Dinitrostilbene-2,2'-disulfonate Research Articles

Solubility of disodium 4,4′-dinitrostilbene-2,2′-disulfonate and sodium 4-nitrotoluene-2-sulfonate in aqueous organic solutions and its application feasibility in oxidation stage of DSD acid synthesis

Solid–liquid equilibrium (SLE) measurements for disodium 4,4′-dinitrostilbene-2,2′-disulfonate (DNSNa) and sodium 4-nitrotoluene-2-sulfonate (NTSNa) in aqueous ethylene glycol monoethyl ether solution and aqueous ethylene glycol monobutyl ether solution were conducted using a dynamic method over the temperature range from (280 to 335)K. A synergistic effect on DNSNa solubility was observed with the maximum solubility at solute-free mass fraction of ethylene glycol monoethyl ether w30=0.4000 and solute-free mass fraction of ethylene glycol monobutyl ether w40=0.5999, respectively. The solubility data were correlated using the thermodynamic electrolyte non-random two-liquid (E-NRTL) model and model parameters were determined simultaneously. Aqueous ethylene glycol monobutyl ether solution at solute-free mass fraction of ethylene glycol monobutyl ether w40=0.2000 was found to be suitable solvent medium for the oxidation of 4-nitrotoluene-2-sulfonic acid (NTS) to 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNS) and the conclusion was confirmed using the static analytical method combined with UV–VIS spectrophotometer.

Measurement and Correlation of the Solubility of Disodium 4,4′-Dinitrostilbene-2,2′-disulfonate in Aqueous Organic Solutions

The solubility data of disodium 4,4′-dinitrostilbene-2,2′-disulfonate (DNSNa) in aqueous organic solutions (ethanol + water) and (ethylene diglycol + water) were investigated over the temperature range from (280 to 323) K using a dynamic method. The mole fraction of water in the two different solvent mixtures ranged from 0.2221 to 1.0000 and 0.5959 to 1.0000, respectively. The electrolyte nonrandom two-liquid (E-NRTL) model proposed by Chen was applied to model the solubility data of DNSNa in the above systems. The binary interaction parameters of the E-NRTL model were obtained via regression of the experimental solubility data. The root-mean-square deviations of determined equilibrium temperature and the calculated equilibrium temperature varied from (0.40 to 1.22) K.

Photochemical study of 4,4′-dinitrostilbene-2,2′-disulfonate (DSD) degradation in water

When 4,4′-dinitrostilbene-2,2′-disulfonate (DSD) is submitted to UV or visible light in aqueous solution, the main photochemical process is a reversible trans–cis isomerisation, the cis form absorbs at shorter wavelength than the trans isomer. The UV spectrum of cis form may be obtained not only by Fischer’s method but also by using HPLC with UV detection at isosbestic point (311nm). Quantum yields were evaluated at 0.30 and 0.24 for trans→cis and cis→trans isomerisation, respectively.Photolysis also occurs but only with a very low quantum yield [(2.2−2.4)×10−4]. For this reason photocatalysis may be useful to accelerate the elimination of DSD from waste waters. Photocatalytic transformation is more efficient with TiO2 Degussa P25 than with TiO2 Millennium PC50 in spite of pure anatase composition of the latter. Immobilization of TiO2 on pumice stone is an interesting method to eliminate the problem of filtration implied by the use of slurries. To evaluate disappearance by UV detection it is recommended to detect at isosbestic point to do not interfere with isomerisation.

Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.

AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonate) and DNDS (4,4'-dinitrostilbene-2,2'-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes upon noncovalent binding of DNDS. Since conformational changes induced by stilbene disulfonate binding cause anion transport inhibition, identification of the DNDS binding regions may localize the substrate binding region of the protein. Cysteine residues were introduced into 27 sites in the extracellular loop regions of an otherwise cysteineless form of AE1, called AE1C(-). The ability to label these residues with biotin maleimide [3-(N-maleimidylpropionyl)biocytin] was then measured in the absence and presence of DNDS. DNDS reduced the ability to label residues in the regions around G565, S643-M663, and S731-S742. We interpret these regions either as (i) part of the DNDS binding site or (ii) distal to the binding site but undergoing a conformational change that sequesters the region from accessibility to biotin maleimide. DNDS alters the conformation of residues outside the plane of the bilayer since the S643-M663 region was previously shown to be extramembranous. Upon binding DNDS, AE1 undergoes conformational changes that can be detected in extracellular loops at least 20 residues away from the hydrophobic core of the lipid bilayer. We conclude that the TM7-10 region of AE1 is central to the stilbene disulfonate and substrate binding region of AE1.

