Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides

 Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is ɛ₅₂₀=34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells. Accepted: 17 December 1996     

Reduction (dethiolation) of protein mixed-disulfides; distribution and specificity of dethiolating enzymes and N,N'-bis(2-chlorethyl)-N-nitrosourea inhibition of an NADPH-dependent cardiac dethiolase

The S-thiolated proteins phosphorylase b (Phb) and carbonic anhydrase III (CAIII) were prepared with [3H]glutathione in a reaction initiated with diamide. These substrates were used to measure the rate of reduction (dethiolation) of protein mixed-disulfides by enzymes with properties similar to those of thioredoxin and glutaredoxin. This enzyme activity is termed a dethiolase since the identities of the enzymes are still unknown. The dethiolation of either S-[3H]glutathiolated Phb or S-[3H]glutathiolated CAIII was employed in tissue assays and for study of two partially purified dethiolases from cardiac tissue. NADPH-dependent dethiolase activity was most abundant except in rat liver and muscle. Total dethiolase activity was approximately 10-fold higher in neutrophils, 3T3-L1 cells, and Escherichia coli than in other sources. Rat skeletal muscle had 3- to 4-fold higher dethiolase activity than rat heart or liver. These data indicate that protein dethiolase activity is ubiquitous and that normal expression of the two dethiolase activities varies considerably. A partially purified cardiac NADPH-dependent dethiolase acted on Phb approximately 1.5 times faster than CAIII, and a glutathione (GSH)-dependent dethiolase acted on Phb 3 times faster than CAIII. The Km for glutathione for the GSH-dependent dethiolase was 15 microM with Phb as substrate and 10 microM with CAIII. Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 mM). Partially purified cardiac NADPH-dependent dethiolase was inactivated by BCNU (N,N'-bis(2-chloroethyl)-N-nitrosourea) and the GSH-dependent dethiolase was unaffected under similar conditions. In a soluble extract from bovine heart, 200 microM BCNU inhibited NADPH-dependent dethiolase by more than 60% but did not affect GSH-dependent activity. These results demonstrate that BCNU is a selective inhibitor of the NADPH-dependent dethiolase.     

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.     

Picomole-level mapping of protein disulfides by mass spectrometry following partial reduction and alkylation

We have deduced the disulfide bond linkage patterns, at very low protein levels (<0.5 nmol), in two cysteine-rich polypeptide domains using a new strategy involving partial reduction/alkylation of the protein, followed by peptide mapping and tanden mass spectrometry (MS/MS) sequencing on a nanoflow liquid chromatography-MS/MS system. The substrates for our work were the cysteine-rich ectodomain of human Fn14, a member of the tumor necrosis factor receptor family, and the IgV domain of murine TIM-1 (T-cell, Ig domain, and mucin domain-1). We have successfully determined the disulfide linkages for Fn14 and independently confirmed those of the IgV domain of TIM-1, whose crystal structure was published recently. The procedures that we describe here can be used to determine the disulfide structures for proteins with complex characteristics. They will also provide a means to obtain important information for structure-function studies and to ensure correct protein folding and batch-to-batch consistency in commercially produced recombinant proteins.     

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.     

Molecular role of GATA binding protein 4 (GATA-4) in hyperglycemia-induced reduction of cardiac contractility

Diabetes mellitus has emerged as one of the main alarms to human health in the 21st century. Pronounced changes in the human environment, behavior and lifestyle have accompanied globalization, which resulted in escalating rates of both obesity and diabetes, already described as diabesity. This pandemic causes deterioration of life quality with high socio-economic costs, particularly due to premature morbidity and mortality. To avoid late complications of type 2 diabetes and related costs, primary prevention and early treatment are therefore necessary. In this context, effective non-pharmacological measures, such as regular physical activity, are imperative to avoid complications, as well as polymedication, which is associated with serious side-effects and drug-to-drug interactions. Our previous work showed, in an animal model of obese type 2 diabetes, the Zucker Diabetic Fatty (ZDF) rat, that regular and moderate intensity physical exercise (training) is able, per se, to attenuate insulin resistance and control glycaemia, dyslipidaemia and blood pressure, thus reducing cardiovascular risk, by interfering with the pathophysiological mechanisms at different levels, including oxidative stress and low-grade inflammation, which are key features of diabesity. This paper briefly reviews the wide pathophysiological pathways associated with Type 2 diabetes and then discusses in detail the benefits of training therapy on glycaemic control and on cardiovascular risk profile in Type 2 diabetes, focusing particularly on antioxidant and anti-inflammatory properties. Based on the current knowledge, including our own findings using an animal model, it is concluded that regular and moderate intensity physical exercise (training), due to its pleiotropic effects, could replace, or at least reduce, the use of anti-diabetic drugs, as well as of other drugs given for the control of cardiovascular risk factors in obese type 2 diabetic patients, working as a physiological "polypill".     

