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Background The microelectrode array (MEA) was used to investigate the pharmacological relevance of chloride (Cl−) ions in antigen-dependent mast cell activation and the inhibitory effect of disodium cromoglycate (DSCG) on mast cell activation. Methods The movements of ions across the cellular membrane and the potential relationship between Cl− channels and DSCG during immunological activation were investigated using the MEA. The results were then subsequently compared with the amount of histamine released from anti-IgE activated peritoneal mast cells. Results The inclusion of charybdotoxin (ChTX) in Cl−-free buffer showed that the measured field potentials during antigen-stimulated peritoneal mast cell were a combination of Cl− influx and K+ efflux. The delayed onset time of Cl− influx indicated the presence of a delayed outwardly-rectifying Cl− current in the antigen-stimulated peritoneal mast cells. The use of 5-nitro-2-(3-phenylpropylamino) benzoic acid demonstrated that the activated mast cell membrane potential can be stabilised, thereby reducing the amount of histamine released from the anti-IgE activated mast cells. The correlation between the results of the histamine release assay and the electrophysiological measurements demonstrated the importance of Cl− to anti-IgE dependent mast cell activation. The inhibitory effect of DSCG on anti-IgE activated cells, however, did not correlate with the presumed influx of Cl−. Conclusions The MEA data suggest that Cl− influx is crucial to IgE-dependent mast cell degranulation. General significance While the MEA cannot yield information about single channel properties, it is convenient to use and can provide information on the global changes in electrophysiological responses of non-excitable cells.

Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), has been used to titrate D-lactate dehydrogenase (D-LDH), a respiratory flavoenzyme of Escherichia coli. All six of the possible sulfhydryls titrate in the presence of 2% sodium dodecylsulfate, showing that D-lactate dehydrogenase does not contain any -S-S- bridges. In the native state, only two sulfhydryls are accessible in buffer and only one in the presence of lipid. Single-site mutations of each of the six cysteines of D-lactate dehydrogenase have been prepared. Each of the purified mutant proteins has full activity, demonstrating that an -SH group is not essential to the FAD-driven redox reaction. Ellman's titrations of the mutant proteins have led to the identification of cysteines 65, 146, 156, and 256 in the amino-terminal end as those containing the sulfhydryls that are not accessible in buffer or in buffer plus lipid. The cysteine at 422 is titrated only partially in buffer, while in buffer containing lipid, a necessary factor for full enzymatic activity, its sulfhydryl is inaccessible to the reagent. Cysteine 492 has been identified as containing the sulfhydryl that is accessible to the reagent under both conditions.

Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p- chloromercuribenzenesulfonate (PCMBS), N- ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 μM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [3H]bumetanide and [3H]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic and interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 μM, DTONB 141 μM) and for cholate uptake (DTSNB 250 μM, DTONB 115 μM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.

Isolated cells from rat distal colon were investigated with the patch-clamp technique. In cell-attached and cell-excised patches (inside-out) single chloride channels with outward-rectifying properties were observed. In excised patches the single-channel conductance g was 47 ± 5 pS at positive and 22 ± 2 pS at negative clamp potentials ( n = 6 . The Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10 μM) induced fast closing events, whereas 10 μM of 3′,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) had no effect when applied to the cytosolic side. Quinine in the bath inhibited the Cl− channel by reducing its single-channel amplitude and increased open channel noise. With 0.1 mM the current amplitude decreased by 54% and with 1 mM quinine by 67%. Ca2+-dependent nonselective cation channels where observed after excision of the membrane patch. This channel was completely and reversibly inhibited by 100 μM DCDPC. Application of 1 mM quinine to the bath induced flickering and reduced the open-state probability from 0.94 to 0.44. In summary, besides its well established effects on K+ channels, quinine also inhibits nonselective cation channels and chloride channels by inducing fast closing events.

