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At least 703 records · Page 39Linked to original sources
PubMed2000

Ebselen induces apoptosis in HepG(2) cells through rapid depletion of intracellular thiols.

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a synthetic seleno-organic compound with antioxidant capability. In the present study, we systematically examined the ability of ebselen to induce apoptosis in a human hepatoma cell line, HepG(2). Ebselen-induced apoptosis was evaluated by (i) TdT-mediated dUTP nick end labeling assay; (ii) analysis of sub-G1 cells; (iii) cell morphology, including cell size and granularity examination; and (iv) DNA gel electrophoresis. The results showed that ebselen was able to induce typical apoptosis in HepG(2) cells in a dose- and time-dependent manner. In order to explore the possible mechanisms involved in ebselen-induced apoptosis, the effect of ebselen on intracellular thiol concentrations including reduced glutathione (GSH) and protein thiols and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on ebselen-induced apoptosis were investigated. It was found that (i) ebselen rapidly depleted intracellular GSH and protein thiols, moreover, the depletion preceded the occurrence of apoptosis; (ii) NAC, a precursor of intracellular GSH synthesis, significantly alleviated ebselen-induced apoptosis; and (iii) BSO, a specific inhibitor of intracellular GSH synthesis, augmented ebselen-induced apoptosis significantly. Taken together, the present study demonstrates that ebselen is able to induce apoptosis in HepG(2) cells, most probably through rapid depletion of intracellular thiols.

Antioxidants↗
PubMed2000

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein.

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.

Amino Acid Substitution↗
PubMed2000

Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties.

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer-tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern-Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP-enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity.

Acrylamide↗
PubMed2000

Activation of thiol-dependent antioxidant activity of human serum albumin by alkaline pH is due to the B-like conformational change.

Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis.

Antioxidants↗
PubMed1993

Determination and derivatization of protein thiols by n-octyldithionitrobenzoic acid.

n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB), the reaction product of n-octane-1-thiol and Ellman's reagent, can be isolated in crystalline form. Like Ellman's reagent, ODNB can be used for the titration of SH groups in proteins, but with considerable advantages: Due to the absence of one negative charge and the presence of a lipophilic hydrocarbon chain, titrations of protein thiols are significantly faster. Normally, endpoints are reached after 5-30 min, while the corresponding reaction with Ellman's reagent may take 1-2 h. Another advantage of ODNB is that it finds access to thiol groups which normally remain undetected. With myosin S1, for example, ODNB reacts with a thiol group which is not reactive with Ellman's reagent. Although ODNB has detergent properties, reaction of this thiol group as a consequence of denaturation seems unlikely, because ODNB was used below its critical micelle concentration and all other thiols of myosin S1 remained buried. The n-octylthiol derivatives of proteins formed by reaction with ODNB can be used for transient protection of thiols, for example, during purification procedures, or for a reversible blocking of SH groups involved in biochemical reactions.

Animals↗
PubMed1995

A procedure for the determination of monothiols in the presence of dithiothreitol--an improved assay for the reduction of disulfides.

4,4'-Dipyridyl disulfide (4-PDS) and sodium arsenite have been used to develop a rapid and sensitive assay for monothiols in the presence of dithiothreitol (DTT). The procedure is similar to that of Zahler and Cleland (J. Biol. Chem. 243, 716-719, 1968) but involves the use of a lower pH and 4-PDS, which is more effective than 5,5'-dithiobis-(2-nitrobenzoic acid) at low pH. Background reactions that interfere with the determination of slow reacting thiol groups are much reduced in the new assay procedure. The reduction of oxidized glutathione by DTT at three different pH values and a partial and the complete reduction of bovine serum albumin by DTT have been characterized in order to demonstrate the use of this procedure. In the first two cases monothiol groups were readily detected without interference from DTT. Lower than expected numbers of thiol groups in the case of three reduced and denatured model proteins appeared to reflect their partial complexation by arsenite. Equilibrium and kinetic constants for the formation and dissociation of the DTT-arsenite complex from pH 5 to 8 are presented.

Animals↗
PubMed2002

Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds.

A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.

Animals↗
PubMed1996

Ascertaining the number of essential thiol groups for the folding of creatine kinase.

