Rate Of Anion Exchange Research Articles

Spectroelectrochemical Investigations of Soluble Polyaniline Synthesized via New Inverse Emulsion Pathway

A new inverse emulsion protocol has been developed for the synthesis of polyaniline (PANI), which can be dissolved completely in common organic solvents such as chloroform. Benzoyl peroxide is used as the oxidizing agent and toluene + 2-propanol−water is used as the solvent. Dodecylbenzenesulfonic acid (DBSA) has been selected as the dopant because it also functions as the surfactant. Cyclic voltammetry and spectroelectrochemical investigations were carried out to study the electroactivity, UV−Vis response, and metal-to-insulator transition of the chemically synthesized PANI as a function of applied electrode potential. At more positive potentials, cyclic voltammograms of PANI in aqueous acids show two oxidation waves caused by redox processes of PANI as observed with electrochemically prepared PANI. Peak position and shape are influenced by the slow anion exchange rate. Electrical conductivity of the material is relatively high as the minimum resistance value is nearly 10 Ω. SEM investigations show that ...

Factors Influencing Staging during Anion-Exchange Intercalation into [LiAl2(OH)6]X·mH2O (X = Cl-, Br-, NO3-)

A variety of organic carboxylates and phosphonates have been intercalated into the hexagonal and rhombohedral polymorphs of [LiAl2(OH)6]X·mH2O, with X = Cl- , Br-, and NO3-. The two classes of polytype differ in the stacking sequence of the layers (aba and abca, respectively). Both the initial interlayer anion and the stacking sequence of the host are seen to have a profound effect on whether anion-exchange intercalation reactions occur in a one-step direct transformation from the host to the first-stage intercalated product, or whether the reaction proceeds via a second-stage intermediate. Generally, staging is much more prevalent in the hexagonal system, and with the harder to replace Cl- and Br- anions. Overall, the occurrence of staging appears to be strongly correlated with the rate of anion exchange in these materials.

Lipophilic G-quadruplexes are self-assembled ion pair receptors, and the bound anion modulates the kinetic stability of these complexes.

With an eye toward the eventual selective modification of noncovalent structures, we used ESI-MS, X-ray crystallography, and NMR spectroscopy to study the anion's influence on the structure and dynamics of self-assembled ion pair receptors formed from guanosine G 1. We compared five complexes of formula (G 1)(16).2Ba(2+).4A(-) containing different organic anions: 2,4,6-trinitrophenolate (2), 2,6-dinitrophenolate (3), 4-methyl-2,6-dinitrophenolate (4), 4-methoxy-2,6-dinitrophenolate (5), and 2,5-dinitrophenolate (6). Crystallography reveals that anion-nucleobase hydrogen bond geometry is sensitive to both phenolate basicity and structure. For the 2,6-substituted anions 2-5, progressive shortening of anion-nucleobase hydrogen bonds is correlated with increased phenolate basicity. Lipophilic G-quadruplexes with different anions also have much different kinetic stabilities in CD(2)Cl(2) solution. Proton NMR shows that free 6 exchanges faster with G-quadruplex-bound anion than do the 2,6-dinitrophenolates 2-5. The increased lability of 6 is probably because, unlike the 2,6-dinitrophenolates, this anion cannot effectively chelate separate G(8).M(2+) octamers via anion-nucleobase hydrogen bonds. In addition to these structural effects, the anion's basicity modulates the anion exchange rate between its free and bound states. 2D EXSY NMR shows that 3 and 5 exchange about 7 times slower than the less basic picrate (2). The use of 3, a relatively basic dinitrophenolate that hydrogen bonds with the amino groups of the two "inner" G(4)-quartets, resulted in extraordinary kinetic stabilization of the G-quadruplex in CD(2)Cl(2). Thus, no isomerization product (G 1)(8).Ba(2+).(G 1)(8).Sr(2+).4(3) was observed even 2 months after the separate G-quadruplexes (G 1)(16).2Ba(2+).4(3) and (G 1)(16).2Sr(2+).4(3) were combined in CD(2)Cl(2). In sharp contrast, G-quadruplexes containing the isomeric 6 anion have isomerization half-lives of approximately t(1/2) = 30 min under identical conditions. All the evidence indicates that the structure and electronics of the organic anions, bound to the assembly's periphery, are crucial for controlling the kinetic stability of these cation-filled G-quadruplexes.

