The control of hyperhomocysteinemia through thiol exchange mechanisms by mesna

In hyperhomocysteinemic patients, after reaction with homocysteine-albumin mixed disulfides (HSS-ALB), mesna (MSH) forms the mixed disulfide with Hcy (HSSM) which can be removed by renal clearance, thus reducing the plasma concentration of total homocysteine (tHcy). In order to assess the HSS-ALB dethiolation via thiol exchange reactions, the distribution of redox species of cysteine, cysteinylglycine, homocysteine and glutathione was investigated in the plasma of healthy subjects: (i) in vitro, after addition of 35 μM reduced homocysteine (HSH) to plasma for 72 h, followed by MSH addition (at the concentration range 10–600 μM) for 25 min; (ii) in vivo, after oral treatment with methionine (methionine, 200 mg/kg body weight, observation time 2–6 h). In both experiments the distribution of redox species, but not the total amount of each thiol, was modified by thiol exchange reactions of albumin and cystine, with changes thermodynamically related to the pKa values of thiols in the corresponding mixed disulfides. MSH provoked a dose–response reversal of the redox state of aged plasma, and the thiol action was confirmed by in vivo experiments. Since it was observed that the dimesna production could be detrimental for the in vivo optimization of HSSM formation, we assume that the best plasma tHcy lowering can be obtained at MSH doses producing the minimum dimesna concentration in each individual.     

Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.     

Enzymatic assay for total plasma Cys

Patients with vascular disease and end-stage renal disease have significantly higher concentrations of plasma total Cys (tCys) than do healthy individuals. Described here is a nonradioactive, precise, rapid and sensitive enzymatic colorimetric assay for tCys in plasma samples and is homogeneous in that it avoids separation methods. The tCys assay uses only the recombinant enzymes methionine alpha,gamma-lyase (rMETase) and S-adenosylhomocysteine hydrolase (rSAHH) cloned from Pseudomonas putida and Trichomonas vaginalis, respectively. The rSAHH traps homocysteine and the rMETase thereby produces H(2)S exclusively from Cys. The reaction product, H(2)S, is measured colorimetrically following its reaction with N,N-dibutylphenylenediamine (DBPDA). The procedure takes 3-4 h to complete.     

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.     

Mapping the cysteine proteome: analysis of redox-sensing thiols

The cysteine (Cys) proteome includes 214,000 Cys with thiol and other forms. A relatively small subset functions in cell signaling, while a larger number coordinate cell functions in response to redox state. The former are redox-signaling thiols while the latter are defined as redox-sensing thiols. Bulk measurements are not very informative for systems biology because reactivity of thiols in proteins differs by seven orders of magnitude. Proteomic databases contain annotation of Cys, for example, disulfides and zinc fingers, but do not include quantitative information necessary to develop functional models. Complementary databases and Cys proteome maps are needed to describe thiol redox circuits and connect these to functional redox-dependent pathways. This article summarizes progress in quantitative redox proteomics to develop such maps.     

Homocysteine Level and Mechanisms of Injury in Parkinson's Disease as Related to MTHFR,MTR, and MTHFD1 Genes Polymorphisms and L-Dopa Treatment

An elevated concentration of total homocysteine (tHcy) in plasma and cerebrospinal fluid is considered to be a risk factor for Alzheimer''s disease (AD) and Parkinson''s disease (PD). Homocysteine (Hcy) levels are influenced by folate concentrations and numerous genetic factors through the folate cycle, however, their role in the pathogenesis of PD remains controversial. Hcy exerts a neurotoxic action and may participate in the mechanisms of neurodegeneration, such as excitotoxicity, oxidative stress, calcium accumulation, and apoptosis. Elevated Hcy levels can lead to prooxidative activity, most probably through direct interaction with N-methyl-D-aspartate (NMDA) receptors and sensitization of dopaminergic neurons to age-related dysfunction and death. Several studies have shown that higher concentration of Hcy in PD is related to long-term administration of levodopa (L-dopa). An elevation of plasma tHcy levels can also reflect deficiencies of cofactors in remethylation of Hcy to methionine (Met) (folates and vitamin B12) and in its transsulfuration to cysteine (Cys) (vitamin B6). It is believed that the increase in the concentration of Hcy in PD can affect genetic polymorphisms of the folate metabolic pathway genes, such as MTHFR (C677T, A1298C and G1793A), MTR (A2756G), and MTHFD1 (G1958A), whose frequencies tend to increase in PD patients, as well as the reduced concentration of B vitamins. In PD, increased levels of Hcy may lead to dementia, depression and progression of the disease.     

