Characterization of matrix-bound Band 3, the anion transport protein from human erythrocyte membranes

Band 3 (Mr = 95,000), the anion transport protein of human erythrocyte membranes exists primarily as a dimer in solutions of nonionic detergents such as octaethylene glycol mono-n-dodecyl ether (C12E8). The role of the oligomeric structure of Band 3 in the binding of [14C]4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), an inhibitor of anion transport (Ki = 1-2 microM), was studied by characterizing the interaction of BADS with dimers and monomers of Band 3 covalently attached to p-mercuribenzoate-Sepharose 4B. BADS bound to matrix-bound Band 3 dimers with an affinity of approximately 3 microM at a stoichiometry of 1 BADS molecule/Band 3 monomer, in agreement with the BADS binding characteristic of Band 3 in the membrane and in solutions of C12E8. Band 3 dimers could be attached to the matrix via one subunit by limiting the amount of p-chloromercuribenzoate on the Sepharose bead. Matrix-bound monomers were formed by dissociation of the dimers with dodecyl sulfate or guanidine hydrochloride. Complete removal of the denaturants allowed formation of refolded Band 3 monomers since the matrix-bound subunits could not reassociate. These refolded Band 3 monomers were unable to bind BADS. Release of the monomers from the matrix with 2-mercaptoethanol allowed reformation of dimers with recovery of the BADS binding sites. These results suggest that the dimeric structure of Band 3 is required for BADS binding and that the BADS binding sites may be at the interface between the two halves of the Band 3 dimer.     

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.     

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.     

Conformation and stability of the anion transport protein of human erythrocyte membranes

The conformation and stability of purified preparations of band 3, the anion transport protein of human erythrocyte membranes, and its constituent proteolytic subfragments have been studied by circular dichroism. Band 3, purified in the presence of the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8), had an alpha-helical content of 46%. Denaturation of purified band 3 with guanidine hydrochloride occurred in two phases, one reflecting much more resistance to denaturation than the other. Band 3 can be separated into two domains by limited in situ proteolytic cleavage. The carboxyl-terminal membrane-associated domain (Mr 55 000) purified in C12E8 contained 58% alpha-helix and was very resistant to denaturation by guanidine hydrochloride. The purified amino-terminal, cytoplasmic domain (Mr 41 000) contained 27% alpha-helix and was completely converted to a random-coil conformation by 3 M guanidine hydrochloride. The two phases of denaturation observed for intact band 3 corresponded to the two domains of the protein. Irreversible heat denaturation of purified band 3 occurred with half-maximal change in theta 222.5 at 48 degrees C. Covalent attachment of the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate to band 3 had little effect on the circular dichroism spectra of band 3 or the membrane-associated domain but resulted in stabilization of band 3 to heat denaturation (half-maximal change in theta 222.5 = 61 degrees C). Circular dichroism studies of membranes that had been digested extensively with proteolytic enzymes and stripped of all extrinsic fragments revealed that the portions of red cell membrane proteins that are embedded in the lipid bilayer contain a very high (86-94%) content of alpha-helix.     

Hydroxyapatite chromatography of the D-glucose transport protein of human erythrocyte membranes

Absorption, circular dichroism, electron spin resonance and resonance Raman spectra of a blue copper protein, plantacyanin from cucumber peel have been measured and these spectral properties compared with those of other blue copper proteins. From the spectral properties, amino acid analysis and redox potential, we discuss the active site and redox properties of this protein.     

Biosynthesis of the erythrocyte anion transport protein

The biosynthesis of the erythrocyte anion transport protein (Band III) was studied in erythroid precursor cells obtained from the spleens of anemic mice. Newly synthesized Band III was inserted during or immediately after translation into rough endoplasmic reticulum membranes. The asymmetric orientation of Band III in these membranes resembled that of mature Band III in erythrocyte membranes, with the NH2-terminal portion of the molecule facing the cytoplasm. At this stage Band III contained a high mannose core oligosaccharide, which was susceptible to cleavage by endoglycosidase H. During the next 20 to 30 min, this oligosaccharide was processed to a form resistant to endoglycosidase H degradation, presumably in the Golgi complex. The processed Band III was subsequently expressed on the cell surface, at about 30 to 45 min after synthesis. In many respects, therefore, the biosynthesis of Band III resembles that of cotranslationally inserted proteins whose NH2-terminal portions are exposed on the exterior of the cell, like VSV glycoprotein, HLA-A antigens, and glycophorin.     

