and alpha-tocopherol contents were not significantly changed by either hypoxia or reoxygenation. Mitochondria also exhibited oxidative changes but with more pronounced increases in GSSG and mixed disulfides. There was no change in GSH or GSSG efflux into the coronary effluent during hypoxia, although, in parallel with LDH release, both increased after reoxygenation. Diamide (200 microM), t-butylhydroperoxide (20 microM), or purine (2.3 mM) + xanthine oxidase (0.01 U/ml) were infused for 10 min. Except for large diamide-induced changes in protein thiols and mixed disulfides, the magnitude of the changes produced by these oxidants was similar to those produced by hypoxia. These data show that changes consistent with oxidative processes occur in whole heart and mitochondria in response to hypoxia. The absence of marked signs of oxidation at reoxygenation suggest that enzyme release at this time is unrelated to oxidative stress.

Glutathione metabolism and utilization of external thiols by cigarette smoke-challenged, isolated rat and rabbit lungs

The purpose of the present investigation was to understand the acute effects of cigarette smoke on glutathione (GSH) metabolism and on utilization of external thiols by cigarette smoke-exposed, perfused rat and rabbit lungs. Most of the experiments were carried out using freshly drawn cigarette smoke. However, cigarette smoke condensate was used in some perfusions for the comparison of the effects between the types of exposures on utilization of external thiols. Cigarette smoke decreased GSH levels significantly (50%) without any increase in glutathione disulfide (GSSG) in both rabbit and rat lungs. In smoke-exposed rabbit lungs, protein thiol groups (protein-SH) decreased significantly (17%) without a significant change in protein-GSH mixed disulfides. However, in the rat lungs, cigarette smoke did not decrease protein-SH and protein-GSH mixed disulfides, indicating species variation in the effect of cigarette smoke. Cigarette smoke inhibited selenium-dependent and -independent GSH peroxidase activities in the rat lung (33%), but not in the rabbit lung. GSH S-transferase and GSSG reductase activities were not altered in cigarette smoke-challenged rabbit and rat lungs. γ-Glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase activities were significantly lower in smoke-exposed rat lungs as against control lungs, indicating that rat lung enzymes were more susceptible to the effects of cigarette smoke when compared to those of rabbits. N-Acetylcysteine, but not GSH, added to the perfusate significantly protected rabbit lung from smoke-induced GSH depletion. Smoke condensate added to the perfusate also caused GSH depletion in rabbit lung, and GSH or N-acetylcysteine added to the perfusion medium protected the lung indicating that GSH in the media directly interacts with condensate in the media before coming in contact with cellular GSH. These results indicate that acute smoke inhalation decreases pulmonary GSH and that the decreased GSH was not related to disulfide formation. Inhibited GSH synthesis in rat lung could account for the loss of GSH in part after exposure to cigarette smoke. The alternative pathway of GSH utilization could be conjugation with electrophilic smoke components. Thiols, like N-acetylcysteine, were protective against cigarette smoke-induced damage to the rabbit lung. The mechanism could be either by the increased GSH synthesis or by the direct delivery of sulfhydryls from N-acetylcysteine.

Cleavage of lens protein-GSH mixed disulfide by glutathione reductase

Mixed disulfide complexing the bovine lens erystallins and GSH was prepared by incubating the soluble proteins with GSSG. GSH could be released from the mixed disulfide in the presence of NADPH or NADH. Mixed disulfide was purified using DEAE-Sephadex A-50 column chromatography and was found to be free of glutathione reductase. In such preparation of mixed disulfide NADPH failed to release GSH. However, addition of glutathione reductase along with NADPH released substantial amounts of GSH from the mixed disulfide. Bovine serum albumins also form mixed disulfide with GSH and the mixed disulfide could be cleaved by glutathione reductase. Alkylation of protein sulfhydryl groups by N-ethylmaleimide prior to the addition of GSSG for the preparation of mixed disulfide, almost completely prevents the formation of mixed disulfide indicating that the formation of mixed disulfide involves protein sulfhydryl groups. Evidence is presented that the cleavage of mixed disulfide is not mediated by GSH with cyclic reduction of GSSG by glutathione reductase, but rather proceeds enzymically through glutathione reductase. Using the borohydride method, small amounts of GSH were found to be bound to normal human lens proteins and a substantial amount of GSH was found to be bound to lens proteins from senile cataracts. Only a very small amount of GSH was bound to bovine lens proteins. Glutathione reductase failed to release a significant amount of GSH bound to lens proteins in human cataractous and normal lens. The significantly greater increase in the protein thiols after the borohydride treatment of lens proteins from normal and cataractous human lens and from bovine lens cannot be accounted for by release of GSH bound to proteins. This indicates significant intramolecular and/or intermolecular disulfide bridges in lens proteins.