Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate.

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents. Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a cross-linking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport, Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the noninhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control anion transport.     

Anion transport across the erythrocyte membrane,in situ proteolysis of band 3 protein,and cross-linking of proteolytic fragments by 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonate

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents.Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a crosslinking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport.Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the non-inhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control of anion transport.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

Anion transport in relation to proteolytic dissection of band 3 protein

Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55 000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.     

Effect of stilbenedisulfonate binding on the state of association of the membrane-spanning domain of band 3 from bovine erythrocyte membrane

The membrane-spanning domain of bovine band 3, the anion transport protein of erythrocyte membrane, was purified in the presence of nonaethyleneglycol lauryl ether (C12E9) and the effect of a covalent attachment of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a potent transport inhibitor, on the state of association of the domain isolated (the 58 kDa fragment) was studied via gel filtration, gel electrophoresis and sedimentation velocity experiments. It was indicated that the DIDS-unlabeled fragment in C12E9 solution forms heterogeneous aggregates which are larger in size than the dimer. This contrasted with the behavior that bovine band 3 is present as dimers or tetramers in the same medium (Nakashima and Makino (1980) J. Biochem. 88, 933-947). When DIDS was covalently attached, the fragment was present as a single molecular species which was indicated to be a dimer by molecular weight determination. The secondary structure of the fragment was not affected by DIDS. The change in the state of association caused by the DIDS-binding was also found in the presence of sucrose monolaurate (SE12), which was a more potent detergent for extraction of the 58 kDa fragment from membranes than C12E9. However, the complex with SE12 was extremely unstable.     

Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal: arg stoichiometry of 2:1. Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were above 10. Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments. The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 10 micrometers, only 50% of the band 3 molecules were labeled with [3H]-DIDS during 90 min at 38 degrees C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules.     

The minimal structure containing the band 3 anion transport site. A 35Cl NMR study

35Cl NMR, which enables observation of chloride binding to the anion transport site on band 3, is used in the present study to determine the minimal structure containing the intact transport site. Removal of cytoskeletal and other nonintegral membrane proteins, or removal of the 40-kDa cytoskeletal domain of band 3, each leave the transport site intact. Similarly, cleavage of the 52-kDa transport domain into 17- and 35-kDa fragments by chymotrypsin leaves the transport site intact. Extensive proteolysis by papain reduces the integral red cell membrane proteins to their transmembrane segments. Papain treatment removes approximately 60% of the extramembrane portion of the transport domain and produces small fragments primarily in the range 3-7 kDa, with 5 kDa being most predominant. Papain treatment damages, but does not destroy, chloride binding to the transport site; thus, the minimal structure containing the transport site is composed solely of transmembrane segments. In short, the results are completely consistent with a picture in which the transport site is buried in the membrane where it is protected from proteolysis; the transmembrane segments that surround the transport site are held together by strong attractive forces within the bilayer; and the transport site is accessed by solution chloride via an anion channel leading from the transport site to the solution.     

Analysis of integral membrane protein contributions to the deformability and stability of the human erythrocyte membrane

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.     

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.     

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.     

Inhibition of anion transport in the red blood cell membrane by anionic and non-anionic arginine-specific reagents

Arginine specific reagents are found to be powerful inhibitors of anion exchange in the red blood cell membrane. Some of these inhibitors such as cyclohexandione, phenylglyoxal and 2, 3-butandione are found to produce their inhibition by interacting covalently with band 3. In contrast to the action of these compounds, the inhibition caused by the phenylglyoxal derivative 4-hydroxy-3-nitrophenyl-glyoxal has been found to be completly reversible. In extending the studies on the mode of action of these compounds on sulfate exchange and to get some more information about their binding site, the degree of inhibition caused by different phenylglyoxal derivatives which have a similar core but differ in their substituent groups have been compared. The interaction between the binding sites of these compounds and other anion transport inhibitors have also been studied.     

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 dinitrofluoro-benzene (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, 1974). 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. The effect of the irreversibly binding inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: (1) binding sites common for DNFB and SITS which are probably related to inhibition, (2) other common sites which are not related to inhibition and (3) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population (1) was estimated to be 0.8–1.2 ± 10⁶/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4′-isothiocyanato-2,2′-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity.     

Protein-mediated chloride-phosphate and lactate-lactate exchange in cytoskeleton-free vesicles budded from rabbit erythrocytes

Spectrin-free budded vesicles from rabbit erythrocytes (Leonards, K.S. and Ohki, S. (1983) Biochim. Biophys. Acta 728, 383-393) exchange intravesicular L-[14C]lactate for extravesicular L-lactate and intravesicular [36C]chloride for extravesicular phosphate with inhibitor sensitivity consistent with what is seen in intact cells. The time-course of these fluxes is faster than for intact cells, but is somewhat slower than predicted from surface to volume ratios. Labelling with tritiated 4,4'-diisothiocyanyl-2,2'-dihydrostilbenedisulfonate (H2DIDS) at concentrations which selectively inhibit inorganic anion exchange or specific lactate exchange supports the involvement of a 93-110 kDa (band 3) polypeptide in anion transport and a 40-50 kDa polypeptide in lactate transport across these vesicle membranes. Since the budded vesicles have a markedly simplified protein profile on electrophoresis, their isolated membranes represent a preliminary stage in the purification of these transport proteins in which structure and function appear to be preserved.     

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.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.     

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.     

Comparison of murine band 3 protein-mediated Cl- transport as measured in mouse red blood cells and in oocytes of Xenopus laevis

Murine band 3 protein was expressed in oocytes of Xenopus laevis after microinjection of the mRNA from the spleens of anemic mice. The 36Cl- efflux from the oocytes was compared with the chloride fluxes measured in murine red cells. In both oocytes and red cells, the band 3-mediated chloride transport showed the following features: the selective inhibitor of band 3-mediated anion transport, 4,4'-dinitrostilbene-2,2'-disulfonate exerts its effects only when applied to the outside and not when applied to the inside of the membrane. The K1/2 for inhibition by external 4,4'-dinitrostilbene-2,2'-disulfonate was of the order of 1.5 to 2.0 mumol/l. Flufenamate and persantine also produce similar inhibitory effects. Decreasing the pH from 7.4 to 6.0 leads to some inhibition. It is concluded that essential features of the mode of action of murine erythroid band 3 protein in the plasma membrane of the oocyte are similar to the mode of action in the bilayer of the red blood cell of the mouse.     

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.     

Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements. Inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 · 10⁵ or 6.0 · 10⁵ molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4′-diisothiocyano-1,2-diphenyl-ethane-2,2′-disulfonic acid (H₂DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.