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Antimicrobial Agents and Chemotherapy Feb 1973An oxidation product of indole-3-acetic acid, 3-methyleneoxindole, is highly reactive with free sulfhydryl groups. It has been shown to be a potent bactericidal agent in...
An oxidation product of indole-3-acetic acid, 3-methyleneoxindole, is highly reactive with free sulfhydryl groups. It has been shown to be a potent bactericidal agent in actively growing cultures. The bactericidal action of 3-methyleneoxindole was found to be independent of protein or deoxyribonucleic acid synthesis but to require the capacity for ribonucleic acid synthesis. The reactions underlying the bactericidal effect of 3-methyleneoxindole are not unique to it among compounds which can bind free SH groups, since N-ethylmaleimide appeared to have a similar mode of action.
Topics: Chloramphenicol; DNA, Bacterial; Escherichia coli; Ethylmaleimide; Indoles; Oxindoles; RNA, Bacterial; Sulfhydryl Reagents
PubMed: 4597712
DOI: 10.1128/AAC.3.2.157 -
The Journal of Biological Chemistry Nov 1996Phenylalanine 1489 in the inactivation gate of the rat brain IIA sodium channel alpha subunit is required for stable inactivation. It is proposed to move into the...
Phenylalanine 1489 in the inactivation gate of the rat brain IIA sodium channel alpha subunit is required for stable inactivation. It is proposed to move into the intracellular mouth of the pore and occlude it during inactivation, but direct evidence for movement of this residue during inactivation has not been presented. We used the substituted cysteine accessibility method to test the availability of a cysteine residue substituted at position 1489 to modification by methanethiosulfonate reagents applied from the cytoplasmic side. Mutation of Phe-1489 to Cys results in a small (8%) fraction of noninactivating current. Ag+ and methanethiosulfonate reagents irreversibly slowed the inactivation rate and increased the fraction of noninactivating current of F1489C but not wild-type channels. Single channel analysis showed that modification slowed inactivation from both closed and open states and destabilized the inactivated state. Depolarization prevented rapid modification of Cys-1489 by these reagents, and the voltage dependence of their reaction rate correlated closely with steady-state inactivation. Modification was not detectably voltage-dependent at voltages more negative than channel gating. Our results show that, upon inactivation, Phe-1489 in the inactivation gate moves from an exposed and modifiable position outside the membrane electric field to a buried and inaccessible position, perhaps in or near the intracellular mouth of the channel pore.
Topics: Animals; Brain; Cysteine; Ion Channel Gating; Motion; Movement; Mutagenesis, Site-Directed; Oocytes; Rats; Recombinant Proteins; Sodium Channels; Structure-Activity Relationship; Sulfhydryl Reagents; Xenopus laevis
PubMed: 8940085
DOI: 10.1074/jbc.271.48.30971 -
Chemico-biological Interactions Sep 2008Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase,...
Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase, cholinesterase 2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). We created the mutants C310A, C466A, C310A/C466A and C310A/F312I to assess the roles of the two cysteines and a proposal that the increased rate of inactivation previously found in an F312I mutant was due to increased access of sulfhydryl reagents to Cys310. Our results indicated that both of the cysteines could be involved in inactivation by sulfhydryl reagents, but that the cysteine near the acyl pocket was more accessible. We speculated that the inactivation of aphid AChEs by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 and proposed that this residue could be a target for a specific insecticide. Here we reconsider this proposal.
Topics: Acetylcholinesterase; Animals; Cholinesterase Inhibitors; Drug Design; Humans; Insecticides; Invertebrates; Sulfhydryl Reagents
PubMed: 18384763
DOI: 10.1016/j.cbi.2008.02.006 -
The Journal of General Physiology Nov 1948Oxidizing agents of sulfhydryl groups such as iodosobenzoate, alkylating agents such as iodoacetamide, and mercaptide-forming agents such as cadmium chloride, mercuric...
Oxidizing agents of sulfhydryl groups such as iodosobenzoate, alkylating agents such as iodoacetamide, and mercaptide-forming agents such as cadmium chloride, mercuric chloride, p-chloromercuribenzoate, sodium arsenite, and p-carboxyphenylarsine oxide, added in small concentrations to a suspension of sea urchin sperm produced an increase in respiration. When the concentration was increased there was an inhibition. These effects are explained by postulating the presence in the cells of two kinds of sulfhydryl groups: soluble sulfhydryl groups, which regulate cellular respiration, and fixed sulfhydryl groups, present in the protein moiety of enzymes. Small concentrations of sulfhydryl reagents combine only with the first, thus producing an increase in respiration; when the concentration is increased, the fixed sulfhydryl groups are also attacked and inhibition of respiration is the consequence. Other inhibitors of cell respiration, such as cyanide and urethanes, which do not combine with -SH groups, did not stimulate respiration in small concentration.