Molecular Dynamics Study of the Ionic Liquid 1-n-Butyl-3-methylimidazolium Hexafluorophosphate

We report the results of a molecular dynamics study of the ionic liquid 1-n-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF6], a widely studied ionic liquid. An all-atom force field is developed using a combination of density functional theory calculations and CHARMM 22 parameter values. Molecular dynamics simulations are carried out in the isothermal−isobaric ensemble at three different temperatures. Quantities computed include infrared frequencies, molar volumes, volume expansivities, isothermal compressibililties, self-diffusivities, cation−anion exchange rates, rotational dynamics, and radial distribution functions. Computed thermodynamic properties are in good agreement with available experimental values.

Acute and chronic influence of temperature on red blood cell anion exchange.

Unidirectional (36)Cl(-) efflux via the red blood cell anion exchanger was measured under Cl(-) self-exchange conditions (i.e. no net flow of anions) in rainbow trout Oncorhynchus mykiss and red-eared freshwater turtle Trachemys scripta to examine the effects of acute temperature changes and acclimation temperature on this process. We also evaluated the possible adaptation of anion exchange to different temperature regimes by including our previously published data on other animals. An acute temperature increase caused a significant increase in the rate constant (k) for unidirectional Cl(-) efflux in rainbow trout and freshwater turtle. After 3 weeks of temperature acclimation, 5 degrees C-acclimated rainbow trout showed only marginally higher Cl(-) transport rates than 15 degrees C-acclimated trout when compared at the same temperature. Apparent activation energies for red blood cell Cl(-) exchange in trout and turtle were lower than values reported in endothermic animals. The Q(10) for red blood cell anion exchange was 2.0 in trout and 2.3 in turtle, values close to those for CO(2) excretion, suggesting that, in ectothermic animals, the temperature sensitivity of band-3-mediated anion exchange matches the temperature sensitivity of CO(2) transport (where red blood cell Cl(-)/HCO(3)(-) exchange is a rate-limiting step). In endotherms, such as man and chicken, Q(10) values for red blood cell anion exchange are considerably higher but are no obstacle to CO(2) transport, because body temperature is normally kept constant at values at which anion exchange rates are high. When compared at constant temperature, red blood cell Cl(-) permeability shows large differences among species (trout, carp, eel, cod, turtle, alligator, chicken and man). Cl(-) permeabilities are, however, remarkable similar when compared at preferred body temperatures, suggesting an appropriate evolutionary adaptation of red blood cell anion exchange function to the different thermal niches occupied by animals.

Parametric studies on the performance of mixed-bed ion exchange at ultralow concentrations –1. Multicomponent system–

We experimentally evaluated the effects of multicomponent systems of divalent cations on the performance of mixed-bed ion exchange in the concentration range used for ultrapure water processing. Data were collected in the batch and continuous column systems with Dowex Monosphere resins used commercially for ultrapure water applications. The presence of divalent cations in the systems affects significantly the capacity of the units to remove a univalent cation. However, the anion exchange rate increases slightly in the multicomponent systems. The equilibrium and the breakthrough curves of ions, plotted as the ratio of the effluent to influent concentration versus run time or solution volume passed through the experimental column, give detailed results about the effects of the existence of other ionic species on the fate of each ion in MBIE units. The results of the present study are expected to be used to develop and simulate mathematical models and to understand the performance of mixed-bed ion exchange at ultralow concentrations.