Although the unfolding and refolding of proteins have been extensively studied in the literature, relatively few attempts have been made to see how many residues of the total residues of a certain amino acid in an enzyme can be modified without seriously affecting its folding. Based on a statistical analysis of the quantitative relationship between the extent of modification of protein functional groups and the decrease in their biological activity, a method proposed by Tsou (Sci. Sin. 1962, 11, 1535-1558) is widely used to determine the number of residues essential for the catalytic activity of modified proteins. In the present paper, Tsou's method is applied to determine the number of cystein residues essential for the folding of creatine kinase. The thiol groups of the cysteine residues in fully unfolded creatine kinase were modified by 2-chloromercuri-4-nitrophenol (MNP). The relationship between the number of MNP-groups introduced and the recovery of activity after refolding was determined. Quantitative treatment of the data by Tsou's plot shows that among the cystein residue modified in each subunit of creatine kinase, only three are essential for its folding.

Animals↗
PubMed1998

Effects of hydroxyl radical and sulfhydryl reagents on the open probability of the purified cardiac ryanodine receptor channel incorporated into planar lipid bilayers.

We examine the effect of hydroxyl radical on the ion permeability of the ryanodine receptor, a calcium releasing channel of sarcoplasmic reticulum. The cardiac ryanodine receptor, purified from pig heart, was reconstituted to proteoliposomes and then incorporated into a planar bilayer membrane. A single channel activity with a conductance of 724 pS in 900/200 mM (cis/trans) KC1 and an ion selectivity of PK:PCl = 1:0.08 was observed. These characteristics are similar to those observed by the incorporation of the channel through sarcoplasmic reticulum vesicles. Hydroxyl radicals chemically generated by the reaction of H2O2 and Cu(ethylenediamine)2 at the cis compartment increased the open probability of the channel. Treatment with SH oxidizing reagents from the cis compartment also increased the open probability, and dithiothreitol, a SH reducing agent, reversed the effect. These findings suggest that hydroxyl radicals react with some SH groups of the ryanodine receptor and increase the Ca2+ release from sarcoplasmic reticulum through the ryanodine receptor.

Adenosine Triphosphate↗
PubMed1999

Modification of the mitochondrial sulfonylurea receptor by thiol reagents.

The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor.

ATP-Binding Cassette Transporters↗
PubMed1999

Differential temperature dependency of chemical stressors in HSF1-mediated stress response in mammalian cells.

Expression of stress proteins is generally induced by a variety of stressors. To gain a better understanding of the sensing and induction mechanisms of stress responses, we studied the effects of culture temperature on responses to various stressors, since the induction of hsp70 in mammalian cells by heat shock is somehow modulated by culture temperature. Hsp70 was not induced by treatment with sodium arsenite, azetidine-2-carboxylic acid, or zinc sulfate at the level of heat shock factor (HSF) 1 activation in cells incubated at low temperature, although these treatments induced hsp70 in cells incubated at 37 degrees C. The repression of sodium arsenite or zinc sulfate-induced HSF1 activation by low temperature was not simply due to the inhibition of protein synthesis. On the other hand, heat shock and iodoacetamide induced HSF 1 activation in cells incubated at either temperature. Thus, there seem to be two kinds of stressors that induce HSF1 activation independently of or dependent on culture temperature. Furthermore, the reduction of glutathione level seemed to be essential for HSF1 activation by chemical stressors.

Animals↗
PubMed1996

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

2,2'-Dipyridyl↗
PubMed1998

S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase decreases the enzyme affinity to the erythrocyte membrane.

The effects of nitric oxide (NO) or related molecules on the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the red blood cell (RBC) membrane were investigated. It was demonstrated that submillimolar concentrations of the NO donor sodium nitroprusside (SNP) not only strongly inactivated GAPDH by S-nitrosylation of the enzyme thiols but also decreased the binding affinity of GAPDH for the RBC membrane. In fact, the incubation with SNP for 60 min at 30 degrees C and at a concentration > 50 microM induced the dissociation of the native GAPDH from the white unsealed membranes (standard ghosts) in a concentration-dependent manner with a partial recovery of the enzyme activity and thiols when SNP concentrations higher of 1 mM were used. Binding experiments under saturating conditions indicate a Ka value for the nitrosylated GAPDH of 3.5 +/- 0.8 x 10(6) M-1, which was more than 50% less than the Ka value of 7.6 +/- 0.6 x 10(6) M-1 observed for the native enzyme. These data were also confirmed in reassociation experiments under nonsaturating conditions. Dithiothreitol (DTT), which at concentrations of less than 1 mM catalyzed the S-nitrosylation of GAPDH and the consequent modification of the binding properties described above, the concentrations higher than 5 mM restored both the enzyme activity and the binding properties. Furthermore, the enzyme-membrane association induced before the incubation step afforded at least partial protection from the loss of titrable thiols and from the inactivation induced either spontaneously or by SNP. Taken together, these data not only confirm the key role of the active site cysteine residues in the catalytic function of GAPDH but also suggest that they may be involved in the NO-dependent regulation of GAPDH binding to the RBC membrane.

