Chloromercuri Benzenesulfonate Research Articles

Molecular Identification and Functional Characterization of Rat Multidrug and Toxin Extrusion Type Transporter 1 as an Organic Cation/H+Antiporter in the Kidney

We have cloned and functionally characterized the rat ortholog of multidrug and toxin extrusion type transporter 1 (rMATE1). The mRNA of rMATE1 was strongly expressed in kidney and detectable in the various tissues such as brain, stomach, colon, lung, liver, spleen, skeletal muscle, and prostate. When stably expressed in HEK293 cells, rMATE1 could mediate the transport of tetraethylammonium (TEA) and cimetidine under the condition where the membrane potential was disrupted by a high concentration of potassium ion and intracellular pH was reduced by NH(4)Cl pretreatment. When extracellular pH was changed from 5.5 to 8.5, the transport of TEA by rMATE1 was greatest at pH 7.5. Kinetic analyses showed that the transports of TEA and cimetidine mediated by rMATE1 were both saturable with a K(m) of 260 +/- 10 and 3.01 +/- 0.21 muM, respectively. It was found that cimetidine is the most potent inhibitor of rMATE1, and many other organic cations, such as 1-methyl-4-phenylpyridinium, amiloride, imipramine, and quinidine, are also effective as inhibitors. Pretreatment of the cells expressing rMATE1 with p-chloromercuribenzene sulfonate significantly reduced TEA transport, but this effect was totally reversed by subsequent treatment with dithiothreitol. These results indicate that the functional nature of rMATE1 is consistent with that of the hypothetical organic cation/H(+) antiporter system in the brush-border membrane of the renal tubular epithelial cells. Accordingly, these results suggest that rMATE1 is an electroneutral and multispecific organic cation transporter energized by the trans-proton gradient, and plays a physiological role in renal secretion of organic cations, including clinically used cationic drugs.

Na +- and anion-dependent Mg 2+ influx in isolated hepatocytes

Hepatocytes, which were Mg 2+-depleted during isolation, took up Mg 2+ during reincubation. Mg 2+ uptake was dependent on the concentration of extracellular Mg 2+, Na +, Cl −, bicarbonate and phosphate, Li + and choline + did not substitute for extracellular Na + in Mg 2+ influx. Mg 2+ influx was maximal when all three anion species were present, and did not occur when these anions were replaced by gluconte. Bicarbonate, phosphate and Cl − could substitute for each other. Mg 2+ uptake in hepatocytes was inhibited by p- chloromercuribenzene sulfonate, ouabain, gramicidin D, amiloride and verapamil. The results were explained by the assumption that the net Mg 2+ influx in hepatocytes is operating via electroneutral Na +, Mg 2+/anion cotransport driven by the Na + gradient. However, electrogenic Mg 2+ uptake gated by extracellular Na + and anions could not be excluded.

Platelets in tumor metastasis: generation of adenosine diphosphate by tumor cells is specific but unrelated to metastatic potential

Human 253J urinary carcinoma cells and the F1 (low-metastatic) and F10 (high-metastatic) variants of the B16 murine melanoma cell line have been shown to activate heparinized human platelets by an adenosine diphosphate (ADP)-dependent mechanism based on inhibition by creatine phosphate/creatine phosphokinase and the identification of aggregating concentrations (1 to 2 mumol/L) of ADP in cell-free culture supernatants by high-performance liquid chromatography. Aggregation did not occur in citrated samples, and hirudin was without effect. Studies were carried out to determine whether extracellular ADP arose from nonspecific cell damage during cell isolation and manipulation or was a specific process under control of the tumor cells themselves. Tumor cell damage during harvesting was shown not to be a factor because the amounts of ADP produced by the three cell lines (a) were inversely related to the appearance of lactic dehydrogenase in the culture supernatants and (b) were similar when measured in confluent monolayers, either in tumor cells after detachment and resuspension or after crossover studies involving culture in, alternatively, Hanks' balanced salt solution and minimal essential medium. Metabolic control of ADP production was indicated by the fact that (a) it was not dependent on cell number, which suggests feedback inhibition; (b) it was reduced 60% when tumor cells were treated with p-chloromercuribenzene sulfonate; and (c) it was completely abolished in those treated with iodoacetic acid, which might be expected to increase nonspecific leakage. These studies indicate that ADP production by these three lines does not arise due to leakage induced by nonspecific membrane damage during cell harvesting and manipulation but is a discrete process under metabolic control of the tumor cells. Moreover, in B16 murine melanoma cells the ability to produce ADP and to support platelet aggregation appears to be unrelated to metastatic potential insofar as identical results were obtained with the F1 and F10 variants.