Low pH-induced hemolysis of erythrocytes is related to the entry of the acid into cytosole and oxidative stress on cellular membranes

HCl-induced lysis of mammalian erythrocytes, pretreated with DIDS, which is a specific inhibitor of the anion transport in their membranes, was markedly delayed. After acidification of a suspension of DIDS-inhibited cells, hemolysis was initiated by addition of a protonophore (Na-salicylate) at any moment chosen by will. These findings revealed that low-pH hemolysis depended on the rate of the transfer of acid equivalents into cytosole. Erythrocyte acid resistance was studied in a group of mammals and found to be inversely related to the rate of monovalent anion exchange in membranes which supported the above observations. In human erythrocytes, the critical level of cytosole acidification was found to be about pH 5.7 by measuring the acid equivalent absorbed by cells prior to hemolysis. HCl-induced hemolysis was also studied in human erythrocyte ghosts resealed with one-sixth of the initial hemoglobin content of cells. During the prelytic interval the ghosts suspended in isotonic NaCl/sucrose media shrunk, indicating an increase in ion permeability. The increase in prelytic permeability and hemolysis were strongly delayed in ghosts prepared from DIDS-treated cells, suggesting a uniform mechanism of lysing in cells and their ghosts. The prelytic increase in ion permeability was measured by the corresponding rate of ghost shrinkage and was found to be pH-dependent, with a high value below pH 3.4 and a very low one above pH 4.0. Compared to cells, the prelytic barrier impairment in ghosts had more mild character although it required greater concentration of cytosolic H +. While finally complete, hemolysis of cells was strongly delayed in the presence of catalase (500–1500 U/ml) and superoxide dismutase (200–600 U/ml) in hemolytic media. In conclusion, the acid-induced hemolysis could be associated with an oxidative injury of membranes, mainly triggered by the entry of acid equivalents into the cytosole.

Enhanced Oxalate Transport in Nucleated Red Blood Cells

Objective: This study was aimed to determine the presence of oxalate exchange/binding protein in the nucleated red blood cells (RBC). Materials and Methods: Intact RBC, nuclei, RBC membrane, nuclear membrane and histones were isolated and ¹⁴C-oxalate exchange/binding was studied. Results: The rate of oxalate anion exchange by nucleated erythrocytes was found to be higher (K = 1.24/min) than that of human RBC (K = 0.51/min) but less than that of nuclei (K = 1.55/min). Oxalate flux rate was pH- and temperature-dependent. Oxalate exchange was reduced by 35% for both chicken RBC and nuclei in presence of sulfate, bicarbonate, phosphate and succinate. Both plasma membrane and nuclear membrane showed maximal oxalate binding activity at pH 7.4, while in the nuclear basic protein fraction it was at pH 4.5. 4,4′-Diisothiocyanostilbene-2,2′-disulfonate, an anion transport inhibitor, inhibited the oxalate exchange and binding activity in both erythrocytes as well as nucleus. Conclusion: Nucleated RBC can be useful model system for studying the interaction of oxalate with nuclear components.

The mixed-bed ion exchange performance at ultralow concentrations 1. Variable feed concentration and incomplete mixing of resins

Experimental data were obtained to evaluate the performance of mixed-bed ion exchange for the cases of variable feed concentration and incomplete mixing of anion and cation resins observed in large scale industrial units. For variable feed concentration, step changes in feed concentration were arbitrarily introduced into a test column. For incomplete mixing, only anion resin was loaded in the upper 20% of the column and more cation resin in the lower portion. Feed concentrations of 5.0× 10−5−2.0×10-4 M NaCl were used for the experiments, with flow rates of 0.665-7.0 ml/sec. The effluent from the column was collected periodically and analyzed using on-line/off-line ion chromatography. The step changes in feed concentration affect the breakthrough times of sodium and chloride. Sodium breakthrough curve is more sensitive to the step changes than chloride breakthrough curve. With the same volumes of cation and anion resins, incomplete mixing of resins increases the cation exchange rate slightly and decreases the anion exchange rate slightly. As the cation resin volume increases, the effect of the incomplete mixing of resins decreases. The breakthrough curves of both ions, plotted as the ratio of effluent to the influent concentration versus run time in hour, give some detailed results about the effects of the conditions.

