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At least 19 recordsLinked to original sources

PubMed1989

p-chloromercuribenzoate causes Ca2+-dependent exocytotic catecholamine secretion from cultured bovine adrenal medullary cells.

Incubation of cultured bovine adrenal medullary cells with p-chloromercuribenzoate (50-500 microM), a sulfhydryl-reacting agent, caused an increase in the secretion of catecholamines, p-Chloromercuriphenyl sulfonate, a p-chloromercuribenzoate analogue that poorly penetrates the cell membrane, caused a similar increase in catecholamine secretion. In both cases, catecholamine secretion was dependent on extracellular Ca2+. Furthermore, p-chloromercuribenzoate caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to p-chloromercuribenzoate concentration. The time courses of the increased secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration by p-chloromercuribenzoate were also quite similar. The stimulation of catecholamine secretion by p-chloromercuribenzoate was reversed by washing the cells with dithiothreitol-containing medium, but not by dithiothreitol-free medium. When the cells were treated with p-chloromercuribenzoate, dopamine-beta-hydroxylase, an enzyme present in the chromaffin granules along with catecholamines, was also released. However, p-chloromercuribenzoate did not cause release of phenylethanolamine-N-methyltransferase, an enzyme present in the cytoplasm. These results indicate that catecholamine secretion due to p-chloromercuribenzoate occurs by Ca2+-dependent exocytosis.

Adrenal Medulla↗

PubMed1989

The activation of glucose dehydrogenase by p-chloromercuribenzoate.

p-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.

Animals↗

PubMed1983

Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range.

The effects of nonsaturating amounts (5-60 nmol/mg membrane protein) of p-chloromercuribenzoate on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher p-chloromercuribenzoate concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled p-chloromercuribenzoate. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to p-chloromercuribenzoate. Other reagents including N-ethylmaleimide, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.

Chloromercuribenzoates↗

PubMed1982

Effect of diamide and reduced glutathione on the elevated levels of cyclic AMP in rat pancreatic islets exposed to glucose, p-chloromercuribenzoate and aminophylline.

In rat pancreatic islets the effects of diamide, which has been shown to decrease islet levels of reduced glutathione (GSH), and of exogenous GSH were investigated on cyclic AMP as increased by glucose, p-chloromercuribenzoate, and aminophylline. In addition the effect of diamide on islet ATP level, low Km and high Km phosphodiesterases was studied. Diamide (0.1 mM) inhibited the increase of cyclic AMP (cAMP) in response to glucose (16.7 mM), and p-chloro-mercuribenzoate (1 mM) in the presence of 5.6 mM glucose. No inhibitory effect of diamide could be demonstrated when cAMP was raised by 10 mM aminophylline in the presence of 5.6 mM glucose. The glucose (27.7 mM) stimulated increase of cAMP was further augmented by GSH (0.4 mM) whereas GSH in the presence of 5.6 mM glucose had no such effect. Diamide neither affected islet high Km nor low Km cAMP-phosphodiesterases. Diamide (0.1 mM) as used in this study did not affect islet AMP levels. A concentration dependent decrease of ATP was observed, however, with higher concentrations of diamide (0.25, 0.5 and 1.0 mM). It is suggested that the accumulation of islet cAMP in response to glucose and para-chloromercuribenzoate depends on the redox state of islet thiols. Since thiol oxidant diamide neither affected cAMP-phosphodiesterase activities nor inhibited aminophylline induced accumulation of cAMP in the presence of low glucose the possibility is raised that in pancreatic islets the formation of cAMP rather than its degradation depends on the redox state of islet thiols.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1976

Effects of dextran-linked chloromercuribenzoic acid on insulin release from microdissected pancreatic islets.

Insulin release in response to dextran-linked p-chloromercuribenzoic acid was studied in microdissected pancreatic islets of non-inbred ob/ob-mice. No contamination of the dextran-linked mercurial with free chloromercuribenzoic acid was detected before or after the incubation with islets. In comparison with free mercurial, of the same thiol-blocking activity, the dextran-linked compound had a weak insulin-releasing action with a different dose vs. response relationship. The dextran-linked mercurial had no demonstrable effect on the islet content of cyclic AMP. The results support the hypothesis that free organic mercurials mainly stimulate insulin release by blocking thiol ground that are embedded within the beta-cell plasma membranes beneath their surfaces.