Platelets in Tumor Metastasis: Generation of Adenosine Diphosphate by Tumor Cells Is Specific but Unrelated to Metastatic Potential

Abstract Human 253J urinary carcinoma cells and the F1 (low-metastatic) and F10 (high-metastatic) variants of the B16 murine melanoma cell line have been shown to activate heparinized human platelets by an adenosine diphosphate (ADP)-dependent mechanism based on inhibition by creatine phosphate/creatine phosphokinase and the identification of aggregating concentrations (1 to 2 mumol/L) of ADP in cell-free culture supernatants by high-performance liquid chromatography. Aggregation did not occur in citrated samples, and hirudin was without effect. Studies were carried out to determine whether extracellular ADP arose from nonspecific cell damage during cell isolation and manipulation or was a specific process under control of the tumor cells themselves. Tumor cell damage during harvesting was shown not to be a factor because the amounts of ADP produced by the three cell lines (a) were inversely related to the appearance of lactic dehydrogenase in the culture supernatants and (b) were similar when measured in confluent monolayers, either in tumor cells after detachment and resuspension or after crossover studies involving culture in, alternatively, Hanks' balanced salt solution and minimal essential medium. Metabolic control of ADP production was indicated by the fact that (a) it was not dependent on cell number, which suggests feedback inhibition; (b) it was reduced 60% when tumor cells were treated with p- chloromercuribenzene sulfonate; and (c) it was completely abolished in those treated with iodoacetic acid, which might be expected to increase nonspecific leakage. These studies indicate that ADP production by these three lines does not arise due to leakage induced by nonspecific membrane damage during cell harvesting and manipulation but is a discrete process under metabolic control of the tumor cells. Moreover, in B16 murine melanoma cells the ability to produce ADP and to support platelet aggregation appears to be unrelated to metastatic potential insofar as identical results were obtained with the F1 and F10 variants.

Effects of temperature on water diffusion in human erythrocytes and ghosts — nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 °C up to 24 h or at 37 °C for 30 min did not change the water exchange time significantly, even if Mn 2+ was present in the medium. This indicates that no significant penetration of Mn 2+ is taking place under such conditions. (2) In case of cells previously incubated at 37°C for longer than 30 min with concentrations of p- chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn 2+ for more than 10 min at 37°C. (3) When the arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn 2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permability and the activation energy of water diffusion ( E a,d ) were noticed. The mean value of E a,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of E a,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for E a,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37°C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the value of inhibition being 46% and 21%, respectively.

Redox reactions of plasma membranes isolated from soybean hypocotyls by phase partition

Ninety-five percent pure plasma membranes were isolated from the microsomal fraction of soybean hypocotyls ( Glycine max L. Merr. cv. Wayne) by phase partition in an aqueous polymer system. It was found that isolated plasma membranes could reduce ferricyanide, cytochrome c or various iron complexes with NADH as an electron donor. The rates of these reactions varied from 300 nmol (mg protein) −1 min −1 for NADH-ferricyanide reductase to 21 nmol (mg protein) −1 min −1 for NADH-cytochrome c reductase. The flavin content on these membranes was 2.4 nmol flavin/mg protein. The membranes also showed evidence of a b- type cytochrome in the oxidized minus reduced difference spectrum ( λ max 561 nm) upon addition of dithionite. The Soret band peak for this b- type cytochrome was at 428 nm in the difference spectrum. The cytochrome content of the isolated plasma membranes was 0.5 nmol/mg protein. The reductase activity of plasma membranes isolated from soybean hypocotyls by two-phase partition was stimulated from 88 to 175% by low concentrations of Triton X-100. The reductase reactions in these membranes were inhibited from 33 to 83% by several nucleotides, manganese and zinc ions, p- chloromercuribenzene sulfonate (PCMBS) and 4-nitrophenyl acetate. These inhibitions were affected differently by Triton X-100 in the assay.

