Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress

Changes in the concentrations of protein-mixed disulfides (XS-SP) of glutathione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in human platelets treated with diamide and t-BOOH in timecourse experiments (time range, 1-30 min) in order to understand the contribution of minor thiols CSH and CGSH to the regulation of glutathione-protein mixed disulfides (GS-SP). Diamide was much more potent than t-BOOH in altering the platelet thiol composition of XS-SP (threshold dose: diamide, 0.03 mM; t-BOOH, 0.5 mM) and caused reversible XS-SP peaks whose magnitude was related to the concentration of free thiols in untreated cells. Thus maximum levels of GS-SP (8 min after 0.4 mM diamide) were about 16-fold higher than those of controls (untreated platelets, GS-SP = 0.374 nmol/10(9) platelets), whereas those of CS-SP and CGS-SP were only 4-fold increased (untreated platelets, CS-SP = 0.112 nmol/10(9) platelets; CGS-SP = 0.024 nmol/10(9) platelets). The greater effects of diamide with respect to t-BOOH were explained on the basis of the activities of fast reactive protein SH groups for diamide and glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t-BOOH. The addition of cysteine (0.3 mM, at 4 min) after treatment of platelets with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to normal values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased gamma-glutamyltranspeptidase activity was found in platelets treated with diamide. Moreover, untreated platelets were found to release and hydrolyze GSH to CGSH and CSH. Ratios of thiols/disulfides (XSH/XSSX) and activities of GR and G-6PDH were also related to a high reducing potential exerted by GSH but not by minor thiols. The lower mass and charge of minor thiols is a likely requisite of the regulation of GS-SP levels in platelets.     

A comparison of protein S-thiolation (protein mixed-disulfide formation) in heart cells treated with t-butyl hydroperoxide or diamide

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide, and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30-45 min. Cells stopped beating 25 min after the addition of t-butyl hydroperoxide, and beating did not resume. t-Butyl hydroperoxide caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42 and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment. S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by t-butyl hydroperoxide or diamide, and that the specificity of the response depends on the agent used.     

Formation of mixed disulfide of cystatin-beta in cultured macrophages treated with various oxidants

Macrophage cell cultures were treated with menadione, zymosan, or phorbol myristate acetate (PMA), and changes in productions of superoxide anion and hydroperoxide, and in glutathione oxidation and S-thiolation of cystatin-beta (formation of a mixed disulfide of cystatin-beta and glutathione) were examined. All three compounds promoted production of superoxide anion and hydroperoxide, but only menadione caused extensive oxidation of glutathione. Menadione caused S-thiolation of cystatin-beta in a dose-dependent fashion, but the other two compounds did not. Removal of menadione promptly reduced the oxidation of glutathione and S-thiolation of cystatin-beta induced by menadione. Inhibition of catalase by aminotriazol caused slight increase in the GSSG content in both menadione- and zymosan-treated cells, but not in S-thiolation of cystatin-beta in zymosan-treated cells. None of the three compounds influenced appreciably the activity of glutathione peroxidase, glutathione reductase, or superoxide dismutase in cultured cells. These results indicate that S-thiolation of cystatin-beta occurs in cells in response to oxidative challenge by menadione but not by zymosan or by the tumor promoter PMA. Dethiolation of cystatin-beta by purified thiol transferase and protein disulfide isomerase in the presence of different concentrations of GSH was examined in vitro. Both enzymes catalyzed dethiolation of cystatin-beta at a much lower level of GSH than that required for the non-enzymatic reaction, suggesting the importance of enzymatic catalysis of S-thiolation and dethiolation of cystatin-beta in cells.     