Experimental determination of F-OH interdiffusion in tremolite and significance to fluorine-zoned amphiboles

A hydrothermal-analytical procedure has been developed that allows for the quantitative study of anion exchange rates in hydrous silicates. This new procedure has provided the first opportunity to quantitatively investigate the kinetics of halogen-OH exchange reactions at subsolidus conditions. Handpicked, chemically homogeneous tremolite crystals were exposed to either F-rich or F-poor fluids at temperatures ranging from 500–800°C at 200 MPa (2000 bars), and the resulting concentration profiles were measured with either an electron microprobe or an ion microprobe. Critical to this procedure is the use of an aqueous silica-buffering assemblage (either quartz or forsterite + diopside + tremolite) in order to maintain the chemical and physical stability of the exposed crystal surfaces. Scanning electron microscope (SEM) photomicrographs taken before and after hydrothermal treatment show no evidence of dissolution reactions, consistent with a diffusion-controlled exchange process. For temperatures between 500 and 800°C, the Arrhenius relations for diffusion parallel to the c-crystallographic direction have an activation energy (Q) = 41 (±5) kJ/mole and (D0) = 3.4(−1.9+4.1) × 10−17 m2/s. Transport rates parallel and perpendicular to the c-axis are the same within the estimated uncertainties. Calculations indicate that F metasomatism by volume diffusion in amphiboles is relatively slow and may only play an important role in the evolution of a given hydrothermal system in the absence of faster processes such as chemical reaction. Knowledge of the Arrhenius relationships for F-OH interdiffusion allows one to extract information about the maximum duration of metasomatic or thermal events from F-zoned amphiboles. This procedure can be adapted to the study of other halogen-OH exchange reactions provided that the mineral stability is controlled by the dissolution of silica.

The Role of Cysteine Residues in the Erythrocyte Plasma Membrane Anion Exchange Protein, AE1

AE1 (Band 3), a congruent to 110-kDa integral plasma membrane protein, facilitates the electroneutral movement of Cl- and HCO3- across the erythrocyte membrane and serves as the primary attachment site for the erythrocyte spectrin-actin cytoskeleton. In this investigation, we have characterized the role of native cysteines in the function of AE1. We have constructed a mutant version of human AE1 (AE1C-) in which all five cysteines of AE1 were replaced with serines. Wild-type and AE1C- cDNAs were expressed by transient transfection of human embryonic kidney cells. Two of the mutated cysteines in AE1C- are in a region involved in ankyrin binding, and ankyrin binding has previously been shown to be sensitive to the oxidation state of these cysteines. However, the KD values for ankyrin binding by AE1 and AE1C- were indistinguishable, suggesting that AE1 cysteines are not essential components of the ankyrin-binding site. Using size exclusion chromatography, both AE1 and AE1C- were found to associate as a mixture of dimers and high molecular mass complexes. The rate of anion exchange by AE1C-, as measured in a reconstituted microsome sulfate transport assay, was indistinguishable from that by AE1 and was inhibited by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate. We conclude that the cysteines of AE1 are not required for the anion exchange or cytoskeletal binding roles of the protein.

Different regulation by pHi and osmolarity of the rabbit ileum brush‐border and parietal cell basolateral anion exchanger.