The topology of the major band 4.5 protein component of the human erythrocyte membrane: characterization of reactive cysteine residues

A preparation of band 4.5 protein of the red cell membrane, containing largely the sugar transporter, was labelled with the sulfhydryl reagent N- ethyl[ 14 C]maleimide . In preparations denatured with sodium dodecyl sulfate (SDS), all five sulfhydryl groups present in the peptide, M r 45 000 to 60 000, react with the alkylating agent within 20 min at 37°C. If the peptide is reconstituted in lipid vesicles and cleaved with trypsin before extraction and denaturation with SDS, three sulfhydryl groups are found in a 30 kDa fragment and two in a 19 kDa fragment. In ‘native’ reconstituted protein only three groups react, even after two hours of exposure, two in the 30 kDa fragment and one in the 19 kDa fragment. Thus, one sulfhydryl group is cryptic, inaccessible to N- ethylmaleimide in each fragment. In intact cells, the single reactive group of the 19 kDa fragment can be protected against reaction with N- ethylmaleimide by the impermeant sulfhydryl reagent, p- chloromercuribenzene sulfonate (PCMBS). It is, therefore, considered to be exposed on the outer face of the membrane. The two reactive groups of the 30 kDa fragment are not protected by PCMBS and are, therefore, not considered to be exposed to the outside medium. Cytochalasin B, a competitive inhibitor of sugar transport affords temporary protection of the exofacial group of the 19 kDa against reaction with N- ethylmaleimide , and affords longer term protection of one of the reactive groups of the 30 kDa fragment. These findings allow conclusions about the topology of the sugar transport protein in the bilayer. Both proteolytic fragments must cross the bilayer. One of three reactive sulfhydryl groups is exofacial and two may be cytoplasmic. The two cryptic groups may be located within the bilayer.

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, p- chloromercuribenzene sulfonate , osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton T 1 NMR and obtained a value of 4.0 · 10 −3 cm · s −1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of N- phenyl-1- naphthylamine by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.

Site of red cell cation leak induced by mercurial sulfhydryl reagents

It has been suggested that the human red cell anion transport protein, band 3, is the site not only of the cation leak induced in human red cells by treatment with the sulfhydryl reagent pCMBS ( p- chloromercuribenzene sulfonate ) but is also the site for the inhibition of water flux induced by the same reagent. Our experiments indicate that N- ethylmaleimide , a sulfhydryl reagent that does not inhibit water transport, also does not induce a cation leak. We have found that the profile of inhibition of water transport by mercurial sulfhydryl reagents is closely mirrored by the effect of these same reagents on the induction of the cation leak. In order to determine whether these effects are caused by band 3 we have reconstituted phosphatidylcholine vesicles containing only purified band 3. Control experiments indicate that these band 3 vesicles do not contain ( Na + + K +)- ATPase as measured by ATP dephosphorylation. pCMBS treatment caused a significant increase in the cation leak in this preparation, consistent with the view that the pCMBS-induced cation leak in whole red cells is mediated by band 3.

Relationship between calcium ion transport and (Ca 2+ + Mg 2+)-ATPase activity in adipocyte endoplasmic reticulum

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca 2+ transport in this preparation were characterized and compared to those of (Ca 2+ + Mg 2+)-ATPase activity. The time course of Ca 2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca 2+/mg protein after 3–4 min of incubation. The rate of Ca 2+ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca 2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent K m values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent K m values for Mg 2+ and Ca 2+ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ( Ca 2+ + Mg 2+ )-ATPase. The presence of potassium was required for maximum Ca 2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca 2+ transport and ( Ca 2+ + Mg 2+- ATPase activity was K + > Na + = NH 4 + > Li + . Ca 2+ transport and ( Ca 2+ + Mg 2+)- ATPase activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM p- chloromercuribenzene sulfonate. The results indicate that Ca 2+ transport in adipocyte endoplasmic reticulum is mediated by a ( Ca 2+ + Mg 2+)-ATPase and suggest an important role for endoplasmic reticulum in control of intracellular Ca 2+ distribution.