Localization of thiol and disulfide groups in guinea pig spermatozoa during maturation and capacitation using bimane fluorescent labels

The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups. In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide (NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0. Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation; thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium. However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates of the SS/SH ratio.     

Capillary electrophoresis in the evaluation of aminothiols in body fluids

Thiols play a fundamental role in cell biology, biochemistry and pharmacology. Altered thiol levels in body fluids are linked to specific pathological conditions. Glutathione is the most abundant intracellular low-molecular-mass thiol, playing an essential role in protecting cells from toxic species; other relevant thiol-containing compounds are homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly). Plasma aminothiols can be bound to proteins but they also occur free in the disulfide (symmetrical and mixed) and in the reduced forms. The simultaneous determination of these aminothiols, their precursor and metabolites is a useful tool in studying oxidative stress, metabolic and redox regulation. Many capillary electrophoresis methods have been proposed for this purpose, the aim of the present review is to support researchers in the choice of suitable methods for the determination of thiols in body fluids evaluating the different approaches and technologies proposed from the literature.     

Determination and characterization of cysteine, glutathione and phytochelatins (PC₂₋₆) in Lolium perenne L. exposed to Cd stress under ambient and elevated carbon dioxide using HPLC with fluorescence detection

Metal-binding thiols, involved in detoxification mechanisms in plant and other organism under heavy metal stress, are receiving more and more attentions, and various methods have been developed to determine related thiols such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs). In present study, an HPLC method was established for simultaneous determination of Cys GSH and PC(2-6) after treatment with disulfide reductant of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and thiolyte reagent of monobromobimane (mBBr). The separation of thiol derivatives was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 30 mm, 1.8 μm) with a linear gradient elution of 0.1% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 0.8 mL min(-1). The temperature of the column was maintained at 25°C. The excitation and emission wavelengths were set at 380 and 470 nm, respectively. The thiol derivatives were well separated in 19 min, and the total analysis time was 30 min. The established method was proved selective, specific and reproducible, and could be applicable to determine Cys, GSH and PC(2-6) and to evaluate their roles in detoxification mechanisms in Cd-treated Lolium perenne L. under ambient and elevated carbon dioxide (CO(2)). It was found that the total SH contents and proportions of thiols in roots and shoots were dependent on Cd concentration, whereas the total SH contents decreased and the proportions of thiols altered without significance at elevated CO(2) level.     

Thiol‐disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free —SH groups. This allowed the oriented conjugation of alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.     

Opposite effect of methionine-supplemented diet, a model of hyperhomocysteinemia, on plasma and liver antioxidant status in normotensive and spontaneously hypertensive rats

Hyperhomocysteinemia is often associated with an increase in blood pressure. However our previous study has shown that methionine supplementation induced an increase in blood pressure in Wistar-Kyoto (WKY) rats and a decrease in blood pressure in spontaneously hypertensive rats (SHR) with significant differences in plasma homocysteine (Hcy) metabolites levels. Previously liver antioxidant status has been shown to be decreased in SHR compared to WKY rats. It has been suggested that oxidative stress may predispose to a decrease in NO bioavailability and induce the flux of Hcy through the liver transsulfuration pathway. Thus the aim of this study was 1) to investigate the effect of methionine supplementation on NO-derived metabolites in plasma and urine 2) to investigate whether abnormalities in Hcy metabolism may be responsible for the discrepancies observed between WKY rats and SHR concerning blood pressure and 3) to investigate whether a methionine-enriched diet, differently modified plasma and liver antioxidant status in WKY rats an SHR. We conclude that the increase in blood pressure in WKY rats is related to high plasma cysteine levels and is not due to a decrease in NO bioavailability and that the decrease in blood pressure in SHR is associated with high plasma GSH levels after methionine supplementation. So GSH synthesis appears to be stimulated by liver oxidative stress and GSH is redistributed into blood in SHR. So the great GSH synthesis can be rationalized as an autocorrective response that leads to a decreased blood pressure in SHR.     