Inducible expression of erythrocyte band 3 protein.

A permanent cell line with inducible expression of the human anion exchanger protein 1 (hAE1) was constructed in a derivative of human embryonic kidney cells (HEK-293). In the absence of the inducer, muristerone A, the new cell line had no detectable hAE1 protein by Western analysis or additional 36Cl flux. Increasing dose and incubation time with muristerone A increased the amount of protein (both unglycosylated and glycosylated). The 4,4'-dinitrostilbene-2, 2'-disulfonate (DNDS)-inhibitable rapid Cl exchange flux was increased up to 40-fold in induced cells compared with noninduced cells. There was no DNDS-inhibitable rapid flux component in noninduced cells. This result demonstrates inducible expression of a new rapid Cl transport pathway that is DNDS sensitive. The additional transport of 36Cl and 35SO4 had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 microM DNDS, 2) activation of 36Cl efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3) activation of 36Cl efflux by external anions that was selective in the order NO3 = Cl > Br > I, and 4) activation of 35SO4 influx by external protons. Under the assumption that the turnover numbers of hAE1 were the same as in erythrocytes, there was good agreement (+/-3-fold) between the number of copies of glycosylated hAE1 and the induced tracer fluxes. This is the first expression of hAE1 in a mammalian system to track the kinetic characteristics of the native protein.

Self-association of Band 3, the human erythrocyte anion exchanger, in detergent solution

Dimeric Band 3 purified in n-dodecyl octaethyleneglycol (C 12E 8) underwent an irreversible, temperature-dependent association, resulting in a complex with a Stokes radius slightly larger than a native tetramer, before forming a higher molecular weight aggregate. Self-association occurred with a half-time of about 1 h at 37°C but did not occur at 0°C after several days. No change in the secondary structure of Band 3, as observed by circular dichroism, occurred during the association process. However, self-association of Band 3 was accompanied by loss of the stilbene disulfonate inhibitor binding site. No association or loss of inhibitor binding occurred with the dimeric membrane domain under similar incubation conditions. The membrane domain dimer was also stable over a wide range of pH (5.5–9.5) and buffer conditions, while Band 3 aggregated below pH 6.5. Inhibitors of anion transport, which stabilize the membrane domain, slowed the association. Band 3, depleted of phospholipids by extensive washing of resin-bound protein with detergent or, incubated with excess detergent, was more prone to aggregation. The membrane domain also showed some aggregation when depleted of lipids. Preparations could be stabilized by adding dimyristoylphosphatidylcholine (DMPC) prior to the 37°C incubation. The effect of inhibitors and DMPC was additive, with a combination of 1 mM 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) and 1:1 (wt/wt) DMPC:Band 3 stabilizing 90% of the protein to a 24-h incubation at 37°C. The results suggest that self-association of Band 3 dimers is promoted by the cytoplasmic domain but results in alterations to the membrane domain involving the loss of essential phospholipids. Addition of phospholipid or inhibitors to Band 3 results in a stable preparation of the intact protein that may be suitable for crystallization studies.

Towards the localization of the essential arginine residues in the band 3 protein of human red blood cell membranes

The effects of 4-hydroxy-3-nitrophenylglyoxal (HNPG), on the binding of eosin-5-maleimide (EMA), and diethyl pyrocarbonate (DEPC) to the anion transport system in the human red blood cell membrane, have been investigated. HNPG is a reversibly binding, arginine-specific, anion transport competitive inhibitor, known to act on the anion binding site. The EMA reaction site is an external facing lysine residue (Lys-430) in the 17 kDa transmembrane segment. The DEPC reaction site is an intracellular histidine (His-819) in the 35 kDa fragment. The results show that inhibition of the transport system with EMA increases in presence of HNPG to about 2.3 times. This finding suggests a positive cooperativity between the HNPG and EMA binding site and give evidence that the essential arginine is either nearby or allosterically linked to Lys-430. The inhibition of the cells with DEPC was nearly unchanged or slightly decreased in the presence of 10 mM HNPG. These results suggest that the intracellular His-residue which reacts with DEPC is not a part of the transport pathway. Our experiments with 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) have shown that its affinity to the transport system does not change after pre-treatment with phenylglyoxal (PG). We also found that the binding of [ 14C]phenylglyoxal (PG) to band 3 reduces significantly in presence of chloride. This is another evidence for the direct involvement of arginine residues in substrate binding.

Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance

We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.