Three-dimensional structure of immunoglobulins.

Publisher Summary This chapter discusses a study analyzing the three-dimensional structure of immunoglobulins. Heavy atom derivatives were obtained. The compounds used were (1) p -chloromercuribenzene sulfonate (PCMBS), (2) mercuric cyanide Hg(CN) 2 , and (3) chloroplatinate ion, (PtCl 6 −− ). X-ray diffraction data were measured for the native crystals and each of the heavy atom derivatives to a Bragg spacing of 6 A. Electron micrographs were prepared of sections of the Dob crystals, cut perpendicular to the short c axis. The crystals were fixed with glutaraldehyde, washed, postfixed in osmium tetroxide, and embedded in Maraglas prior to sectioning. The sections were further stained with uranyl acetate and lead citrate solutions. The method of optical integration was applied in which the periodicity of the crystal was used to reduce the background noise and reveal the molecular outline. An electron density map was calculated with phases obtained from the heavy atom derivatives. The examination of the map showed that one pair of asymmetric units (containing one complete molecule) had density corresponding to three globular regions. One region lay on the twofold axis relating the two halves of the Dob molecule.

Effect of membrane modification on cell fusion of hen erythrocytes induced by dimethyl sulfoxide

Fusion of hen erythrocytes is inhibited by millimolar concentrations of bifunctional reagents glutaraldehyde, formaldehyde and dimethyl suberimidate and by low concentrations of detergents Triton X-100 and sodium dodecylsulfate, whereas fusion is activated by organomercurials p -chloromercuribenzene sulfonate and p -chloromercuribenzoate. The effects are interpreted in terms of changes in membrane stability produced by these reagents.

Effect of p-Chloromercuribenzene Sulfonate on Lens Cation Transport

The effect of p -chloromercuribenzene sulfonate (PCMBS) on the lens cation transport system was studied. PCMBS was used since this reagent has been reported to act primarily on the s

Stimulation of Na + transport across the toad urinary bladder by [formula omitted] sulfonate

The sulfhydryl reagent p- chloromercuribenzene sulfonate increased the I sc across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na +. In the absence of substrate, the rise in I sc was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p- chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na + transport apparatus.

Involvement of sulfhydryl groups in the action of insulin and radiation on thymocyte Na +-dependent amino acid transport

1. 1. p- Chloromercuribenzene sulfonate concentrations <10 −5 M stimulate the the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations > 10 −5 M of this reagent, transport is impaired and cell viability is affected. In contrast, 5,5′-dithiobis-(2-nitrobenzoate) between 10 −4 and 10 −6 M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. 2. Treatment of thymocytes with 10 −4 M 5,5′-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 · 10 7−7 · 10 7/ cell , activates 2-aminoisobutyrate transport to the same extent as does p- chloromercuribenzene sulfonate. Cells treated with 10 −6 M insulin showed a 30–50% reduction in the number of sulfhydryl groups that could be titrated with 5,5′-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10 −6 M p- chloro[ 203Hg] mercuribenzene sulfonate, addition of 10 −6 or 10 −9 M insulin before treatment with the sulfhydryl reagent again reduces the number of titrable SH groups by 20%. 3. 3. Insulin (10 −10−10 −6 M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not addictive. 4. 4. Insulin, but not p- chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by γ-irradiation. Treatment of cells with p- choloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. 5. γ-irradiation reduces the number of 5,5′-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.

Three-dimensional structure of immunoglobulins.