Protein thiol modifications visualized in vivo

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H₂O₂-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H₂O₂ and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated.     

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. Am J Physiol Endocrinol Metab 301: E000-E000, 2011. First published September 20, 2011; 10.1152/ajpendo.00345.2011.-We carried out a (1)H-NMR metabolomic analysis of sera from vitamin B(12)-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 μM in control rats to 402 μM in vitamin B(12)-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B(12)- and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B(6)-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine β-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways.     

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei—the causative agent of Human African Trypanosomiasis—by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)₂), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)₂ redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC–MS/MS analysis was established to quantify intra-cellular CYN, T(SH)₂, GSH, as well as GS-CYN and T(S-CYN)₂ adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)₂ pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)₂ redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)₂ redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.     

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO(-)) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO(-). The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO(-) showed that ICAT labeling of Cys(118) was decreased by 47%, whereas Cys(80) was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys(118) by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras.     

Determination of the oxido-redox status of plasma albumin in hemodialysis patients

The oxido-redox status of plasma albumin in patients treated with hemodialysis was characterized with LC-ESI-MS/MS and was compared with models of oxidative stress. Oxidised albumin was characterized by sulfonation (SO3-) of the SH at Cys 34, unfolding and acidification of the molecule. Albumin in hemodialysis patients presented, instead, only intermediate oxidation products such as sulfenic (SO2), sulfonic (SO)and methionine sulfoxide (C5H9NO2S) involving Cys 165-269 and Met 329-548 but did not present SO3- at Cys 34. Absence of charge and structural alterations compared to the oxidised templates was also confirmed with electrophoretic titration and calorimetry. In conclusion, the oxido-redox status of plasma albumin in hemodialysis patients lacks the hallmarks of the advanced oxidation products. LC-ESI-MS/MS was crucial to characterize albumin in conditions of oxidation stress; surrogate techniques can mirror conformational changes induced by oxidation.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E_{GSSG/2 GSH}) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E_{CySS/2 Cys}). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (E_{thiol–disulphide}), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E_{CySS/2 Cys} to E_{thiol–disulphide} in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Molecular Mechanisms of Glutaredoxin Enzymes: Versatile Hubs for Thiol–Disulfide Exchange between Protein Thiols and Glutathione

The tripeptide glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) constitute a key redox couple in cells. In particular, they partner protein thiols in reversible thiol–disulfide exchange reactions that act as switches in cell signaling and redox homeostasis. Disruption of these processes may impair cellular redox signal transduction and induce redox misbalances that are linked directly to aging processes and to a range of pathological conditions including cancer, cardiovascular diseases and neurological disorders. Glutaredoxins are a class of GSH-dependent oxidoreductase enzymes that specifically catalyze reversible thiol–disulfide exchange reactions between protein thiols and the abundant thiol pool GSSG/GSH. They protect protein thiols from irreversible oxidation, regulate their activities under a variety of cellular conditions and are key players in cell signaling and redox homeostasis. On the other hand, they may also function as metal-binding proteins with a possible role in the cellular homeostasis and metabolism of essential metals copper and iron. However, the molecular basis and underlying mechanisms of glutaredoxin action remain elusive in many situations. This review focuses specifically on these aspects in the context of recent developments that illuminate some of these uncertainties.     

Thioredoxin and related proteins in procaryotes

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.