1. The purpose of this study was to look for evidence of a pH-sensitive modifier site on the parietal cell basolateral anion exchanger, determine the pH range in which allosteric regulation takes place, investigate the effect of the osmolarity on internal pH (pHi) dependence and compare it with that of the ileum brush-border anion exchanger. 2. When the pHi in parietal cell basolateral membrane (BLM) vesicles was increased, the rate of Cl(-)-gradient-driven 36Cl- uptake increased from 6.03 +/- 2.24 to 38.09 +/- 3.33 nmol (mg protein)-1 with the steep increase in anion exchange rates occurring within a narrow pH range between pHi 7.0 and 7.5. This was due to allosteric activation by internal OH- and not due to a change in driving force, since the driving force for maximal exchange rates was provided by the outwardly directed Cl- gradient. 3. The pHi dependency curve of parietal cell BLM anion exchange rates was shifted to the left by 0.25 pH units by increasing the osmolarity of the intra- and extravesicular solutions from 300 to 380 mosmol l-1. Thus cell shrinking may activate the parietal cell anion exchanger without a change in pHi and without phosphorylation of the anion exchanger protein. 4. In ileum brush-border membranes, the pHi-dependent increase in the rate of Cl(-)-gradient-driven 36Cl- uptake was more gradual and the half-maximal anion exchange rate was attained at lower pHi (pH 6.5). Increasing the osmolarity from 300 to 500 mosmol l-1 had no effect on pH dependence. 5. We conclude that the parietal cell basolateral and ileum brush-border anion exchangers possess an internal modifier site for allosteric activation by OH-, but the pH range in which allosteric regulation occurs differs between the two exchangers, as does the effect of an increase in osmolarity. Since current evidence suggests that both the parietal cell basolateral and the ileum brush-border anion exchanger are encoded by the AE2 gene, the differences in pHi dependence between the two may be due to alternative splicing, post-transcriptional modification, or the different membrane environment. 6. The pHi range for allosteric activation found in this study would suggest that for both the ileum and the parietal cell anion exchanger, but especially for the latter, a potentiating effect of the allosteric activation and the HCO3- availability occurs within the physiological pHi range and can cause dramatic increases in maximal anion exchange rates with increasing pHi.

Mechanism of Control of Erythrocyte Shape: A Possible Relationship to Band 3

A mechanism of control of erythrocyte shape, differing significantly from those previously proposed, is hypothesized. It was inferred from previous observations on the states of association of Band 3, the anion exchange protein. Its salient points are as follows. (i) The membrane skeleton is a pliable protein meshwork structure used to generate different erythrocyte shapes. (ii) Band 3 is a dodecamer bound by one of its subunits to ankyrin which is itself bound to the mid region of filamentous spectrin. (iii) Spectrin, by virtue of its high flexibility, folds and unfolds and its folding links the two actin protofilaments bound at its ends to Band 3 through the intermediary of Band 4.1 and glycophorin A. (iv) The alternate influx and efflux of anions mediated by Band 3 respectively folds and unfolds spectrin. (v) The ratio of Band 3 molecules with outward-facing conformation to those with inward-facing conformation controls the extents of folding of the skeleton spectrin molecules. This ratio is governed by the Gibbs-Donnan equilibrium ratio of anions and protons and by the Band 3 anion affinities and exchange rates.

PHi and HCO3- dependence of proton extrusion and Cl(-)-base exchange rates in isolated rabbit parietal cells.

In many cell types, the regulation of intracellular pH (pHi) is different in the presence vs. absence of HCO3-. We investigated the pHi and HCO3- dependence of proton extrusion and anion exchange rates in isolated rabbit parietal cells loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In Cl(-)-depleted parietal cells, the dependence of maximal proton efflux rate on pHi showed a strong inverse correlation but was identical in the presence and absence of HCO3-. Amiloride and Na+ removal inhibited proton extrusion rates to a similar degree with or without HCO3-, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) had no effect. This suggests that a Na(+)-H+ exchanger is the major acid extrusion ion transporter under these experimental conditions. In Cl(-)-containing cells, there was also some Na(+)-independent, extracellular HCO(3-)- and intracellular Cl(-)-dependent, DIDS-inhibitable pHi recovery from an acid load, most likely due to intracellular Cl(-)-extracellular HCO3- exchange. Recovery from an alkaline load was primarily mediated by anion exchange, and the dependence of maximal anion exchange rates on pHi was very different in the absence and presence of HCO3-. In its absence, maximal anion exchange (Cl(-)-OH-) rates increased slowly over the tested pHi range from 6.4 to 7.8. In the presence of HCO3-, however, there was an S-shaped dependence of maximal flux rates on pHi, with a steep increase in flux rates between 6.8 and 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Basolateral Na(+)-independent Cl(-)-HCO3- exchange in primary cultures of rat IMCD cells.