Radiolytic reduction of protein and nonprotein disulfides in the presence of formate: a chain reaction

We have reported recently that the disulfide groups in bovine serum albumin can be reduced by a radiolytic chain reaction which occurs in deoxygenated solutions containing formate ions. This reaction, which involves the reduction of disulfide groups by hydrated electrons and carbon dioxide radical anions, has now been studied in greater detail and compared with an analogous reaction in small, disulfide containing molecules over a range of pH values and substrate concentrations. A two-step reaction is proposed to account for the reduction of disulfides in reactions which can have chain lengths of 20 or more. Thiols produced by the disulfide reduction are stable to the conditions of the reaction. For example, a biological assay showed that the integrity of glutathione was maintained even at radiation doses much larger than those required to achieve complete reduction of glutathione disulfide. It was found that the extent of disulfide reduction could easily be controlled by varying the radiation dose delivered to the solutions. Radiolytic reduction is a very useful way of reducing protein and low molecular weight disulfides without the use of excess quantities of reagents such as dithiothreitol. In many cases, the reaction solutions could be used directly for subsequent reactions and this may be of considerable value in modifying the structure of hormones, enzymes, membrane receptors, and other disulfide containing proteins. If ammonium formate is used, freeze drying is an effective way to remove the formate salt, should this be required.     

Thioredoxin-1 and protein disulfide isomerase catalyze the reduction of similar disulfides in HIV gp120

HIV-1 enters cells via interaction of the viral glycoprotein gp120, the host cell surface receptor CD4 and the co-receptors CCR5 or CXCR4. For entry, gp120 undergoes conformational changes that depend on the reduction of one or more disulfides. Previous studies indicate that protein disulfide isomerase (PDI), thioredoxin-1 (Trx1), and glutaredoxin-1 (Grx1) catalyze gp120 reduction, but their specific disulfide targets are not known. Here, it was demonstrated that PDI and Trx1 have similar gp120 disulfide targets as determined by labeling after reduction, but with some pattern differences, including overall stronger labeling with Trx1 than with PDI. Furthermore, uneven labeling of the residues of a disulfide may reflect altered accessibility by conformational changes upon the reduction process. Since both PDI and Trx1 may be involved in viral entry, compounds that target the host redox system or the viral gp120 were tested in vitro to investigate whether redox regulation is a target for anti-HIV therapy. Carbohydrate binding agents (CBAs), previously shown to bind gp120 and inhibit HIV entry, were now demonstrated to inhibit gp120 disulfide reduction. Auranofin, an inhibitor of thioredoxin reductase 1 (TrxR1), also showed inhibitory activity towards HIV infection, although close to its cytotoxic concentration. Our results demonstrate that both the host redox system and the viral surface glycoproteins are of interest for the development of new generations of anti-HIV therapeutics.     

Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe)

The effect of U(34) dethiolation on the anticodon-anticodon association between E. coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique. An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C. The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5. Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.     

Simultaneous reduction and alkylation of protein disulfides in a centrifugal ultrafiltration device prior to two-dimensional gel electrophoresis

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.1-0.2 mS/cm range in preparation for IEF. The detergent 3-(4-heptyl)phenyl 3-hydroxypropyl dimethylammonio propanesulfonate (C7BzO), which favors the solubilization of proteins, but which interferes with SDS equilibration and second dimension PAGE, was effectively removed by ultrafiltration and exchanged with CHAPS without measurable loss of protein. Disparate protein patterns of Rhodopseudomonas palustris lysates were revealed by two-dimensional gel electrophoresis depending on which reagent was used to induce cell lysis.     

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins.