Animals↗

PubMed1979

Limited proteolysis of p-chloromercuribenzoate-dissociated hemoglobin by trypsin.

p-Chloromercuribenzoate-treated hemoglobin was digested by trypsin. The hydrolysate was subjected to gel-filtration on Bio-Gel P-4 and Sephadex G-50 columns, ion-exchange chromatography on CM-Sephadex and DE 52 columns, and paper electrophoresis. Peptides obtained by this procedure were analyzed for amino acid compositions and amino-terminal amino acid sequences. The results showed that p-chloromercuribenzoate-treated hemoglobin was hydrolyzed to a limited extent by trypsin at the bonds involving the carboxyl group of a lysine or arginine residue in planes A--E in the parent hemoglobin, which represent the external region of the parent tetramer. It is concluded therefore that the slight modification of hemoglobin enhances the susceptibility of the protein to proteases and that the hydrolysis of the modified protein is limited.

Amino Acid Sequence↗

PubMedDate not supplied

[Effect of n-chloromercuribenzoate and albumin on Mg2+, Ca2+-ATPase activity of plasma membranes of smooth muscle cells].

It is shown that Mg2+, Ca2+-ATPase activity of plasma membrane fragments from the rat small intestine myocytes is inhibited by p-chloromercuribenzoate and dithionitrobenzoate (50 and 90%, respectively). The effect of p-chloromercuribenzoate inhibition is removed by serum albumin promoting a rise in the ATPase activity of the plasma membranes and shifting the temperature maximum point up to 32 degrees C (in the norm the maximum is observed at 36 degrees C). According to the data presented, albumin changes the composition and phase state of the lipid surrounding of the membrane enzymes.

Albumins↗

PubMed1978

Synthesis of combined protein antigens. Cross-linking of Hg-(p-chloromercuribenzoate) and--SH(thiolactone) substituted proteins.

The method of coupling of -Hg (p-chloromercuribenzoate) substituted proteins with protein substituted by -SH (thiolactone) groups was designed to obtain AnBm type copolymers not associated with An or Bm homopolymers. Reaction of p-chloromercuribenzoate with human serum albumin (HSA) was performed in 1.47 M imidazole. The conjugate was treated with thiolated proteins (human immunoglobulin G or human serum albumin). Disulfide linking of thiolated proteins was negligible under the conditions used. The total yield of polymerization reached the value 35%-75% of proteins taken for the reaction. It depended on the amount of reactive groups substituted and ratio of proteins A/B, used for cross-linking. The results were confirmed by theoretical calculations based on Bernoulli distribution.

Antigens↗

PubMed1984

p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of beta-ketoadipate enol-lactone hydrolase and succinyl CoA: beta-ketoadipate CoA transferase.

beta-Ketoadipate enol-lactone hydrolase (EC 3.1.1.24) and succinyl CoA: beta-ketoadipate transferase (EC 2.8.3.6) catalyze consecutive metabolic reactions in bacteria. The enzymes appear to be members of different families of related proteins. Enzymes within the enol-lactone hydrolase family appear to have diverged so extensively that common ancestry sometimes is not directly evident from comparison of NH2-terminal amino acid sequences of the proteins. Amino acid sequences at or near the active sites of the enzymes are likely to have been conserved, and hence a chemical proble that reacted specifically near the active sites of the enzymes might identify regions of amino acid sequence in which evolutionary affinities among widely divergent proteins could be identified. p-Chloromercuribenzoate appears to be such a probe because enol-lactone hydrolases and CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida were completely inhibited by stoichiometric quantities of the compound which appears to modify selectively cysteinyl side chains at or near the active sites of the enzymes. Stoichiometric inhibition of P. putida enol-lactone hydrolase was observed in the presence of excess dithiothreitol; therefore the reactive cysteinyl residue in this enzyme appears to be nucleophilic. The hydrolase is inhibited by beta-ketoadipate, but the compound must be supplied at 10 mM concentrations in order to achieve 50% inhibition, so the product inhibition is unlikely to be significant under physiological conditions.

Acinetobacter↗

PubMed1981

Time dependence of the effect of p-chloromercuribenzoate on erythrocyte water permeability: a pulsed nuclear magnetic resonance study.

Pulsed nuclear magnetic resonance spectroscopy is employed to determine the time dependence of the change in erythrocyte water permeability following exposure to p-chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonic acid (PCMBS). pH variation was used to examine the environment of the sulfhydryl groups reactive to these drugs. PCMB reacted with at least two sulfhydryl groups which affect water permeability. This was shown by the double exponential character of the change in erythrocyte diffusional permeability with time after PCMB addition. However, only one inhibition rate process could be distinguished following PCMBS exposure, suggesting that one site bound by PCMB is not accessible to PCMBS. This site is postulated to be located in a hydrophobic region of the membrane, whereas the site reached by both drugs is located in the normal anion permeation channel. The effect of pH on the degree of inhibition due to each component and the inhibition rates is explained in terms of its effect on solubility of the reagents in the membrane and variation of the dissociated-to-undissociated ratio of PCMB.