The role of anion exchange in the regulation of intracellular pH (pHi) under base load and steady-state conditions was investigated in confluent monolayers of rat inner medullary collecting duct (IMCD) cells in primary culture using the pH-sensitive fluoroprobe 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF). Recovery of pHi after imposition of a base load induced either by replacement of HCO3-/CO2 by N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at the same extracellular pH (pHo) or deletion of Cl- from a HCO3-/CO2-buffered solution had an absolute requirement for Cl-, was Na+ independent, and was inhibited approximately 90% by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). When pHo was decreased by lowering HCO3- concentration in the constant presence of 5% CO2, the rate of decrement in pHi was significantly blunted in the absence of Cl-. Imposition of a positive or negative diffusion potential of equal but opposite magnitude did not modify the anion exchange rate, confirming the electroneutrality of the process. Under steady-state conditions, pHi of cells bathed in a HCO3-/CO2-buffered solution was 7.33 +/- 0.06, significantly lower than that of cells bathed in a nominally HCO3-/CO2-free buffer (7.50 +/- 0.04), indicating that under physiological conditions the pathway functions as a base extruder. In studies performed on cells grown on permeable supports, the anion exchange pathway was found to be confined exclusively to the basolateral-equivalent cell surface. In summary, confluent monolayers of rat IMCD cells in primary culture possess a Na(+)-independent, DIDS-inhibitable electroneutral Cl(-)-HCO3- exchange pathway that is confined to the basolateral cell surface. The transporter is an important determinant of steady-state pHi and is the predominant mechanism whereby the cell recovers from imposed elevations in pHi.

Internal alkalinization by reversal of anion exchange in human neutrophils: regulation of transport by pH

When steady-state human neutrophils bathed in 148 mM Cl- are transferred to a Cl(-)-free medium containing 0.5 mM HCO3- and 148 mM glucuronate or aspartate as nominally inert replacement ions, there is a rapid efflux of 36Cl- from the cells. The accelerated loss of Cl- is accompanied by an intracellular alkalinization of 0.7-0.9 pH units. Both the Cl- efflux and intracellular pH (pHi) transient are dependent on extracellular HCO3- and are sensitive to inhibition by SITS and alpha-cyano-4-hydroxycinnamate, which block anion exchange, thereby indicating that these processes are due to the countertransport of internal Cl- for external HCO3-. Rate of anion exchange is strongly influenced by pH, which functions to regulate carrier activity; alkalinization stimulates the transport velocity, whereas acidification inhibits it. The relationship to pHi follows a Hill equation with pK' approximately 7.40 and Hill coefficient of 3.3, thereby suggesting that approximately 3 HCO3- may be required to bind to the modifier site. Neutrophils placed in glucuronate medium progressively shrink during the first 7.5 min of incubation due to the net loss of osmotically active particles through Cl(-)-HCO3- exchange. However, between 7.5 and 30 min, cells regain their normal cell size. This volume recovery phase correlates with the time course of 22Na+ and [14C]glucuronate influxes, whose kinetics can be dissociated from that of anion exchange. Uptake of glucuronate is largely Na+ dependent (whereas Cl(-)-HCO3- exchange is not), is resistant to amiloride, and can be blocked by furosemide, which suggests that glucuronate probably enters via a volume-activated pathway such as Na+ + glucuronate cotransport.

Ion exchange kinetics in ultra pure water systems

AbstractThe continuous production of ultra pure water on an industrial scale uses ion exchange mixed beds as a major method of reducing ionic impurities to sub‐μg dm−3 concentrations. Under such demanding operating conditions, particularly the high flow rate and high volume requirements of condensate purification in the power generation industry, it is the kinetics rather than the equilibria of the exchange processes that becomes the main controlling factor. The interactions between anion and cation exchange rates have been studied, particularly with respect to changes in the aqueous phase pH within a bed of exchange resins. The cause and effects of cation exchange resin fouling, both in laboratory tests and in practical applications, have been investigated. The most suitable choice of ion exchange system for ultra pure water production, whether mixed or separate beds, is discussed.

Phosphorylation sites in human erythrocyte band 3 protein

The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253–260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750–2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [γ- 32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were bound on the cytoplasmic domain, RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1–56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn 2+ but also by Mg 2+. Tyrosine-8 was the main phosphate acceptor residue (50–70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg 2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was copurified with whole band 3 protein which dephosphorylates specifically P-Tyr-8, indicating a highly exchangeable phosphate. The membrane-spanning fragment was only faintly labeled.