Publisher Summary This chapter discusses a study analyzing the three-dimensional structure of immunoglobulins. Heavy atom derivatives were obtained. The compounds used were (1) p -chloromercuribenzene sulfonate (PCMBS), (2) mercuric cyanide Hg(CN) 2 , and (3) chloroplatinate ion, (PtCl 6 −− ). X-ray diffraction data were measured for the native crystals and each of the heavy atom derivatives to a Bragg spacing of 6 A. Electron micrographs were prepared of sections of the Dob crystals, cut perpendicular to the short c axis. The crystals were fixed with glutaraldehyde, washed, postfixed in osmium tetroxide, and embedded in Maraglas prior to sectioning. The sections were further stained with uranyl acetate and lead citrate solutions. The method of optical integration was applied in which the periodicity of the crystal was used to reduce the background noise and reveal the molecular outline. An electron density map was calculated with phases obtained from the heavy atom derivatives. The examination of the map showed that one pair of asymmetric units (containing one complete molecule) had density corresponding to three globular regions. One region lay on the twofold axis relating the two halves of the Dob molecule.

Purification and properties of dihydroorotase from Escherichia coli B

Dihydroorotase (4,5- l-dihydroorotate amidohydrolase, EC 3.5.2.3) from Escherichia coli B was purified 145-fold. Following the terminal purification procedure, polyacrylamide gel electrophoresis showed only one protein band. In contrast to dihydroorotase from Zymobacterium oroticum, the enzyme is neither inhibited by EDTA nor activated by divalent metals. The enzyme is specific for its natural substrates l-ureidosuccinate and l-dihydroorotate and will not catalyze the hydrolysis of p- nitrophenylacetate . The N- carbamyl derivatives of glycine, dl-serine, and l-glutamic acid are competitive inhibitors. The enzyme is stablized by dithiolthreitol and is reversibly inhibited at pH 5.8 by the mercaptide-forming reagents AgNO 3 and p- chloromercuribenzene sulfonate but not the alkylating agent iodoacetamide. Phosphate and arsenate buffers appear to specifically increase the initial rates of dihydroorotase catalyzed reaction. The molecular weight of E. coli dihydroorotase in 0.05 M sodium phosphate buffer (pH 7.0) is estimated from gel filtration to be 76 000 ± 10%.

Mercury-induced reversible increase in 2,3-diphosphoglycerate phosphatase and concomitant decrease in mutase activity of animal phosphoglycerate mutases

A reversible transformation induced by HgCl 2 of phosphoglycerate mutase (EC 2.7.5.3) into 2,3-diphosphoglycerate phosphatase (EC 3.1.3.13) has been discovered and is documented. Maximum effects are obtained at pH 7 and above and at 3 mM Hg 2−. The interconversion occurs with all animal phosphoglycerate mutases tested, i.e. chicken breast, rabbit muscle and pig heart. All these phosphoglycerate mutases contain SH groups and require 2,3-diphosphoglycerate. Interconversion does not occur with yeast phosphoglycerate mutase which contains no SH groups. Also the phosphoglycerate mutases which are non-2,3-diphosphoglycerate dependent (EC 5.4.2.1) are neither inhibited nor the 2,3-diphosphoglycerate phosphatase activity increased with SH reagents. This reversible Hg 2+-induced phosphoglycerate mutase-2,3-diphosphoglycerate phosphatase interconversion is not carried out by other heavy metals such as Cd 2+ and Pb 2+ nor by organic mercurials such as p- hydroxymercuribenzoate and p- chloromercuribenzene sulfonate which are believed to be more specific for mercaptide formation than HgCl 2; indeed N- ethylmaleimide cannot convert the phosphoglycerate mutase into a 2,3-diphosphoglycerate phosphatase. The Hg 2+-induced reversible phosphoglycerate mutase-2,3-diphosphoglycerate phosphatase transformation does not entail changes in quaternary structure of the protein as shown by ultracentrifuge measurements. However, Hg 2+ seems to modify the tertiary structure as shown by spectropolarimetric and fluorimetric measurements. Mercury modifies the active site sufficiently so that the Michaelis constant for 2,3-diphosphoglycerate decreases for the phosphoglycerate mutase and increases for the 2,3-diphosphoglycerate phosphatase.