Animals↗
PubMed1975

The radiobiology of DNA strand breakage.

The yield of single-strand breaks in lambda DNA within lysogenic host bacteria was measured after exposure to 4-MeV electrons (50 msec) and rapid transfer (45 msec) to alkaline detergent. In nitrogen anoxia the yield was 1.2 X 10(-12) DNA single-strand breaks per rad per dalton, and under full oxygenation the yield increased to 5 X 10(-12) breaks per rad per dalton. A search for the presence of fast repair of strand breaks operating within a fraction of a second. Strand breaks produced in the persence of oxygen were repaired in 30-40 sec, while breaks produced under anoxia were rejoined even slower. A functional product from the po[A] gene was needed for the rejoining of the broken molecules. Intermediate levels of DNA strand breakage seen at low concentrations of oxygen are dependent on the concentration of cellular sulfhydryl compounds, suggesting that in strand breakage oxygen donors compete for reactions with radiation-induced transients in the DNA. Intercomparisons of data on radiation-induced lethality of cells and single-strand breaks in episomal DNA allow the distinction between two classes of radiation-induced radicals, R-1 and R-2, with different chemical properties; R-1 reacts readily with oxygen and N-oxyls under formation of potentially lethal products. The reactivity of oxygen in this reaction is 30-40 times higher than that of TMPN. R-2 reacts 16 times more readily that R-1 with oxygen under formation of single-strand breaks in the DNA. R-2 does not react with N-oxyls.

Aerobiosis↗
PubMed1986

Covalent reactions in the toxicity of SO2 and sulfite.

Toxic effects of SO2 and sulfite such as bronchitis and bronchoconstriction have been well documented. SO2 has also been suggested to potentiate carcinogenic effects of PAH. However, the molecular basis of these toxic effects is unclear. We have examined the covalent reaction of SO2 and sulfite with cellular proteinacious and nonproteinaceous sulfhydryl compounds using rat liver, and lung and human lung derived A549 cells. Reactions of sulfite and protein in rat and human lung cells reveals at least three proteins with sulfite-reactive disulfide bonds. Besides fibronectin and serum albumin, which had been reported to contain sulfonated products following exposure to sulfite, we have found one other protein with sulfite-binding capabilities. Since the integrity of disulfide bonds is crucial to the tertiary structure and thus protein function, the disruption of protein structure by sulfitolysis may result in altered cellular activities leading to biochemical lesions. Using carefully controlled conditions, reproducible GSH contents can be found in cultured cells and used as an experimental basis for studying alterations in the GSH and GSSG content of cells. Sulfitolysis of GSSG results in the formation of GSSO3H in A549 cells, and possibly in the lung. GSSO3H can be reduced enzymatically by GSSG reductase. However, the Km of GSSO3H is high compared to that of GSSG, suggesting the existence of a transient concentration of GSSO3H once it is formed. Cysteine S-sulfonate is, however, not reduced by cytosolic extracts in the presence of NADPH and would have to be eliminated from the cell by other means. GSSO3H is a strong competitive inhibitor of GST in rat liver and lung and A549 cells, using 1-chloro-2,4-dinitrobenzene as a substrate. It also inhibits the formation of GSH conjugates of BP 4,5-oxide, anti and syn BPDE, but to a lesser extent. These results suggest that SO2 may affect the detoxification of xenobiotic compounds by inhibiting, via formation of GSSO3H, the enzymatic conjugation of GSH and reactive electrophiles. Since GSH conjugation represents the major pathway of elimination of BP epoxides in the lung, our results offer a possible explanation for the cocarcinogenicity of SO2 with PAHs. These data suggest that the sulfitolysis reaction of sulfite is the common reaction mechanism mediating the underlying biochemical reactions leading to both the toxic and cocarcinogenic properties of SO2. Quantitation of sulfitolysis products and their interaction with cellular processes should provide a coherent scheme relating SO2 and sulfite toxicity among animal species and humans.

Animals↗