Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385-389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4′-dichloromercuric-2,2,2′,2′-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385–389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.     

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.     

New evidence for the essential role of arginine residues in anion transport across the red blood cell membrane

2,3-Butanedione, in the dark and in the presence of borate, reacts rapidly to inactivate the sulfate equilibrium exchange across the human red cell membrane. Reactivation occurs spontaneously after the removal of borate, indicating the reaction of butanedione with essential arginine residues. The inactivation of the transport system depends on the concentration of the reagent, on the incubation time and exhibits pseudo-first-order kinetics. Chloride ions are able to protect the transport system against inactivation with the reagent. This would suggest the participation of the modified residue in the substrate binding site. When the transport system is inhibited to 50-60% by butanedione, the transporter can still bind covalently the anion transport inhibitor 2H2DIDS up to 85 +/- 12% of its total binding capacity. 3H2DIDS concentration was either 3.15, 10 or 20 microM. Modification of resealed ghosts with 50 mM butanedione under conditions where the transport system is to more than 75% inhibited, causes a reduction of only about 30% of the reversibly bound 3H2DIDS.     

Binding of DTNB to band 3 in the human red cell membrane

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 KI 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.     

Thiamine triphosphatase from Electrophorus electric organ is anion-dependent and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'disulfonic acid

Thiamine triphosphatase (TTPase) from membranes isolated from the main electric organ of E. electricus is activated about 8 fold by NO3-, I- and SCN- while SO42- is inhibitory. Activating anions shift the pH optimum of the enzyme from 5.0 to 8.0. The enzyme is irreversibly inactivated by low concentrations of 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS), an inhibitor of anion transport. Anions protect from DIDS inactivation. These and other results suggest that the membrane-bound TTPase activity is tightly controlled, possibly through mechanisms involving anion transport.     

A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.     

Properties of a new calcium ion antagonist on cellular uptake and mitochondrial efflux of calcium ions.

Compound YS 035 [NN-bis-(3,4-dimethoxyphenethyl)-N-methylamine] is a new synthetic compound capable of inhibiting Ca2+ uptake by different cells. The inhibition of Ca2+ uptake by muscle cells isolated from chicken embryo is dose-dependent in the compound YS 035 concentration range 10-30 microM. The new compound also inhibits Ca2+ entry into rat brain synaptosomes and less effectively into baby-hamster kidney cells. Compound YS 035 partially inhibits the slow Ca2+ release induced by Ruthenium Red and the rapid Na+-dependent efflux from heart mitochondria. The inhibition of the Na+/Ca2+ exchange appears to be of a non-competitive type with an apparent Ki of 28 microM. The new Ca2+ antagonist totally inhibits the Ca2+ efflux from liver mitochondria induced by Ruthenium Red, but it does not affect the release induced by uncoupler, respiratory inhibitor or chelator, nor the mitochondrial ATP synthesis and membrane potential. The properties shown by the new compound indicate it to be a Ca2+ antagonist and a useful tool for studies on the mitochondrial Ca2+ transport.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asymmetry of the red cell anion exchange system. Different mechanisms of reversible inhibition by N-(4-azido-2-nitrophenyl)-2- aminoethylsulfonate (NAP-taurine) at the inside and outside of the membrane

In the dark, the photoaffinity reagent, N-(4-azido-2-nitrophenyl)-2- aminoethylsulfonate (NAP-taurine), acts as a reversible inhibitor of red cell anion exchange when it is present either within the cell or in the external solution. A detailed analysis of the inhibition kinetics, however, reveals substantial differences in the responses to the probe at the two sides of the membrane. On the inside of the cell, NAP- taurine is a relatively low affinity inhibitor of chloride exchange (Ki = 370 microM). Both the effects of chloride on NAP-taurine inhibition and the affinity of NAP-taurine for the system as a substrate are consistent with the concept that internal NAP-taurine competes with chloride for the substrate site of the anion exchange system. External NAP-taurine, on the other hand, is a far more potent inhibitor of chloride exchange (Ki = 20 microM). It acts at a site of considerably lower affinity for chloride than the substrate site, probably the modifier site, at which halide anions are reported to cause a noncompetitive inhibition of chloride transport. NAP-taurine therefore seems to interact preferentially with either the substrate or modifier site of the transport system, depending on the side of the membrane at which it is present. It is suggested that the modifier site is accessible to NAP-taurine only from the outside whereas the transport site may be accessible from either side.     

Control of red cell urea and water permeability by sulfhydryl reagents

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.     

N-(Hydroxyaminocarbonyl)phenylalanine: a novel class of inhibitor for carboxypeptidase A

N-(Hydroxyaminocarbonyl)phenylalanine (1) was designed rationally as a new type of inhibitor for carboxypeptidase A (CPA). The designed inhibitor was readily prepared from phenylalnine benzyl ester in two steps and evaluated to find that rac-1 inhibits CPA in a competitive fashion with the Ki value of 2.09 microM. Surprisingly, inhibitor 1 having the D-configuration is more potent (Ki = 1.54 microM) than its antipode by about 3-fold. A possible explanation for the stereochemistry observed in the inhibition of CPA with 1 is presented.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by stilbene derivatives and the identification of an inhibitor-binding protein

The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H₂DIDS bound to the vesicles was determined by using [³H]-H₂DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 μmol/g protein. SDS-polyacrylamide gel electrophoresis of the vesicles treated with [³H]H₂DIDS showed that about 10% of the total bound H₂DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H₂DIDS-binding protein was about 0.5 μmol/g protein. These results suggest that the H₂DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.     

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS; Ki = 10 microM)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4'-aminostilbene 2,2'-disulfonate (BADS; Ki = 2 microM), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4 degrees C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4 degrees C, the weakly bound form was slowly converted to the strongly bound form. At 37 degrees C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37 degrees C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37 degrees C.     

The amino acid conjugate formed by the interaction of the anion transport inhibitor 4,4′-diisothiocy ano-2,2′- stilbenedisulfonic acid (DIDS) with band 3 protein from human red blood cell membranes

The specific anion transport inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and its reduced analog (H₂DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the ?-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.     

Relationship of net chloride flow across the human erythrocyte membrane to the anion exchange mechanism

The parallel effects of the anion transport inhibitor DIDS (4,4'- diisothiocyanostilbene-2,2'-disulfonate) on net chloride flow and on chloride exchange suggest that a major portion of net chloride flow takes place through the anion exchange system. The "slippage" model postulates that the rate of net anion flow is determined by the movement of the unloaded anion transport site across the membrane. Both the halide selectivity of net anion flow and the dependence of net chloride flux on chloride concentration over the range of 75 to 300 mM are inconsistent with the slippage model. Models in which the divalent form of the anion exchange carrier or water pores mediate net anion flow are also inconsistent with the data. The observations that net chloride flux increases with chloride concentration and that the DIDS- sensitive component tends to saturate suggest a model in which net anion flow involves "transit" of anions through the diffusion barriers in series with the transport site, without any change in transport site conformation such as normally occurs during the anion exchange process. This model is successful in predicting that the anion exchange inhibitor NAP-taurine, which binds to the modifier site and inhibits the conformational change, has less effect on net chloride flow than on chloride exchange.     

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.