Hemoglobin S-thiolation during peroxide-induced oxidative stress in chicken blood

Protein S-thiolation or protein-glutathione mixed disulfide (PSSG) occurs when cells are exposed to oxidative stress, and has been implicated in several cellular functions. The S-thiolation of hemoglobin as well as other abundant proteins is proposed to participate as a redox buffer, being part of the antioxidant protection system of the cell during the oxidative challenge. We studied the oxidative stress caused by peroxides (H(2)O(2), cumene and tert-butyl hydroperoxide) on chicken blood by measuring the thiol/disulfide status. Chicken blood under peroxide treatment showed a time- and concentration-dependent increase in glutathione disulfide (GSSG) and PSSG. GSSG peaked immediately after treatment (1 min), while PSSG increased progressively over time, showing a maximum after about 30 min. The system recovered after 140 min of incubation, with GSSG and PSSG then barely reaching control values. The S-thiolation of hemoglobin was monitored under nondenaturing PAGE, and the fraction of S-thiolated hemoglobin, or Hb A1, rose in a dose-dependent fashion and was proportional to total S-thiolation, measured as PSSG. This significant correlation indicates that hemoglobin is the major S-thiolated protein in chicken erythrocytes treated with peroxides. The present work shows the behavior of chicken blood under peroxide treatment; it anticipated that chicken hemoglobin thiol groups can actively participate in the redox processes of erythrocytes exposed to oxidative stress, and that hemoglobin is the major S-thiolated protein. This further corroborates the hypothesis that abundant proteins, such as hemoglobin, may take part in the cellular antioxidant defense system.     

Glutathione system adaptation to acute stress in the heart of rats during different regimes of hypoxia training

The intensity of lipid peroxidation (LPO), reduced and oxidized glutathione (GSH and GSSG) contents, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase (G-6-PDH), and NADP-isocitrate dehydrogenase (NADP-IDH) activities were studied in the heart of male rats exposed to two modes of intermittent hypoxic training (IHT): I-breathing in normobaric chamber with 7% O2 gas mixture for 5 min with 15 min normoxic intervals 4 times daily during 3 weeks; II-breathing by 12% O2 gas mixture in the same manner). After adaptation to hypoxia, the rats were subjected to 6h-immobilization stress. It has been shown that stress action after IHT (regime I) caused the increase in LPO and the shift of GSH/GSSG to disulfides. A disbalance in antioxidative defense system was determined by the decrease in glutatione peroxidase, G-6-PDH activities, and GSH content. The support of glutathione reductase activity under stress in this group with simultaneous decrease of enzyme activity in the pentose phosphate pathway was realized through the participation of NADP-IDH. Hypoxic training in regime II induced LPO decrease in the heart tissue after stress. The increase in the heart GSH content, optimal balance of glutathione-related enzymes in this group evidences for the dependence of adaptation effects on the vigor of hypoxic exposition. Our results suggest the active participation of glutathione system in the formation of adaptation reactions under the extreme factor influences through the action on intracellular red/ox potential as well as effectiveness of antioxidant defense.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status

Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.     

Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamide

In the present study, the effect of thiol redox and its possible role in the inhibitory effect of nicotinamide on renal brush-border membrane (BBM) phosphate uptake was examined. Addition of thiol reducing agent, dithiothreitol (DTT, 5 mM), caused an increase, while addition of thiol oxidant, diamide (DM, 5 mM) caused a reversible decrease in sodium-dependent BBM phosphate uptake. Kinetic analyses revealed an increase in both Vmax and Km by DTT, and a decrease in Vmax by DM. These results suggest that thiol redox influences BBM phosphate uptake with sulfhydryl (SH) groups relate to its capacity and disulfide (SS) groups to its affinity for phosphate. Since changes in cytosolic NAD levels may affect BBM thiol redox through changes in redox states of NADP and glutathione systems, we have examined such possibility by studying the effect of nicotinamide (NM). Incubation of proximal tubules with NM (10 mM) induced an oxidative effect on redox states of cytosolic NAD, NADP systems as inferred from decreased cellular lactate/pyruvate, malate/pyruvate, respectively. Measurements of cytosolic glutathiones and BBM thiols also revealed that NM pretreatment shifted the cytosolic glutathione redox (GSH/GSSG) and BBM thiol redox (SH/SS) toward more oxidized state. On the other hand, incubation of proximal tubules with NM suppressed phosphate uptake by the subsequently isolated BBM vesicles. The lower phosphate uptake by NM-pretreated BBM vesicles was reversed by DTT and was resistant to the inhibitory effect of DM. These results thus suggest that BBM thiol oxidation may be involved in the inhibitory effect of NM on BBM phosphate uptake.     