1. 1.|All sulfhydryl groups of a purified Ca 2+ -ATPase (ATP phosphohydrolase, EC 3.6.1.3) vesicle preparation of sarcoplasmic reticulum from rabbit skeletal muscle were reactive with 5,5′-dithobis(2-nitrobenzoate) (DTNB). In the absence of substrates, four groups per ATPase molecule (115 000 daltons) reacted with a pseudo first-order constant of 0.18 min -1 (0.5 mM DTNB, pH 7.5, I =0.11, and 20°C), while the remaining 15 groups were characterized by a rate constant of 0.016 min -1 . Reaction rates were markedly enhanced by a rise of pH and ionic strength. 2. 2.|At 0.1 mM ATP, ADP and Mg · ADP, two of the rapidly reacting groups disappeared, and at 1 mM nucleotide there was in addition a decrease of the modification rate of slowly reacting groups. ATPase activity decreased linearly with the number of modified groups, complete inactivation occurring after reaction of 12 groups. Protection by ATP against DTNB inactivation was accompanied by a reduction in modification rate. 3. 3.|Ca 2+ and Mg 2+ did not appear to affect modification rates except at high concentrations (0.5–1 mM), where the reaction was stimulated 4. 4.|During phosphorylation (simultaneous presence of Ca 2+ , Mg 2+ and 0.1 mM ATP) the number of rapidly reacting groups increased from two to three or four, while the modification rate of slowly reacting groups was reduced 5. 5.|Reaction of DTNB with the low molecular weight substrate, glutathion, was found to be 350–4000 times faster than with the sulfhydryl groups of ATPase. The reaction was similarly affected as that of ATPase by ionic strength and pH, but not by the substrates of ATPase. 6. 6.|It is suggested that the disappearance of two rapidly reacting sulfhydryl groups by adenine nucleotide is caused by a masking effect of bound nucleotide, while the decrease in modification rate of slowly reacting groups during phophorylation and at high adedine nucleotide concentration is attribute to conformation changes of the protein.

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14--16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.

Inhibition of red cell water transport by the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported by Naccache and Sha'afi ((1974) J. Cell Physiol. 84, 449–456) but other investigators have not been able to confirm this observation. Brown et al. ((1975) Nature 254, 523–525) have shown that, under appropriate conditions, DTNB binds only to band 3 in the red cell membrane. We have made a detailed investigation of DTNB binding to red cell membranes that had been treated with the sulfhydryl reagent N -ethylmaleimide (NEM), and our results confirm the observation of Brown et al. Since this covalent binding site does not react with either N -ethylmaleimide or the sulfhydryl reagent pCMBS ( p -chloromercuribenzenesulfonate), its presence has not previously been reported. This covalent site does not inhibit water transport nor does it affect any transport process we have studied. There is an additional low-affinity (non-covalent) DTNB site that Reithmeier ((1983) Biochim. Biophys. Acta 732, 122–125) has shown to inhibit anion transport. In N -ethylmaleimide-treated red cells, we have found that this binding site inhibits water transport and that the inhibition can be partially reversed by the specific stilbene anion exchange transport inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS), thus linking water transport to anion exchange. DTNB binding to this low-affinity site also inhibits ethylene glycol and methyl urea transport with the same K I as that for water inhibition, thus linking these transport systems to that for water and anions. These results support the view that band 3 is a principal constituent of the red cell aqueous channel, through which urea and ethylene glycol also enter the cell.

Treatment of the adenosylcobalamin-requiring enzyme ethanolamine ammonia-lyase (EC 4.3.1.7) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) discloses three classes of -SH groups: a rapidly reacting class (2 -SH groups/enzyme molecule), a slowly reacting class (6 -SH groups/molecule) and a class which does not react unless the enzyme is denatured (7 -SH groups/molecule (Kaplan, B.H. and Stadtman, E.R. (1968) J. Biol. Chem. 243, 1794)). The enzyme is inactivated by Nbs2 at a rate similar to the rate at which Nbs2 reacts with the slowly reacting class of -SH groups. Inactivation of the enzyme is retarded by adenosylcobalamin but not by ethanolamine. Once inactivated, the enzyme cannot be reactivated with mercaptoethanol. These observations provide further evidence for the importance of -SH groups in catalysis by adenosyl-cobalamin-requiring enzymes.

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber in order to identify and localize chloride conductances. The [Cl-] was rapidly reduced (substituted with isethionate) in the apical or basolateral perfusate and the transepithelial potential difference (Vt) and transepithelial resistance (Rt) were monitored continuously. In addition, agents known to inhibit anion transport in other epithelia were applied. The direction of all initial changes in Vt and Rt due to Cl- substitutions were consistent with the presence of ionic conductances for Cl- on both sides of the epithelium. The time-courses and magnitudes of the fall in Vt and increase in Rt during apical [Cl-] steps in the presence and absence of basolateral bumetanide were monophasic and identical in the two cases. The response of Vt to basolateral [Cl-] steps was biphasic and the initial response was greatly attenuated by bumetanide. These findings demonstrate that the largest conductance for Cl- is in the basolateral cell membrane, but that the paracellular and/or apical pathway also possess a finite Cl- conductance. All three agents tested, 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), caused an increase in Vt. NPPB and DIDS were more effective from the apical side. DCDPC and DIDS administered from the apical side led to a decrease in Rt. These results suggest that these agents act in this tissue by enhancing a conductive pathway on the apical membrane rather than blocking the basolateral Cl- conductance.

The uptake of [32P]phosphate into human red blood cells was inhibited (Ki = 0.6 mM) by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor (Ki = 7 mM). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[14C]-benzamido-4'-aminostilbene-2,2'-disulfonate, a potent inhibitor of anion transport (Ki = 1-2 microM), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.