Topics: Animals; Cell Respiration; Male; Proteins; Respiration; Sea Urchins; Spermatozoa; Sulfhydryl Compounds; Sulfhydryl Reagents; Tissues
PubMed: 18891144
DOI: 10.1085/jgp.32.2.179 -
Journal of Bacteriology Aug 1962Schaechter, M. (College of Medicine, University of Florida, Gainesville) and K. Santomassino. Lysis of Escherichia coli by sulfhydryl-binding reagents. J. Bacteriol....
Schaechter, M. (College of Medicine, University of Florida, Gainesville) and K. Santomassino. Lysis of Escherichia coli by sulfhydryl-binding reagents. J. Bacteriol. 84:318-325. 1962-Washed suspensions of gram-negative rods were lysed by low concentrations of some sulfhydryl-binding and oxidizing reagents, but not by reducing agents. Some kinetic aspects of this phenomenon were studied with p-chloromercuribenzoate and Escherichia coli B/r. Structures resulting from the action of this reagent consisted of impure cell walls. These could be purified by treatment with trypsin. Cell walls prepared mechanically and cell membranes obtained by lysing protoplasts were not overtly affected by this chemical.
Topics: Bacteriolysis; Escherichia coli; Indicators and Reagents; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 14497913
DOI: 10.1128/jb.84.2.318-325.1962 -
International Journal of Molecular... Jan 2023The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated....
The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [H]-carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in . Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.
Topics: Humans; Animals; Rats; Patulin; Escherichia coli; Cysteine; Sulfhydryl Reagents; Carnitine; Glutathione; Membrane Transport Proteins
PubMed: 36768549
DOI: 10.3390/ijms24032228 -
Journal of Food Protection Feb 2001This study was undertaken to determine the effects of reducing conditions (L-cysteine) and seryl (phenylmethylsulfonyl fluoride) and sulfhydryl (divalent cadmium) group...
This study was undertaken to determine the effects of reducing conditions (L-cysteine) and seryl (phenylmethylsulfonyl fluoride) and sulfhydryl (divalent cadmium) group inhibitors on aflatoxin B1 (AFB1) degradation by Flavobacterium aurantiacum. High-performance liquid chromatography was used to determine AFB1 concentrations in 72-h cultures of F. aurantiacum. The addition of 0.1, 1, or 10 mM L-cysteine did not have any significant effect on AFB1 degradation by these cultures after incubation for 4, 24, or 48 h (P > 0.05). The addition of 0.1 mM phenylmethylsulfonyl fluoride did not significantly decrease AFB1 degradation (P > 0.05), but 1 mM phenylmethylsulfonyl fluoride significantly decreased AFB1 degradation after 4, 24, and 48 h of incubation (P < or = 0.05). No significant difference in AFB1 degradation was obtained with 0.1 mM Cd2+ after 4, 24, or 48 h of incubation (P > 0.05). The addition of 1 and 10 mM Cd2+ significantly decreased AFB1 degradation compared with the cells containing AFB1 alone after 4 and 24 h (P < or = 0.05). The addition of chelators, 1 mM EDTA and 1 mM o-phenanthroline, did not result in removal of inhibition of AFB1 degradation observed with 1 and 10 mM Cd2+. Higher concentration of chelators (>1 mM) are necessary to overcome the inhibitory effect. Further work on the cellular fractions and/or crude enzyme preparations is necessary to determine if indeed sulfhydryl and seryl groups of the enzymes are involved in AFB1 degradation (by maintaining either the structure or function of the enzyme).
Topics: Aflatoxin B1; Cadmium; Chelating Agents; Chromatography, High Pressure Liquid; Cysteine; Flavobacterium; Oxidation-Reduction; Phenylmethylsulfonyl Fluoride; Sulfhydryl Reagents; Time Factors
PubMed: 11271780
DOI: 10.4315/0362-028x-64.2.268 -
Balkan Medical Journal Apr 2017Oxidative stress is implicated as one of the main molecular mechanism underlying silicosis.
BACKGROUND
Oxidative stress is implicated as one of the main molecular mechanism underlying silicosis.
AIMS
In this study, our aim was to asses the redox status in occupationally silica-exposed workers, by evaluating the dynamic thiol-disulphide homeostasis.