4-Chloromercuribenzenesulfonate↗

PubMed1987

p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.

Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.

Actins↗

PubMed1992

Effect of p-chloromercuribenzoate on Clostridium perfringens beta toxin.

p-Chloromercuribenzoate (PCMB) was shown to bind to Clostridium perfringens beta toxin. Treatment of the toxin with N-ethylmaleimide (NEM), 5,5'-dithio-bis(2-nitro-benzoic acid) (DTNB), o-iodosobenzoate (OIBA) and metal ions such as Cu2+ and Ag+ decreased the lethal activity, but PCMB did not affect the lethal activity. On the other hand, the binding of PCMB to the toxin was inhibited by DTNB and NEM in a dose-dependent manner. Furthermore, the lethal activity of beta toxin pretreated with PCMB was not blocked by treatment with NEM, DTNB, OIBA, Cu2+ and Ag+. However, the PCMB-treated toxin treated with reduced glutathione, dithiothreitol, 2-mercaptoethanol, liver homogenate or serum from mice was inactivated by NEM.

Animals↗

PubMed1983

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate.

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.

Chloromercuribenzoates↗

PubMed1984

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle.

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.

Actomyosin↗

PubMed1984

Further improvement in preparation and some properties of native alpha and beta chains from canine hemoglobin treated with p-chloromercuribenzoate.

The alpha and beta chains were prepared from canine hemoglobin by a modification of the method of Bucci and Fronticelli ((1965) J. Biol. Chem. 240, PC551-552) which involved treatment of hemoglobin with an excess of p-chloromercuribenzoate (pCMB), separation of the mercurated chains by chromatography on a DE-32 column with a salt gradient at pH 8.6, and regeneration of sulfhydryl groups of the chains with 2-mercaptoethanol. The SH titer was two per heme for both the regenerated alpha and beta chains. The titer decreased to four per tetramer of hemoglobin after equimolar recombination of both chains. Measurements of the absorption spectrum and oxygen binding showed that the chains were in a native state.

Amino Acids↗

PubMed1994

Selective cysteine-->serine replacements in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens allow the unambiguous assignment of Cys211 as the site of modification by spin-labeled p-chloromercuribenzoate.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains five sulfhydryl groups per subunit. Cysteine-->serine replacements show that the thiols are not essential for catalysis. The increased dissociation constant for FAD in mutant Cys158Ser suggests that Cys158 is important for the solvation of the pyrophosphate moiety of the prosthetic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivated by mercurial compounds. Inactivation by a spin-labeled derivative of p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Incorporation of the spin label in the other Cys-->Ser mutants strongly impairs substrate binding without affecting the catalytic properties of the FAD. The results are discussed with respect to previous tentative assignments from chemical modification studies and in light of the 3-D structure of the enzyme-substrate complex.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1983

The effects of p-chloromercuribenzoate on muscarinic receptors in the cerebral cortex.

The action of p-chloromercuribenzoate (PCMB) on the ligand binding properties of the muscarinic receptors in the rat cerebral cortex has been examined. At low concentrations, PCMB produces a selective change in the binding of agonists without any effect on the binding of antagonists. At higher concentrations, the structure-binding profile for binding antagonists is changed. The affinity of agonists is greatly reduced and the heterogeneity of binding eliminated. The effects of both high and low concentrations of PCMB can be reversed by dithiothreitol. Inactivation of receptors proceeds in parallel and is kinetically complex. It can only be partially reversed by dithiothreitol. Evidence is presented connecting the low affinity agonist binding site with the high affinity pirenzepine binding site. The changes produced by PCMB have been interpreted in terms of the modification of receptor conformation.

Animals↗

PubMed1983

The effect of p-chloromercuribenzoate on structure-binding relationships of muscarinic receptors in the rat cerebral cortex.

Muscarinic receptors in the rat cerebral cortex, reacted with p-chloromercuribenzoate (PCMB) under different conditions (Phase I and II), have modified binding sites. These exhibit remarkable changes in the structural dependence of the binding of drugs. In Phase I, the structure-binding profile of agonists for both the high and low affinity agonist sites are altered. In Phase II, the structure-binding profile of antagonists is also observed. In Phase II, the ability of potent agonists to discriminate between sub-classes of agonist binding sites is eliminated. There is also a loss of heterogeneity in the binding of the selective antagonist pirenzepine. Of the 16 agonists examined, only pilocarpine has a heterogeneous binding profile in Phase II, the dispersity of binding being increased. The changes in binding properties of the receptors are discussed in terms of general theories of drug-receptor interactions.

Animals↗