Reversible DIDS binding to Band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([³H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS-Band 3' complex at 0° C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k₊₁     

Affinity labeling of erythrocyte band 3 protein with pyridoxal 5-phosphate. Involvement of the 35,000-dalton fragment in anion transport

Transport of pyridoxal 5-phosphate (PLP) into erythrocytes was inhibited by inhibitors of anion transport including stilbene disulfonate compounds, indicating that it is mediated by Band 3 protein. When erythrocytes were treated with PLP and large amounts of free lysine and NaBH4, two membrane-spanning fragments of Band 3 (Mr = 17,000 and 35,000) were specifically labeled. When the cells were pretreated with 4,4'-dinitrostilbene 2,2'-disulfonate, the labeling in the 35,000-dalton fragment was inhibited. Erythrocytes labeled by PLP in both the 17,000- and 35,000-dalton fragments transported PLP at a decreased rate, whereas the cells labeled in only the 17,000-dalton fragment had essentially the same transport activity as the control when 4,4'-dinitrostilbene 2,2'-disulfonate was removed. The extent of inhibition of transport of inorganic phosphate in the labeled cells was similar to that of PLP. The results indicate that the 35,000-dalton fragment participates in the anion transport of the cell membrane.     

Oligomeric structure and the anion transport function of human erythrocyte band 3 protein

Conclusions Evidence from many laboratories using several different techniques strongly suggests that, in the intact red cell, band 3 exists as dimers which can associate with other dimers to form tetramers. The kinetics of anion transport inhibition by stilbenedisulfonates indicate that irreversible inhibition of one subunit does not detectably affect anion transport by the other subunit. This does not imply that monomeric band 3 could necessarily transport anions; the native conformation of each subunit may require stabilizing interactions with another subunit, as indicated by the recent work of Boodhoo and Reithmeier [10]. A more detailed understanding of the structure of the band 3 dimer/tetramer will require information on which specific segments of the primary structure are involved in subunit-subunit contact. The combination of chemical cross-linking with proteolysis [136] is a promising approach to this problem.     

Reconstitution of band 3, the erythrocyte anion exchange protein

Band 3, the erythrocyte membrane protein thought to be responsible for anion transport, was purified to near homogeneity using a Concanavalin A affinity column. Band 3 was then combined with egg lecithin, erythrocyte lipid, cholesterol, and glycophorin, the major erythrocyte sialoglycoprotein, to form vesicles capable of rapid sulfate transport. The transport activity was sensitive to prior treatment of the erythrocytes with pyridoxal phosphate-NaBH₄, a potent inhibitor of anion transport in these cells.     

Analysis of the oligomeric state of Band 3, the anion transport protein of the human erythrocyte membrane, by size exclusion high performance liquid chromatography. Oligomeric stability and origin of heterogeneity

The oligomeric state of human Band 3 (Mr = 95,000), the erythrocyte membrane anion exchanger, was examined by size exclusion high performance liquid chromatography in solutions containing the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). Band 3 was heterogeneous with respect to oligomeric composition, the predominant (70%) species being a dimer that bound 0.57 mg of C12E8/mg of protein (Stokes radius = 78 A, s20,w = 6.9 S). Variable amounts of larger oligomers were also present; however, no evidence for equilibration between oligomeric species was observed in detergent solution. Analytical and large zone size exclusion chromatography showed that Band 3 could not be dissociated to monomers, other than by protein denaturation. The membrane domain of Band 3 (Mr = 52,000) was also dimeric, but without evidence for higher oligomeric forms, which implies that the interactions responsible for higher associations involve the cytoplasmic domain. Prelabeling of Band 3 with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate had no effect upon the oligomeric state of either intact Band 3 or its 52-kDa membrane domain. Band 3 oligomeric state could be reversibly changed in the membrane by altering the pH of the solution. The fraction of Band 3 not associated with the cytoskeleton was almost entirely dimeric. Band 3 purified from erythrocytes separated by density gradient centrifugation revealed that older red cells contained a larger proportion of higher oligomers than did younger cells. We conclude that Band 3, in the membrane and in C12E8 solution, exists as a mixture of dimers and larger oligomers. The higher oligomers interact with the cytoskeleton, increase in amount with cell age, and are held together by interactions of the cytoplasmic domain.     