Internal pH-sensitive site couples Cl-(-)HCO3- exchange to Na+-H+ antiport in lymphocytes

Parallel exchange of Na+-H+ and Cl-(-)HCO3- is thought to be central to the translocation of electrolytes and water during cell volume regulation and in transepithelial transport. Coupling between these transporters is thought to be indirect, through changes in the concentration of HCO3-, which result from alterations in cytosolic pH (pHi). The possibility of a more direct, HCO3-(-)independent interaction between Cl-(-)HCO3- exchange and the Na+-H+ antiport was studied in rat thymic lymphocytes. Measurements of radioactive Cl- flux and of HCO3-(-)dependent pHi changes demonstrated the presence of a Na+-independent, 4,4'diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl-(-)HCO3- exchanger. At constant external pH, the rate of Cl-(-)HCO3- exchange was markedly accelerated by increasing pHi between 7.0 and 7.4. This activation was not related to variations in the concentration of HCO3- and is likely caused by a direct effect of intracellular H+ (OH-) on the exchanger. Osmotic shrinking of the cells induced a cytoplasmic alkalinization, due to activation of the Na+-H+ antiport. Concomitantly, the rate of anion exchange also increased. The stimulation of Cl-(-)HCO3- exchange was eliminated when the alkalinization caused by Na+-H+ exchange was precluded. These observations suggest that the exquisite pHi sensitivity of the Cl-(-)HCO3- exchange system provides a mechanism whereby the rates of cation and anion transport are closely coupled.

Catecholamine-induced transport systems in trout erythrocyte. Na+/H+ countertransport or NaCl cotransport?

It has previously been shown (Baroin, A., F. Garcia-Romeu, T. Lamarre, and R. Motais. 1984a, b. Journal of Physiology. 350:137, 356:21; Mahé, Y., F. Garcia-Romeu, and R. Motais. 1985. European Journal of Pharmacology. 116:199) that the addition of catecholamines to an isotonic suspension of nucleated red blood cells of the rainbow trout first stimulates a cAMP-dependent, amiloride-sensitive Na+/H+ exchange. This stimulation seems to be transient. It is followed by a more permanent activation of a coupled entry of Na+ and Cl-, which is inhibited by amiloride but also by inhibitors of band 3 protein (DIDS, furosemide, niflumic acid). The coupled entry of Na+ and Cl- could therefore result from the parallel and simultaneous exchange of Na+out for H+in (via the cAMP-dependent Na+/H+ antiporter) and Cl- out for HCO3- in (via the anion exchange system located in band 3 protein). However, in view of the following arguments, it had been proposed that NaCl uptake does not proceed by the double-exchanger system but via an NaCl cotransport: (a) Na+ entry requires Cl- as anion (in NO3- medium, the Na uptake is strongly inhibited, whereas NO3- is an extremely effective substitute for Cl- in the anion exchange system); (b) Na uptake is not significantly affected by the presence of HCO3- in the suspension medium despite the fact that in red cells, Cl-/HCO3- exchange occurs more readily than the exchanges of Cl- for basic equivalents in a theoretically CO2-free medium (the so-called Cl-/OH- exchanges). The purpose of the present paper was a reassessment of the two models by using monensin, an ionophore allowing Na+/H+ exchange. From this study, it appears that NaCl entry results from the simultaneous functioning of the Na+/H+ antiporter and the anion exchange system. The apparent Cl dependence is explained by the fact that, in these erythrocytes, NO3- clearly inhibits the turnover rate of the Na+/H+ antiporter. As Na+/H+ exchange is the driving component in the salt uptake process, this inhibition explains the Cl requirement for Na entry. The lack of stimulation of cell swelling by bicarbonate is explained by the fact that the rate of anion exchange in a CO2-free medium (Cl-/OH- exchange) is roughly equivalent to that of Na+/H+ exchange and thus in practice is not limiting to the net influx of NaCl through the two exchangers.(ABSTRACT TRUNCATED AT 400 WORDS)