Sulfate Equilibrium Exchange Research Articles

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.

Comparison of the Action of an Organophosphorus Insecticide and Its Metabolite on Chloride and Sulfate Transport in Erythrocyte Membrane

The effect of the organophosphorus insecticide methylparathion and its main metabolite methylparaoxon on chloride and sulfate equilibrium exchange in pig erythrocytes was investigated using an isotope labelling technique. Efflux of both radioactive isotopes with time followed a single exponential. Methylparathion and methylparaoxon inhibited the chloride equilibrium exchange in erythrocyte ghosts in a dose- and time-dependent manner. There was no difference between effects evoked by these two compounds. Methylparathion and methylparaoxon inhibited sulfate efflux from resealed ghosts. The effect was also dose- and time-dependent. Again, there was no difference between the action of both agents. Dixon analysis revealed a non-competitive character of the inhibition of the exchange of both anions with apparent Ki values 183 and 184 microM for methylparathion and methylparaoxon, respectively in the case of chloride transport; for sulfate exchange these values were 675 and 648 microM. It was suggested that structural similarity between the parent agent and its metabolite accounts for their identical effects. Methylparathion and methylparaoxon might inhibit the anion exchange indirectly by changing the fluidity of the erythrocyte membrane or directly by binding to the band 3 protein and evoking conformational changes that lead to the inhibition of the anion transport. The insecticides, due to their ability to phosphorylate, might also disturb some regulation processes in the band 3 protein and affect anion transport in this way.

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 2H 2DIDS up to 85 ± 12% of its total binding capacity. 3H 2DIDS concentration was either 3.15, 10 or 20 μM. 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 3H 2DIDS.

Anion transport in red blood cells and arginine-specific reagents. Interaction between the substrate-binding site and the binding site of arginine-specific reagents

Phenylglyoxal is found to be a potent inhibitor of sulfate equilibrium exchange across the red blood cell membrane at both pH 7.4 and 8.0. The inactivation exhibits pseudo-first-order kinetics with a reaction order close to one at both pH 7.4 and 8. The rate constant of inactivation at 37°C was found to be 0.12 min −1 at pH 7.4 and 0.19 min −1 at pH 8.0. Saturation kinetics are observed if the pseudo-first order rate constant of inhibition is measured as a function of phenylglyoxal concentration. Sulfate ions as well as chloride ions markedly decrease the rate of inactivation by phenylglyoxal at pH 7.4, suggesting that the modification occurs at or near to the binding site for chloride and sulfate. The decrease of the rate of inactivation produced at pH 8.0 by chloride ions is much higher than that produced by sulfate ions. Kinetic analysis of the protection experiments showed that the loaded transport site is unable to react with phenylglyoxal. From the data it is concluded that the modified amino acid(s) residues, presumably arginine, is (are) important for the binding of the substrate anion.

Anion exchange in human erythrocytes has a large activation volume

Sulfate equilibrium exchange in human red cells has an activation volume of +150±20 cm 3/mol over the pressure range 0.1 to 83 MPa (15 to 12000 lb/in 2) at 30°C. This value greatly exceeds the expected contribution from sulfate binding to the anion exchanger. We suggest that the activation volume reflects conformational changes during the transport cycle.

Anion transport in red blood cells and arginine-specific reagents: The location of [ 14C]Phenylglyoxal binding sites in the anion transport protein in the membrane of human red cells

The reaction of phenylglyoxal, a reagent specific for arginine residues, with erythrocyte membrane at pH 7.4 results in complete inhibition of sulfate equilibrium exchange across human red cells. The inactivation was found to be concentration and time depenent. The binding sites of this reagent in the anion transport protein (band 3) under these conditions were determined by using [ 14C]phenylglyoxal. The rate of incorporation of the radioactivity into band 3 gave a good correlation with the rate of inactivation. Under conditions where the transport is completely inhibited about 6 mol [ 14C]phenylglyoxal are incorporated into 1 mol band 3. Treating the [ 14C]phenylglyoxalated ghosts at different degrees of inactivation with extracellular chymotrypsin showed that about two-thirds of these binding sites are located on the 60 kDa fragment.

Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts

Varying concentrations of trypsin were sealed into human red cell ghosts and the effects on membrane proteins and sulfate equilibrium exchange were studied. After incubation for 45 min at 37 °C, pH 7.2, the following observations were made: above 10 ng/ml the ghosts undergo fragmentation without lysis. Dodecyl sulfate gel electrophoresis shows that the digestion of spectrin and of the protein in band 2.1 (nomenclature of Steck (1974) J. Cell. Biol. 62, 1–19) is nearly complete at 50 ng/ml, that of the protein in band 3 at 25 μg/ml. After digestion at 25 μg/ml, about 60% of the total protein of the membrane is released and the original bands of conventional dodecyl sulfate gel electropherograms of the remaining protein are nearly completely abolished. In their place three new bands appear representing peptides with molecular weights of 58 000, 48 000 and 34 000, respectively. Sometimes a fourt peptide with a molecular weight of approx. 13 000 is also observed. Using a radioactive labeling technique it is shown that the two peptides with the highest molecular weights are derived from the protein in band 3. Labeling with diazo[ 3 5S]sulfanilic acid indicates that in addition to the peptides in the described four Coomassie blue-stainable bands, other peptides with molecular weights up to 100 000 are still present in the exhaustively trypsinized ghosts. External trypsin has no effect on the sulfate equilibrium exchange in ghosts while internal trypsin causes inhibition. Inhibition becomes apparent at trypsin concentrations exceeding those required to produce a complete digestion of spectrin. It remains incomplete, even at the highest intracellular concentrations which cause maximal effects on all membrane proteins, including the protein in band 3. Under these conditions strong further inhibition can be produced by agents which are known to inhibit anion transport in untreated red cells and ghosts. These agents include the penetrating 1-fluoro-2,4-dinitrobenzene and the nonpenetrating phlorizin, 4-acetamido-4′-isothiocyanato stilbene-2,2′-disulfonic acid, 4,4′-diacetamido stilbene-2,2′-disulfonic acid, and 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB). Unlike the other nonpenetrating inhibitors APMB is not only capable of inhibiting at the outer but also at the inner membrane surface. Treatment with internal trypsin does not significantly reduce the inhibition by incorporated APMB. The described observations suggest that after exhaustive tryptic digestion of the major membrane proteins, the receptor sites for typical inhibitors of anion transport continue to exert their function.

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS).

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

Photo-activated inhibition of sulfate equilibrium exchange in human erythrocyte ghosts by A 4-azido-2-nitrobenzoate derivative of phlorizin

Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO 4 2− equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO 4 2− exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular triatated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccesibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.

Photo-activated inhibition of sulfate equilibrium exchange in human erythrocyte ghosts by a 4-azido-2-nitrobenzoate derivative of phlorizin

Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO 4 2− equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO 4 2− exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular tritiated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccessibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.

Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells.

Mono-, di-, and trisulfonic acids, including 4,4'-diacetamido stilbene-2,2'-disulfonic acid (DAS) and 2-(4'-amino phenyl)-6-methylbenzene thiazol-3',7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluorobenzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, '74). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements.

Effect of temperature on sulfate movements across chemically or enzymatically modified membranes of human red blood cells

The effect of temperature on sulfate equilibrium exchange was measured in cells which were modified by papain, pronase, salicylate, or i-fluoro-2,4-dinitrobenzene (FDNB). Following modification at 37°C with the enzymes of FDNB the excess modifiers were removed and sulfate exchange was measured at 4°C and 34°C. The reversibly bound salicylate was also present during the exchange. With increasing concentrations of salicylate of papain, SO 4 2−-permeability shows a saturation type decrease at both temperatures. The apparent activation enthalpy ( E A) does not change significantly. After treatment with FDNB or pronase, saturation type inhibition is observed at 37°C. If measured at 4 °C, sufficiently high concentrations of both modifiers accelerate SO 4 2− transfer. At pH 7.2, E A decreases from 33 to 10 kcal per mole per °K. If measured at 37 °C, in FDNB or pronase treated cells the rate of anion movement decreases with increasing pH. If measured at 4°C, the pH dependence is reversed. E A is correspondingly lowered with increasing pH down to 6.4 kcal per mole per °K at pH 8.3. In contrast, the pH dependence of untreated cells is generally similar at both temperatures. At 37 °C a maximum is observed at pH 6.2. This is shifted to pH 5.8 at 4 °C. Variations of E A with pH are relatively small over the range pH 5.8 to 8.0. A study of the effects of purified pronase subfractions showed that neither aminopeptidase nor carboxypeptidase activity is responsible for the change of E A. Among the endopeptidases those with activity towards casein and the ester p-nitrophenyl acetate were found to be particularly potent.