An oxonol dye is the most potent known inhibitor of band 3-mediated anion exchange

When cells are acutely exposed to the oxonol dye, bis(1,3-dibutylbarbituric acid)pentamethine oxonol (diBA), at 0 degrees C, the concentration that gives half inhibition of Cl- exchange (IC50) is 0.146 +/- 0.013 microM (n = 12) initially, but the inhibition increases with time. These characteristics indicate that a rapid initial binding is followed by a slow conformational change that makes the binding tighter. If diBA is allowed to equilibrate with band 3, the IC50 is only 1.05 +/- 0.13 nM (n = 5), making diBA a more potent inhibitor than 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), for which the IC50 under similar conditions is 31 +/- 6 nM [T. Janas, P. J. Bjerrum, J. Brahm, and J. O. Wieth. Am. J. Physiol. 257 (Cell Physiol. 26): C601-C606, 1989]. Inhibition by diBA is very slowly reversible at 0 degrees C (t1/2 > 50 h), but the effect is more readily reversible at higher temperatures. DiBA competes with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) for inhibition, suggesting an external site of action. In contrast to DIDS and DNDS, however, increasing Cl- concentrations do not decrease the inhibitory effect of diBA, indicating that the inhibition is not competitive. Thus diBA may be useful for investigating conformational changes during anion exchange and for stopping transport without preventing substrate binding. However, when diBA and other oxonols are used to sense membrane potential, they may have undesirable side effects on anion transport processes.

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.

Binding of nitrate to renal brush border membranes studied with 14N nuclear magnetic resonance (NMR)

Renal brush border membranes contain several anion exchangers that may play a role in electrolyte transport and pH regulation. To help characterize the types of exchangers present and the binding properties of these membranes, the binding of nitrate (NO 3 −) to highly purified rabbit kidney brush border membrane vesicles was studied. The method is based on the binding induced quadrupole relaxation of the 14N-NMR signal of nitrate [1,2]. Brush border membrane vesicles caused a relaxation of the 14N-NMR nitrate signal which could be characterized by relatively high affinity sites, K D = 6.7 ± 1.5 mM, as well as nonspecific interactions with the membranes, K D > 150 mM. The anion transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) inhibited 51 ± 6% ( n = 4) of the relaxation due to the high affinity binding sites. The DNDS inhibition could be characterized by a K i of 10–80 μM. Both bicarbonate and formate (HCO 2 −) were found to partially inhibit the high affinity induced relaxation, with maximal inhibition of 37 ± 8% ( n = 3) and 30 ± 2% ( n = 3), respectively. The inhibitory effects of saturating concentrations of bicarbonate and formate were non-additive, suggesting the existence of a stilbene sensitive exchanger that can bind nitrate, as well as both bicarbonate and formate. This study indicates the usefulness of this new method for further investigation of anion exchangers on these and other membranes.

Trace‐level determination of aromatic sulfonates in water by on‐line ion‐pair extraction/ion‐pair chromatography and their behavior in the aquatic environment

AbstractA new HPLC method for the fully automatic determination of aromatic sulfonates in aqueous samples is presented. The analytical procedure consists of an on‐line combination of ion‐pair extraction (IPE) and ion‐pair chromatography (IPC), both using RP‐C18 solid‐phase material and a tetrabutylammonium salt as ion pairing reagent. Experimental details and performance data are given. This method is suited for the trace‐level determination of a wide variety of benzene, naphthalene, anthraquinone and stilbene sulfonates. Detection limits for surface water using a diode‐array detector are in the sub‐ppb range. For naphthalene sulfonates a very good selectivity and minimal detectable limits of 0.02 μg/L or even lower can be achieved. So far, this method has been successfully applied to waste water, river water, bank filtrate, and water from different steps of drinking water production. The fate of several aromatic sulfonates has been studied beginning at the effluents of industrial waste water treatment plants and ending after activated carbon filtration in a water works. Napthalene‐1,5‐disulfonate (NDS, Armstrong acid) and cis‐4,4′‐dinitrostilbene‐2,2′‐disulfonate (DNS) appear in the raw water of the investigated water works and therefore have to be termed as relevant to water works. In contrast to other disulfonates NDS is extremely stable to biodegradation and ozonation and it is even desorbed from a highly loaded activated carbon filter.