The effect of selenium on the function of the anion transporter (Band 3) of erythrocyte membranes.

The transport activity of Band 3 of spectrin-stripped inside-out erythrocyte membrane vesicles (IOVs) or resealed ghosts was enhanced in the presence of trace amounts of Na2SeO3 (0.2-0.5 p.p.m.); however, at higher concentrations of Na2SeO3 (> 4.0 p.p.m.), an inverse result was obtained. Reassociation of spectrin with IOVs has no effect either on the transport activity of Band 3 or on the enhancement of its activity by Na2SeO3. Sulfhydryl reagents (p-chloromercuribenzoic acid and N-ethylmaleimide) could also inhibit Band 3 activity and eliminate the selenium effect. It is suggested that SH groups are involved in anion transport of Band 3 and that the selenium effect is based on the interaction of SH groups of Band 3 with Na2SeO3.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Changes in the structural-functional state of erythrocyte membranes treated with anion transport inhibitors

Effect of 4,4-dyisotiocyanostilben-2,2-disulfonate (DIDS) and 1-ftor-2,4-dinitrobenzol (NDFB) on the rate of phosphate ion transport in erythrocytes, filtrability and thermal stability of erythrocytes and on the structural state of the erythrocyte membrane estimated by UV-fluorescence, PAAG--electrophoresis and measuring of the activity of membrane-bound acetylcholinesterase (AChE) has been studied. Unpenetrating anion transport inhibitor DIDS is shown to induce structural modifications of bands 3 of protein and AChE, while DNFB penetrating the membrane causes a significant reorganization of many membrane proteins (including spectrin) resulting in changes of transport and mechanical properties of erythrocytes.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Functional characterization of anion transport system isolated from human erythrocyte membranes.

The anion transport system of human red blood cells was isolated in vesicles containing the original lipids of the membrane and predominantly the 95,000-dalton polypeptides (Band 3) associated with intralipid particles. The vesicles display various characteristic properties of anion permeation closely resembling those of the native system. The properties include energy of activation, pH dependence, anion sleectivity, sensitivity to specific inhibitors, and exchange and net rates of sulfate transport. Based on these and other criteria, the functional properties of isolated vesicles could be equated with those of the intact cell system. Direct support for the involvement of 95,000-dalton polypeptides in permeation functions is provided.     

The effects of anion transport inhibitors on structural transitions in erythrocyte membranes

Red blood cell membranes have been labeled with several covalent and non-covalent inhibitors of anion transport and their heat capacity profiles determined as a function of temperature. Covalent inhibitors include the amino reactive agents 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, pyridoxal phosphate and 1-fluoro-2,4-dinitro benzene. The non-covalent inhibitors include several well known local anesthetics. The study was undertaken in order to identify regions of the membrane involved in anion transport. Covalent modification in all cases resulted in a large upward shift of the C transition, which is believed to involved a localized phospholipid region. Evidence is presented which indicates that Band III protein and this phospholipid region are in close physical proximity on the membrane. Addition of non-covalent inhibitors affects the membrane in either or both of two ways. In some cases, a lowering and broadening of the C transition occurs; in other the B₁ and B₂ transitions are altered. These latter transitions are believed to involve both phospholipid and protein, including Band III. These results may indicate that the non-covalent inhibitors produce their inhibitory effect on anion transport at least in part by interacting with membrane phospholipid.     

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane.

The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two-dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six-fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two-fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 x 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 x 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation.