Glutathione mixed disulfide inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase

Six glutathione-containing inhibitors of the human NADP-linked 15-hydroxyprostaglandin dehydrogenase have been isolated from placental homogenates. Glutathione disulfide is one of these inhibitors. Although the structures of the other five have not been fully elucidated, all are disulfides. Studies with these compounds and with other mixed disulfides have shown that the glutathione mixed disulfides of β-mercaptopyruvate, mercaptoacetate, and β-mercaptolactate are more effective inhibitors of the enzyme than are the glutathione-containing mixed disulfides isolated from placental homogenates. β-Mercaptolactate is particularly noteworthy because of its low K_j (0.13 μM). The results reported here suggest that the activity of the prostaglandin dehydrogenase may be influenced by various glutathione mixed disulfides.     

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.     

Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems

A series of simple methodologies for the determination of the redox status of low molecular weight and protein thiols in biological systems is described. Based centrally upon the use of monobromobimane, we describe a standard in situ derivatisation procedure simultaneously resulting in maximal recovery of both free, reduced low molecular weight and bromobimane accessible protein thiols as their corresponding bimane adducts from intact biological systems. Test systems include isolated and cultured cells, tissue homogenates and body fluids such as blood plasma. Quantitation of the bimane adducts of cysteine and glutathione is achieved by reversed phase high performance liquid chromatography, whereas quantitation of the corresponding adducts of protein thiols is achieved by fluorescence spectroscopy following protein precipitation. Full validation data for quantitative estimates are described. Additionally we have coupled these procedures to prederivatization denaturation treatments of biological protein samples in order to quantitate pools of protein thiols which are inaccessible to bromobimane in samples of native protein. We have also coupled these procedures with prederivatization reductions of biological systems under study with dithiothreitol, rendering simultaneously both oxidized low molecular weight thiols and oxidized protein thiols accessible to derivatisation with monobromobimane. Thus, we have obtained quantitative determinations of cysteine and glutathione present in mixed disulfides with protein and in soluble low molecular weight disulfides and estimates of intraprotein disulfides in a number of test biological systems.     

Irreversible inhibition of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase/9-ketoprostaglandin reductase by glutathione thiosulfonate

Oxidation of glutathione disulfide by a mixture of performic and hydrochloric acids leads to the formation of several compounds that are stronger inhibitors than glutathione disulfide of the placental enzyme that possess both NADP-linked 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The only one of these inhibitors that has been identified is glutathione thiosulfonate. The others are unstable and may include glutathione sulfinyl sulfone and glutathione disulfone. Since the enzyme appears to have a glutathione binding site in close proximity to its active site and glutathione thiosulfonate reacts with free sulfhydryl groups, the effects of this thiosulfonate on the enzyme were examined in more detail. Glutathione thiosulfonate and methyl methanethiosulfonate cause a time-dependent irreversible inhibition of both the hydroxyprostaglandin dehydrogenase and the ketoprostaglandin reductase activities, presumably by reacting with a free sulfhydryl at the prostaglandin binding site. Experiments with PGA1-glutathione show that this sulfhydryl is not necessary for the catalytic activity of the enzyme as long as the substrate can bind at the glutathione site.     

Irreversible inhibition of the human placental NADP-linked 15-hydroxyrpostaglandin dehydrogenase/9-ketoprostaglandin reductase by glutathione thiosulfonate

Oxidation of glutathione disulfide by a mixture of performic and hydrochloric acids leads to the formation of several compounds that are stronger inhibitors than glutathione disulfide of the placental enzyme that posses both NADP-linked 15-hydroxypyrostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The only one of these inhibitors that has been identified is glutathione thiosulfonate. The others are unstble and may include glutathione sulfinyl sulfone and glutathione disulfone. Since the enzyme appears to have a glutathione binding site in close proximity to its active site and glutathione thiosulfonate reacts with free sulfhydryl groups, the effects of this thiosulfonate on the enzyme were examined in more detail. Glutahione thiosulfonate and methyl methanethiosulfonate cause a time-dependent irreversible inhibition of both the hydroxyprostaglandin dehydrogenase and the ketoprostaglandin reductase activities, presumably by reacting with a free sulfhydryl at the prostaglandin binding site. Experiments with PGA-glutathione show that this sulfhydryl is not necessary for the catalytic activity of the enzyme as long as the substrate can bind at the glutahione site.     

Glutathione-related inhibition of prostaglandin metabolism

Placental homogenates contain a heat-stable, dialyzable fraction which specifically inhibits two placental enzymes, both of which possess 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The inhibition of the two enzymes is the same. The inhibitor has been resolved into two components by gel filtration on a column of Sephadex LH-20. The component which eluted first has been identified as oxidized glutathione (GSSG), the other as a glutathione-containing material (GSX). Inhibition of the 15-hydroxyprostaglandin dehydrogenase activity is competitive with respect to the prostaglandin substrate (K_i^GSSG = 26 μM, K_i^GSX = 1.4 μM). Inhibition of the 9-ketoprostaglandin reductase activity is also competitive with respect to the prostaglandin substrate (K_i^GSSG = 68 μM). The most effective inhibitor of the 15-hydroxyprostaglandin dehydrogenase is the prostaglandin A₁-glutathione adduct (K_i = 0.27 μM). This compound is not a substrate for oxidation of the 15-hydroxyl group but it is the best substrate found to date for reduction of the 9-keto function.     

Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue

The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.     

Polycyclic aromatic hydrocarbon quinones and glutathione thioethers as substrates and inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase

The human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase catalyzes oxidoreduction at the 9- and 15-positions of many prostaglandins, but its catalytic efficiency (i.e. kcat/Km) for these reactions is low (Jarabak, J., Luncsford, A., and Berkowitz, D. (1983) Prostaglandins 26, 849-868). In the present study, we demonstrate that both K-region and non-K-region o-quinones of polycyclic aromatic hydrocarbons are excellent substrates for this enzyme. These compounds are reduced with kcat/Km values ranging from 3 to 20 X 10(6) S-1 M-1. The glutathione thioethers of menadione and toluquinone are reduced with similar catalytic efficiencies. Furthermore, these substances and certain other glutathione thioethers are potent inhibitors of prostaglandin B1 oxidation ([I50] = 7 X 10(-8) to 5 X 10(-6) M); while several glutathione thioethers also inhibit polycyclic aromatic hydrocarbon quinone reduction ([I50] = 1.7-6.5 microM). These findings raise the possibility that the potential toxicity of quinones of polycyclic aromatic hyrocarbons and other xenobiotic substances may be altered in the placenta by an oxidoreductase for which prostaglandins are relatively poor substrates. They also suggest that the presence in placental tissue of certain glutathione thioethers could influence the reduction of these quinones and other xenobiotic substances by this enzyme.     

YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1, protects cells against protein sulfenylation

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently described the oxidative-stress-dependent aggregation of proteins in yajL mutants and the oxidative-stress-dependent formation of mixed disulfides between YajL and members of the thiol proteome. We report here that yajL mutants display increased protein sulfenic acids levels and that formation of mixed disulfides between YajL and its protein substrates in vivo is inhibited by the sulfenic acid reactant dimedone, suggesting that YajL preferentially forms disulfides with sulfenylated proteins. YajL (but not YajL(C106A)) also forms mixed disulfides in vitro with the sulfenylated form of bovine serum albumin. The YajL-serum albumin disulfides can be subsequently reduced by glutathione or dihydrolipoic acid. We also show that DJ-1 can form mixed disulfides with sulfenylated E. coli proteins and with sulfenylated serum albumin. These results suggest that YajL and possibly DJ-1 function as covalent chaperones involved in the detection of sulfenylated proteins by forming mixed disulfides with them and that these disulfides are subsequently reduced by low-molecular-weight thiols.     

A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein

The tripeptide glutathione is the most abundant thiol/disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo.     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.     

Effects of the aminonucleoside of puromycin on kidney cortex sulfhydryl and disulfide-containing components and their respective oxidoreductase activities.

Comparative study of glutathione reductase and of mixed disulfide reducing activity assayed with a bovine serum albumin-glutathione substrate in kidney cortex of normal and of aminonucleoside-nephrotic rats reveals significantly higher activities in the latter. Total acid-soluble sulfhydryl and reduced glutathione (GSH) were also found to be significantly higher in kidney cortex of the aminonucleoside-treated rats. Neither total mixed disulfides nor mixed disulfides in which glutathione is covalently bound by disulfide linkage to kidney cortex protein are significantly altered by the nephrosis-producing aminonucleoside.     

Cooperative interaction between ascorbate and glutathione during mitochondrial impairment in mesencephalic cultures

A decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein-glutathione mixed disulfide (Pr-SSG) levels. The present study further examined ascorbate-glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate-induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH-depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr-SSG formation during mitochondrial impairment in normal and GSH-depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate-treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr-SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.     

Determination of thiols and disulfides using high-performance liquid chromatography with electrochemical detection

Low-molecular-mass thiols, such as glutathione (GSH), and their associated disulfides are ubiquitous in nature, and based upon the many known functions of these compounds, their identification and accurate measurement is essential. Our objectives were to develop a simple method for the simultaneous measurement of thiols and disulfides in biological samples using HPLC with dual electrochemical detection (HPLC-DED). Particular emphasis was placed on the applicability to a wide variety of important GSH-related thiols and disulfides, including γ-Glu-Cys, Cys-Gly, their disulfides, and the mixed disulfide of glutathione and cysteine (CSSG), validation on different types of biological samples, maintenance of chromatographic resolution and reproducibility with routine and extended use, and enhancement of assay sensitivity. To this end, optimal HPLC conditions including mobile phase, column, and electrode polishing procedures were established and the method was applied to, and validated on a variety of biological samples. This improved methodology should prove to be a useful tool in studies on the metabolism of GSH and other thiols and disulfides and their role in cellular homeostasis and disease processes.     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.     

NMR structure of oxidized glutaredoxin 3 from Escherichia coli

A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

The redox state of glutathione, cysteine and homocysteine in the extracellular fluid in the skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.