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.     

Rat lung antioxidant enzyme activities and their specific proteins during hyperoxia

The hyperoxia-induced increases in the activity of lung glucose-6-phosphate dehydrogenase (G-6-P) and glutathione reductase (GR) after exposure of rats to greater than 97% O2 for 6 days were accompanied by equivalent increases in the amount of the respective immunoreactive proteins. Hyperoxia also increased lung glutathione (GSH) + oxidized glutathione (GSSG) content and the magnitude of this hyperoxic response of increased GSH + GSSG, G-6-P, and GR (maximal 1.3- to 1.8-fold) declined as a function of age during the first 3 wk of life. Fetal rat lung explants cultured 4 days in 95% O2 showed increased G-6-P and GR activity and increased levels of the specific proteins 1.5-fold those of explants at 2 days of culture. We conclude that the hyperoxic response of increased rat lung G-6-P and GR activity in vivo and in vitro involves not just alteration of enzyme activity but also specific increases in the proteins catalyzing the reactions.     

Diamide induced shift in protein and glutathione thiol: disulfide status delays DNA rejoining after X-irradiation of human cancer cells

By treating a human tumor cell line with various concentrations of diamide, we explored the relationship between extent and duration of protein and nonprotein thiol oxidation, initiation of DNA double-strand break rejoining after X-rays, and the degree of radiosensitization. We also examined the relationship between protein thiol status and the non-protein thiol, glutathione (GSH). A549 cells were irradiated and incubated postirradiation with 0, 100, 300 or 500 microM diamide for 1 h. The dose of radiation required to give 10% survival decreased from 4.8 Gy to 3.2 Gy with 300 microM and to 2.7 Gy with 500 microM diamide (enhancement ratios of 1.5 and 1.8, respectively) but was not significantly affected by 100 microM diamide. The time of initiation of double-stranded DNA rejoining after X-irradiation (DNA repair) was delayed by 300 and 500 microM diamide. Furthermore, DNA rejoining began only after total cellular protein thiol content recovered to 55% of pretreatment levels for both concentrations. Intracellular GSH/GSSG ratios decreased immediately after diamide addition to less than 1. Large decreases in GSH/GSSG ratio preceded significant loss of protein thiols, but protein-glutathione mixed disulfides accounted for a minor percentage of the total protein thiol oxidized (up to 20%). We believe that diamide-induced protein thiol loss, and not GSH oxidation, is related to the cessation of DNA strand rejoining after X-irradiation, thereby affecting survival.     

Short-term exercise preserves myocardial glutathione and decreases arrhythmias after thiol oxidation and ischemia in isolated rat hearts

The purpose of this study was to determine if exercise (Ex) protects hearts from arrhythmias induced by glutathione oxidation or ischemia-reperfusion (I/R). Female Sprague-Dawley rats were divided into two experimental groups: sedentary controls (Sed) or short-term Ex (10 days of treadmill running). Twenty-four hours after the last session, hearts were excised and exposed to either perfusion with the thiol oxidant diamide (200 μM) or global I/R. Ex significantly delayed the time to the onset of ventricular arrhythmia after irreversible diamide perfusion. During a shorter diamide perfusion protocol with washout, Ex significantly decreased the incidence of arrhythmia, as evidenced by a delayed time to the first observed arrhythmia, lower arrhythmia scores, and lower incidence of ventricular fibrillation. Ex hearts exposed to I/R (30-min ischemia/30-min reperfusion) also showed lower arrhythmia scores and incidence of ventricular fibrillation compared with Sed counterparts. Our finding that Ex protected intact hearts from thiol oxidation was corroborated in isolated ventricular myocytes. In myocytes from Ex animals, both the increase in H(2)O(2) fluorescence and incidence of cell death were delayed after diamide. Although there were no baseline differences in reduced-to-oxidized glutathione ratios (GSH/GSSG) between the Sed and Ex groups, GSH/GSSG was better preserved in Ex groups after diamide perfusion and I/R. Myocardial glutathione reductase activity was significantly enhanced after Ex, and this was preserved in the Ex group after diamide perfusion. Our results show that Ex protects the heart from arrhythmias after two different oxidative stressors and support the hypothesis that sustaining the GSH/GSSG pool stabilizes cardiac electrical function during conditions of oxidative stress.     