Identification and covalent modification of a renal brush-border anion exchanger

Brush-border membrane (BBM) proteins that bind the arginine-specific reagent phenylglyoxal (PG) and interact with stilbene disulfonic derivatives were identified in canine kidney cortex. Pretreatment of BBM vesicles with PG resulted in irreversible inhibition of anion exchange as assayed by 36Cl − influx mediated via Cl − /Cl − exchange. Cl − /Cl − exchange was reversibly inhibited by the disulfonic stilbene 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS). A stilbene-affinity matrix was prepared by immobilizing DNDS in polyacrylamide beads. Elution of the BBM proteins from a disulfonic stilbene (DNDS) affinity matrix revealed two proteins at 160 and 230 kDa that were significantly enriched compared to initial material. Radiolabeling of the eluted mixture with [ 14C]phenylglyoxal demonstrated covalent binding to several proteins, including the 160 kDa protein. Reconstitution of the proteins eluted from the affinity matrix into phosphatydilcholine demostrated DIDS-sensitive 36Cl −-influx mediated via Cl −/Cl − exchange. Pretreatment of the BBM vesicles with PG selectively blocked binding of the 160 kDa protein to the DNDS affinity matrix. Radiolabelling of the PG-pretreated, affinity-purified membrane proteins showed selective prevention of [ 14C]phenylglyoxal binding to the 160 kDa protein. Reconstitution of the PG-pretreated proteins eluted from the affinity matrix demonstrated significant reduction in Cl −/Cl − exchange activity. These results suggest that a 160 kDa protein is a strong candidate for anion exchange transport in kidney proximal tubules.

Kinetics of residual chloride transport in human red blood cells after maximum covalent 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid binding.

Irreversible inhibition, 99.8% of control values for chloride transport in human red blood cells, was obtained by well-established methods of maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The kinetics of the residual chloride transport (0.2%, 106 pmol.cm-2 x s-1) at 38 degrees C, pH 7.2) was studied by means of 36Cl- efflux. The outside apparent affinity, expressed by Ko1/2,c, was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Borders, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemistry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of the residual chloride flux depended on the extracellular chloride concentration, but was not purely competitive. The half-inhibition concentrations at [Cl(o)] = 150 mM in control cells (Ki,o) and covalently DIDS-treated cells (Ki,c) were: DIDS, Ki,c = 73 nM; DNDS, Ki,o = 6.3 microM, Ki,c = 22 microM; phloretin, Ki,o = 19 microM, Ki,c = 17 microM; salicylate, Ki,o = 4 mM, Ki,c = 8 mM; Killer III, Ki,o = 10 microM, Ki,c = 10 microM.

In vivo quantitation of carbonic anhydrase and band 3 protein contributions to pulmonary gas exchange

The contributions to pulmonary gas exchange of red blood cell (RBC) membrane band 3 protein HCO3(-)-Cl- exchange and carbonic anhydrase- (CA) catalyzed HCO3- dehydration have never been determined directly in the whole animal. We utilized an experimental and model approach to measure these by analysis of phase III exhaled CO2 and O2 profiles in anesthetized dogs. In this method, we inhibit RBC membrane band 3 protein and cytoplasmic CA in RBCs passing the pulmonary capillaries and lung vascular luminal membrane-bound CA during a single ventilatory cycle. This is achieved with appropriately timed right atrial infusions of 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to inhibit band 3 protein, ethoxzolamide (a lipophilic CA inhibitor with rapid membrane penetrance) to inhibit RBC and lung tissue CA, and benzolamide (an extremely hydrophilic CA inhibitor with virtually no penetrance into RBC cytoplasm) to inhibit only lung vascular luminal membrane CA. DNDS caused a 15% reduction in CO2 production (VCO2) without any change in O2 consumption (VO2). The addition of benzolamide to DNDS did not cause any further decrease in VCO2. Inhibition of RBC CA by ethoxzolamide caused a 67% reduction in VCO2 and a 11.5% reduction in VO2. Inhibition of lung vascular CA by benzolamide alone caused no statistically significant changes in either VCO2 or VO2. These results are in general agreement with in vitro data and model calculations. The only exceptions are the higher than predicted effect of RBC CA inhibition on VO2 (Bohr effect) and the lack of any contribution to CO2 transfer in the dog by lung vascular CA with access to plasma as a possible consequence of an endogenous plasma CA inhibitor.