STUDY DESIGN
Case-control study.
METHODS
Thirty-six male workers occupationally exposed to silica particles and 30 healthy volunteers, working as office workers were included to the study. Posteroanterior chest radiographs and pulmonary function tests of both groups were evaluated. Also serum thiol disulphide levels were measured using the spectrophotometric method described by Erel and Neşelioğlu.
RESULTS
Among the 36 workers that underwent pulmonary function tests 6 (17%) had obstructive, 7 (19%) had restrictive, 6 (17%) had obstructive and restrictive signs whereas 17 (47%) had no signs. The mean PFTs results of silica-exposed workers were significantly lower than control subjects. The serum disulphide levels of silica-exposed workers were significantly higher than control subjects (23.84±5.89 μmol/L and 21.18±3.44 μmol/L, respectively p=0.02).
CONCLUSION
The serum disulphide levels, a biomarker of oxidative stress, are found to be higher in silica-exposed workers.
Topics: Adult; Biomarkers; Case-Control Studies; Disulfides; Homeostasis; Humans; Male; Middle Aged; Occupational Exposure; Oxidative Stress; Radiography; Respiratory Function Tests; Silicon Dioxide; Statistics, Nonparametric; Sulfhydryl Reagents
PubMed: 28418335
DOI: 10.4274/balkanmedj.2015.1632 -
Journal of Biochemistry Jun 1976Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation...
Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.
Topics: Amino Acids; Animals; Calcium; Circular Dichroism; Enzyme Activation; Kinetics; Macromolecular Substances; Magnesium; Molecular Weight; Myocardium; Optical Rotatory Dispersion; Peptide Fragments; Protein Conformation; Pyruvate Dehydrogenase Complex; Sulfhydryl Reagents; Swine; Thiamine Pyrophosphate; Trypsin
PubMed: 956154
DOI: 10.1093/oxfordjournals.jbchem.a131181 -
Biochimica Et Biophysica Acta Mar 1988The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepatic Ah receptor was demonstrated using a variety of sulfhydryl...
The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepatic Ah receptor was demonstrated using a variety of sulfhydryl (SH)-modifying reagents. Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding was approximately equivalent by 5,5'-dithiobis(2-nitrobenzoate), mersalyl, N-ethylmaleimide, and p-chloromercuriphenylsulfonate, whose inhibition curves were steep in the concentration range close to that of nonprotein SH groups in cytosol (ED50 values 50-200 microM or 13-48 nmol/mg cytosolic protein). Inhibition by p-hydroxymercuribenzoate (PHMB), although exhibiting a lower ED50, was more gradual over this range; iodoacetamide was an order of magnitude less potent. The ability of dithiothreitol to reverse binding inhibition induced by 150 microM (approximately 60 nmol/mg protein) mersalyl diminished with time; it decreased more rapidly in the simultaneous presence of TCDD and mersalyl than when mersalyl was present alone, consistent with increased accessibility of key SH group(s) due to conformational changes attending TCDD-receptor complex formation. Brief exposure of unoccupied receptor to mersalyl prior to TCDD binding caused slower sedimentation of the complex in 0-KCl sucrose gradients and alterations in its elution profiles on DEAE- and DNA-Sepharose suggestive of some impairment of the transformation process. When reagents were added to the transformed TCDD-receptor complex, loss of binding was observed only at concentrations which were an order of magnitude higher than those inhibiting TCDD binding. Loss of binding by each reagent was biphasic, and except for that caused by mersalyl, was not complete even after 6-8 h. Dithiothreitol was able to reverse the effects of mersalyl or PHMB only partially and only if added during the early phase (10-30 min) of binding loss. Mersalyl was much more potent in disrupting the untransformed than the transformed TCDD receptor complex. Physical alteration of the mersalyl-treated TCDD-receptor complex was evident from gel filtration, sucrose gradients, and DNA- and DEAE-Sepharose chromatography. Our results are in striking contrast to the effects of these reagents on steroid receptors, whose bound steroid hormone ligand is rapidly and reversibly displaced by lower concentrations of reagent.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Animals; Cytosol; DNA; DNA-Binding Proteins; Dithiothreitol; Kinetics; Liver; Male; Mersalyl; Oxidation-Reduction; Polychlorinated Dibenzodioxins; Protein Binding; Rats; Receptors, Aryl Hydrocarbon; Receptors, Drug; Structure-Activity Relationship; Sulfhydryl Reagents; Surface Properties
PubMed: 2831991
DOI: 10.1016/0304-4165(88)90037-2