S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography

S-Glutathionylation is emerging as a novel regulatory and adoptive mechanism by which glutathione (GSH or GSSG) conjugation can modify functionally important reactive cysteines in redox-sensitive proteins. The dynamics of generation and reversal of this modification in cells is poorly understood. This study describes the ability and applicability of GSH- and GSSG-affinity matrices to quantitatively bind proteins which harbor reactive cysteines and undergo glutathionylation. We showed that purified proteins, known to be modified by S-thiolation, bind to these matrices, are selectively eluted by dithiothreitol and rapidly incorporate biotin-labeled GSH or GSSG in vitro. Chromatography of extracts from tumor cells that had been treated with oxidants (diamide, H(2)O(2), tert-butyl hydroperoxide) on GSH-Sepharose showed the specific binding of many proteins, whose levels increased transiently (2- to 6-fold) soon after treatments. However, when these cells were post-incubated in drug/oxidant-free media, protein binding decreased gradually to control levels over 3-12h, thereby demonstrating the central role of cysteine redox status in the binding. Immunoblotting of eluates from GSH-Sepharose showed the presence of known (actin, ubiquitin-activating enzyme E1, NF-kappaB, and proteasome) and putative (p53, glutathione-S-transferase P1) targets for glutathionation. After oxidant withdrawal, many of these proteins displayed unique kinetics in their loss of binding to GSH-matrix, reflecting their differential abilities to recover from cysteine redox changes in cellular milieu. Further, we correlated the kinetics of S-thiolation susceptibility of the proteasome and ubiquitin-E1 proteins with altered levels of protein ubiquitination in H(2)O(2)-treated cells. Our study reveals the hitherto underutilized ability of glutathione matrices for analyzing the kinetics of cysteine redox in cellular proteins and allows easy identification of S-thiolatable proteins.     

S-cysteinylation is a general mechanism for thiol protection of Bacillus subtilis proteins after oxidative stress

S-Thiolation is crucial for protection and regulation of thiol-containing proteins during oxidative stress and is frequently achieved by the formation of mixed disulfides with glutathione. However, many Gram-positive bacteria including Bacillus subtilis lack the low molecular weight (LMW) thiol glutathione. Here we provide evidence that S-thiolation by the LMW thiol cysteine represents a general mechanism in B. subtilis. In vivo labeling of proteins with [(35)S]cysteine and nonreducing two-dimensional PAGE analyses revealed that a large subset of proteins previously identified as having redox-sensitive thiols are modified by cysteine in response to treatment with the thiol-specific oxidant diamide. By means of multidimensional shotgun proteomics, the sites of S-cysteinylation for six proteins could be identified, three of which are known to be S-glutathionylated in other organisms.     

