Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS)), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.     

Anion transport inhibitor binding to band 3 in red blood cell membranes

The inhibitor of anion exchange 4,4''-dibenzoamido-2,2''-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.     

Anion transport across the red blood cell membrane and the protein in band 3.

The paper reviews existing evidence for the participation of the protein in band 3 (nomenclature of Steck, [1]) in anion transport across the red cell membrane and discusses the possible role of common binding sites on band 3 for 1-fluoro-2,4-dinitrobenzene, 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid and dihydro 4,4'-diisothiocyanato stilbene-2,2'-disulfonic acid in the transport process.     

The red cell band 3 protein: its role in anion transport

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60% of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.     

An immunological study of band 3, the anion transport protein of the human red blood cell membrane

Band 3, the predominant membrane-spanning polypeptide and purported anion transport protein of human red cells, was isolated by a new procedure which utilized selective solubilization and anion exchange chromatography on Affi-Gel 102 in 0.5% and Triton X-100/0.03% sodium dodecyl sulfate. Rabbit anti-serum prepared against the purified protein reacted with human and monkey band 3 but gave no immunoprecipitate with membrane proteins from several non-primate species. The antiserum was directed solely towards a portion of the cytoplasmic pole of the band 3 polypeptide contained within a 23,000 dalton amino-terminal fragment, as shown by agglutination, absorption, double diffusion and immunoprecipitation techniques. Saturation of both surfaces of resealed erythrocyte ghosts with the anti-band 3 antiserum had no significant effect on chloride transport. Our data define the topographically-limited immunogenicity of human band 3 in rabbits, demonstrate a lack of immunological cross-reactivity of band 3 between primates and non-primates, and support the hypothesis that the cytoplasmic domain of band 3 is not intimately involved in anion transport.     

Inhibition of inorganic anion transport across the human red blood cell membrane by chloride-dependent association of dipyridamole with a stilbene disulfonate binding site on the band 3 protein

The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding.     

Interaction of thiourea with band 3 in human red cell membranes

Summary Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent,pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore,pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence ofpCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea,pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved.     

Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.     

Effect of pCMBS on anion transport in human red cell membranes.

The kinetics of binding of the mercurial sulfhydryl reagent, pCMBS (p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea transport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl- transport, we measured Cl-/NO3- exchange by fluorescence enhancement, using the dye SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase, correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl- binding to band 3 by the pCMBS anion. The ID50 of 2.0 +/- 0.1 mM agrees with other organic sulfonates, but is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracellular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with Ki = 5.5 +/- 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with Ki = 2.1 +/- 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to the cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.     

Some aspects of zinc transport across eel (Anguilla anguilla) red blood cell membranes.

Zinc movement across eel and human red blood cell membranes was measured by atomic absorption spectrophotometry. It was observed that:

• 1) In human red blood cells zinc uptake is twice as rapid as in fish red blood cells over a temperature range of 10-40°C. The low rate of zinc uptake in eel red blood cell may be simply the side effect of different surface area to volume ratios for the differences in cell size or, it may be due to the low permeability of bicarbonate through the red blood cell membranes.
• 2) Zinc uptake measured in eel and human red blood cells treated and untreated with external trypsin shows different features. The zinc uptake was reduced by about 40% in treated eel red blood cells with respect to the total uptake of untreated red blood cells. Human red blood cells treated and untreated with trypsin do not show any differences in the amount of zinc transported.
• 3) In fish red blood cells, zinc uptake in NANO₃ medium is markedly reduced, compared with that measured in NaCl medium. The [Zn²⁺_i slightly increases in the presence of bicarbonate. In human red blood cells in NANO₃ medium the zinc uptake is strongly reduced and the presence of bicarbonate marginally increases the zinc influx.
• 4) In eel red blood cells there seem to be two independent pathways for zinc uptake: one DIDS-sensitive and the other DIDS-insensitive. DIDS 10 μM inhibits only 64% of the total zinc transported. Iincreasing the DIDS concentration did not give more inhibition. In human red blood cells only one DIDS-sensitive pathway for zinc transported seems to exist, because, 2,5 μM DIDS inhibits 97% of zinc uptake.

     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO₄²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane

The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000, 4000 and 4000 daltons by CNBr. The C-terminus of the segment is located in the 7000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by $\text{N-}\text{bromosuccinimide}$ into two fragments of 2000 daltons. The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.     

Chemical properties of the anion transport inhibitory binding site of arginine-specific reagents in human red blood cell membranes

A series of arginine-specific reagents with different size and polarity have been synthesized and their inhibitory potency on sulfate exchange in resealed ghosts has been investigated. The synthesized phenylglyoxal derivatives p-nitro-, p-methyl-, p-hydroxy-, p-carboxy-, p-sulfo-, and p-azido-phenylglyoxal are found to be potent inhibitors of anion transport. The reaction between the cells and azidophenylglyoxal was performed in the dark. Exposure of the modified cells to the light was not followed by an increase in the inhibition. No cross-linking products were visible after gel electrophoresis. The rate of inactivation of sulfate flux with these reagents obeyed pseudo-first-order kinetics and increases with increasing reagents concentration and pH. Prolonged incubation of the cells with these reagents results in almost complete inhibition of the transport system. The positively charged phenylglyoxal derivative 4-(trimethylammonioacetylamido)phenylglyoxal was not able to inhibit the transport system. The hydrophobic character and the electronic properties of these reagents do not correlate with their inhibitory potency. Their electrostatic and steric effects seem to play the major role in their action.     

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.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.