1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitogen-activated protein kinase (MAPK) in cardiac tissues

Mitogen-activated protein kinase (MAPK) has recently emerged as a prominent role player in intracellular signalling in the ventricular myocyte with attention being focussed on its possible role in the development of ventricular hypertrophy. It is becoming clear that MAPK is also active in other cells of cardiac origin such as cardiac fibroblasts and possible functions of this signalling pathway in the heart have yet to be explored. In this report the mammalian MAPK pathway is briefly outlined, before reviewing current knowledge of the MAPK pathway in cardiac tissue (ventricular myocytes, vascular smooth muscle cells and cardiac fibroblasts). New data is also presented on the presence and activity of MAPK in two additional cardiac celltypes namely atrial myocytes and vascular endothelial cells from the coronary microcirculation. (Mol Cell Biochem 157: 49–57, 1996)     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.     

(Non)Parallel developmental mechanisms in vertebrate appendage reduction and loss

Appendages have been reduced or lost hundreds of times during vertebrate evolution. This phenotypic convergence may be underlain by shared or different molecular mechanisms in distantly related vertebrate clades. To investigate, we reviewed the developmental and evolutionary literature of appendage reduction and loss in more than a dozen vertebrate genera from fish to mammals. We found that appendage reduction and loss was nearly always driven by modified gene expression as opposed to changes in coding sequences. Moreover, expression of the same genes was repeatedly modified across vertebrate taxa. However, the specific mechanisms by which expression was modified were rarely shared. The multiple routes to appendage reduction and loss suggest that adaptive loss of function phenotypes might arise routinely through changes in expression of key developmental genes.     

Inhibition of papain by S-nitrosothiols. Formation of mixed disulfides

S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys(25)). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S-NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S-nitrosothiols as the NO donor. Five S-nitroso compounds, S-nitroso-N-acetyl-dl-penicillamine, S-nitrosoglutathione, S-nitrosocaptopril, glucose-S-nitroso-N-acetyl-dl-penicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time- and concentration-dependent manner with second-order rate constants (k(i)/K(I)) ranging from 8.9 to 17.2 m(-1) s(-1). The inhibition of papain by S-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF(4). Incubation of the enzyme with a fluorescent S-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain by S-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys(25)) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain.     

Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiotheritol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid, 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be indepedent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.     

Protein disulfides and protein disulfide oxidoreductases in hyperthermophiles

Disulfide bonds are required for the stability and function of a large number of proteins. Recently, the results from genome analysis have suggested an important role for disulfide bonds concerning the structural stabilization of intracellular proteins from hyperthermophilic Archaea and Bacteria, contrary to the conventional view that structural disulfide bonds are rare in proteins from Archaea. A specific protein, known as protein disulfide oxidoreductase (PDO) is recognized as a potential key player in intracellular disulfide-shuffling in hyperthermophiles. The structure of this protein shows a combination of two thioredoxin-related units with low sequence identity which together, in tandem-like manner, form a closed protein domain. Each of these units contains a distinct CXXC active site motif. Due to their estimated conformational energies, both sites are likely to have different redox properties. The observed structural and functional characteristics suggest a relation to eukaryotic protein disulfide isomerase. Functional studies have revealed that both the archaeal and bacterial forms of this protein show oxidative and reductive activity and are able to isomerize protein disulfides. The physiological substrates and reduction systems, however, are to date unknown. The variety of active site disulfides found in PDOs from hyperthermophiles is puzzling. Nevertheless, the catalytic function of any PDO is expected to be correlated with the redox properties of its active site disulfides CXXC and with the distinct nature of its redox environment. The residues around the two active sites form two grooves on the protein surface. In analogy to a similar groove in thioredoxin, both grooves are suggested to constitute the substrate binding sites of PDO. The direct neighbourhood of the grooves and the different redox properties of both sites may favour sequential reactions in protein disulfide shuffling, like reduction followed by oxidation. A model for peptide binding by PDO is proposed to be derived from the analysis of crystal packing contacts mimicking substrate binding interactions. It is assumed, that PDO enzymes in hyperthermophilic Archaea and Bacteria may be part of a complex system involved in the maintenance of protein disulfide bonds. The regulation of disulfide bond formation may be dependent on a distinct interplay of thermodynamic and kinetic effects, including functional asymmetry and substrate-mediated protection of the active sites, in analogy to the situation in protein disulfide isomerase. Numerous questions related to the function of PDO enzymes in hyperthermophiles remain unanswered to date, but can probably successfully be studied by a number of approaches, such as first-line genetic and in vivo studies.