Delta 5-3 beta-hydroxysteroid dehydrogenase, succinate dehydrogenase and glucose-6-phosphate dehydrogenase in the bovine corpus luteum of estrous cycle: a quantitative histochemical study

Genital organs and blood were obtained from dairy cows at a local abattoir. 3 recently ovulated follicles and 20 corpora lutea of estrous cycle (CLC) were used for the quantitative enzyme histochemical demonstration of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-OHSDH), succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activity, employing a computerized microscope photometer. Progesterone was determined in blood serum by radioimmunoassay. Luteal tissue was grouped into several stages of development according to micromorphological criteria. Activities per volume unit of 3 beta-OHSDH and SDH in large luteal cells (LLC), as well as in small luteal cells (SLC), and luteal tissue (LT), relative amounts of the 3 beta-OHSDH-positive tissue fraction (PLCC), and progesterone concentrations in blood serum exhibited a significant pattern corresponding to the morphological development of the endocrine gland. G-6-PDH showed an increase in activity per volume unit during tissue development lasting until the beginning of regressive changes, and as significant in LLC and LT. Activities per volume unit of 3 beta-OHSDH (p less than or equal to 0.001) and SDH (p less than or equal to 0.01) were higher in LLC than in SLC, indicating superior steroidogenic capacities, while G-6-PDH activity was distinctly higher in the latter (p less than or equal to 0.001). Almost all parameters tested were correlated positively. 3 beta-OHSDH and SDH exhibited a significantly positive correlation in LLC (p less than or equal to 0.01) and LT (p less than or equal to 0.001) during periods of measureable progesterone secretion. In SLC this correlation was nonsignificant (p greater than 0.05). G-6-PDH showed a relative poor correlation to 3 beta-OHSDH (LLC, p less than or equal to 0.05; LT, p less than or equal to 0.01) and SDH (LT, p less than or equal to 0.05). Enzyme activities in LLC as well as in SLC were generally positively correlated (p less than or equal to 0.001). All enzymes tested exhibited a significantly positive correlation with progesterone concentrations in blood serum. This was significant for SDH only during measurable progesterone secretion, and less marked for G-6-PDH.     

Actin S-glutathionylation: evidence against a thiol-disulphide exchange mechanism

Many proteins, including actin, are targets for S-glutathionylation, the reversible formation of mixed disulphides between protein cysteinyl thiol groups and glutathione (GSH) that can be induced in cells by oxidative stress. Proposed mechanisms of protein S-glutathionylation follow mainly two distinct pathways. One route involves the initial oxidative modification of a reduced protein thiol to an activated protein, which may then react with GSH to the mixed disulphide. The second route involves the oxidative modification of GSH to an activated form such as glutathione disulphide (GSSG), which may then react with a reduced protein thiol, yielding the corresponding protein mixed disulphide. We show here that physiological levels of GSSG induce a little extent of actin S-glutathionylation. Instead, actin with the exposed cysteine thiol activated by diamide or 5,5'-dithiobis(2-nitrobenzoic acid) reacts with physiological levels of GSH, incorporating about 0.7 mol GSH/mol protein. Differently, an extremely high concentration of GSSG induces an increased level of S-glutathionylation that causes a 50% inhibition in actin polymerization not reversed by dithiotreitol. In mammalian cells, GSH is present in millimolar concentrations and is in about 100-fold excess over GSSG. The high concentration of GSSG required for obtaining a significant actin S-glutathionylation as well as attendant irreversible changes in protein functions make unlikely that actin may be S-glutathionylated by a thiol-disulphide exchange mechanism within the cell.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

The use of the oxidant"diamide" for studying the non-mitochondrial reducing capacity of Ehrlich ascites tumor cells

Diazenedicarboxylic acid bis(N,N-dimethylamide), (“diamide”) lowered non-mitochondrial NAD(P)H stores in Ehrlich ascites tumor cells in vitro by indirect reactions involving oxidation of glutathione and reduction of GSSG via glutathione reductase. The concentrations of diamide used did not alter the mitochondrial capacity to reduce NAD(P)H under anaerobic conditions. “Endogenous substrates” could be removed by multiple additions of diamide which indirectly inhibited NAD(P)H and GSH regeneration because of a lack of cellular reducing capacity. The regenerative power of the cells was restored by the addition of glucose. We conclude that diamide may prove to be a useful agent for studying the reducing capacity as well as the redox compartmentalization of cells in vitro.