Inhibition of anion transport in the human red blood cell membrane with para- and meta-methoxyphenylglyoxal

The positional isomers para-methoxyphenylglyoxal and meta-methoxyphenylglyoxal were newly synthesized and found to be potent inhibitors of sulfate exchange in the red blood cell membrane. The rate of inactivation of the transport system with both reagents obeys pseudo-first-order kinetics and increases with increasing pH and reagent concentration. The degree of inhibition of the transport system with the meta-isomer exceeds the inhibition caused by the para-isomer. At 2 mM 3-methoxyphenylglyoxal (3-MOPG) and 37°C the half-lifetime of the anion transporter is 5.4 min at pH 8.0. Under the same experimental conditions the half-lifetime of the transporter at 2 mM 4-methoxyphenylglyoxal (4-MOPG) is found to be 24.7 min. The binding site of these reagents is found to be the same as binding site of the reversibly acting phenylglyoxal derivative 4-hydroxy 3-nitrophenylglyoxal (HNPG). Chloride ions are able to protect the transporter against inhibition with both reagents. Anion transport inhibitors like 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) and flufenemate, which are known to act on band 3 protein, are able to interact with the binding of the newly synthesised reagents. Phloretin and phloridzin show no interaction.

Exploration of the functional significance of the stilbene disulfonate binding site in mouse band 3 by site-directed mutagenesis

In 1985, it was shown that microinjection into Xenopus oocytes of mRNA prepared from the spleens of anemic mice leads to biosynthesis of band 3 protein (as shown by immunoprecipitation) and induction of a CIflux, which is not normally seen in the oocytes (Morgan et aI., 1985). This flux can be inhibited by typical inhibitors of band 3-mediated anion exchange, including 4,4' -dinitro stilbene-2,2'-disulfonate (DNDS), dipyridamole, phloretin and H+. These results encouraged us to microinject band 3 (AEl)-encoding cRNA derived from mouse band 3 cDNA (Hanke-Baier et aI., 1988). After biosynthesis of band 3, we microinjected 36CIinto oocytes and placed them individually into a perfusion chamber the bottom of which is formed by the mica window of a Geiger Muller tube. Perfusion of the chamber with a modified Amphibian Ringer solution removes 36CIescaping from the oocyte. The radioactivity inside the oocyte is recorded as a function of time. On a semilog scale, a plot of radioactivity vs. time yields a straight line relationship which persists until 80% of the initial radioactivity has left the oocyte. The slope of the straight line yields the rate constant for CIefflux (Grygorczyk et aI., 1987, 1989; Kietz et aI., 1991a). This efflux is inhibited when the CIof the Ringers solution is replaced by slowly penetrating SO/ions or nonpenetrating gluconate ions, indicating that, as in the red cell, the 36C1efflux can only take place by anion exchange. The K] values for H 2DIDS and DNDS are of the same order of magnitude as in the red cell (Kietz et aI., 1991a; Morgan et aI., 1985). Moreover, again as in the red cell, the hydrophobic domain suffices for the mediation of anion exchange after expression in the oocyte and remains susceptible to inhibition by H 2DIDS or DNDS (Lepke et aI., 1991). The agreement of important features of mouse band 3-mediated anion transport in red cells and oocytes encouraged us to use oligonucleotide-directed mutagenesis of a number of (conserved) lysine residues, Lys 449,558,649,869, to localize the sites of action of a range of covalently binding, inhibitory amino group reactive agents in the known primary structure of band 3 (see Fig 1). Regardless of the mutation, anion exchange is still accomplished. Thus, none of these residues is directly involved in anion binding and translocation. However, the susceptibility to inhibition by covalently and noncovalently binding inhibitors is drastically altered. Substitution by Asn of Lys 558 and Lys 561 (Bartel et aI., 1989b), or of Lys 558 alone (Bartel, 1989a), does not prevent reversible inhibition of CI/CIexchange by H 2DIDS, but prevents the subsequent irreversible reaction. Each of the two isothiocynate (NCS) groups of H 2DIDS is able to react with a different lysine residue in wild type band 3, to form an intramolecular cross-link. Because the NCS groups only react with the deprotonated forms of the lysine NH2 group, the reaction rate depends on pH. One of the NCS groups reacts with a lysine residue with pK ::II: 8.2, the other with a residue with pK '' 10.8 (20°C). In the wild type, the rate of covalent H2DIDS binding varies with pH in a manner which suggests reaction with the lysine residue with the lower pK value. After the mutation K558N, the pH dependence changes to a pattern compatible with a reaction with the lysine residue with the higher pK value (Kietz, 1991b). Pyridoxal phosphate (P-5P; 5 mM, pH 7.6) produces irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues are replaced (NM), irreversible inhibition can no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues 558 or 869 (Wood et aI., 1991; Kawano et aI., 1988). Inhibition by DNDS changes dramatically upon mutation. The Kial'P increases from 6.0 J.LM in the wild type (KK) to 23 J.LM in the mutant NK, to 73 J.LM in the mutant KM, and to 473 J.LM in the mutant NM. The K, value for activation of the transport system is 11 mM both in the wild type (KK) and in the mutant (NM) if measured by isosmotic substitution of CIby gluconate, which is not a substrate of band 3 (Passow et aI., 1992). The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate CIand that at last one of the two is allosterically linked to the substrate binding site. In the mutant NM, the K( value for reversible H2DIDS binding is considerably increased. Nevertheless, at alka-

Sodium-bicarbonate cotransport in retinal Muller (glial) cells of the salamander

An electrogenic Na+/HCO3- cotransport system was studied in freshly dissociated Müller cells of the salamander retina. Cotransporter currents were recorded from isolated cells using the whole-cell, voltage-clamp technique following the block of K+ conductance with external Ba2+ and internal Cs+. At constant pHo, an outward current was evoked when extracellular HCO3- concentration was raised by pressure ejecting a HCO3(-)-buffered solution onto the surface of cells bathed in nominally HCO3(-)-free solution. The HCO3(-)-evoked outward current was reduced to 4.4% of control by 0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate), to 28.8% of control by 2 mM DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), and to 28.4% of control by 2 mM harmaline. Substitution of choline for Na+ in bath and ejection solutions reduced the response to 1.3% of control. Bicarbonate-evoked currents of normal magnitude were recorded when methane sulfonate was substituted for Cl- in bath, ejection, and intracellular solutions. Similarly, an outward current was evoked when extracellular Na+ concentration was raised in the presence of HCO3-. The Na(+)-evoked response was reduced to 16.2% of control by 2 mM DNDS and was abolished by removal of HCO3- from bath and ejection solutions. Taken together, these results (block by stilbenes and harmaline, HCO3- and Na+ dependence, Cl- independence) indicate that salamander Müller cells possess an electrogenic Na+/HCO3- cotransport system. Na+/HCO3- cotransporter sites were localized primarily at the endfoot region of Müller cells. Ejection of HCO3- onto the endfoot evoked outward currents 10 times larger than currents evoked by ejections onto the opposite (distal) end of the cell. The reversal potential of the cotransporter was determined by DNDS block of cotransport current. In the absence of a transmembrane HCO3- gradient, the reversal potential varied systematically as a function of the transmembrane Na+ gradient. The reversal potential was -0.1 mV for a [Na+]o:[Na+]i ratio of 1:1 and -25.2 mV for a Na+ gradient ratio of 7.4:1. Based on these values, the estimated stoichiometry of the cotransporter was 2.80 +/- 0.13:1 (HCO3-:Na+). Possible functions of the glial cell Na+/HCO3- cotransporter, including the regulation of CO2 in the retina and the regulation of cerebral blood flow, are discussed.

Evidence for the development of an intermonomeric asymmetry in the covalent binding of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to human erythrocyte band 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies have identified two oligomeric forms of band 3 whose proportions on gel profiles were modulated by the particular ligand occupying the intramonomeric stilbenedisulfonate site during intermonomeric cross-linking by BS3 [bis-(sulfosuccinimidyl) suberate] [Salhany et al. (1990) J. Biol. Chem. 265, 17688-17693]. When DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) was irreversibly attached to all monomers, BS3 covalent dimers predominated, while with DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) present to protect the intramonomeric stilbenedisulfonate site from attack by BS3, a partially cross-linked band 3 tetramer was observed. In the present study, we investigate the structure of the protected stilbenedisulfonate site within the tetrameric complex by measuring the ability of patent monomers to react irreversibly with DIDS. Our results show two main populations of band 3 monomers present after reaction with DNDS/BS3: (a) inactive monomers resulting from the displacement of reversibly bound DNDS molecules and subsequent irreversible attachment of BS3 to the intramonomeric stilbenedisulfonate site and (b) residual, active monomers. All of the residual activity was fully inhibitable by DIDS under conditions of reversible binding, confirming expectations that all of the monomers responsible for the residual activity have patent stilbenedisulfonate sites. However, within this active population, two subpopulations could be identified: (1) monomers which were irreversibly reactive toward DIDS and (2) monomers which were refractory toward irreversible binding of DIDS at pH 6.9, despite being capable of binding DIDS reversibly. Increasing the pH to 9.5 during treatment of DNDS/BS3-modified cells with 300 microM DIDS did not cause increased irreversible transport inhibition relative to that seen for cells treated at pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)