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.     

Intrinsic segments of band 3 that are associated with anion transport across red blood cell membranes

Summary After treatment of red cell ghosts with chymotrypsin, the predominant intrinsic peptides remaining in the membrane fraction are 15,000 and 9,000 daltons mol wt. After partial extraction with Triton X-100, the residual membrane vesicles have almost no other stained peptides and such vesicles are reported to carry out anion transport activities sensitive to specific inhibitors. In vesicles derived from cells treated with DIDS(4,4-diisothiocyano-2,2-stilbene disulfonic acid), an irreversible inhibitor of anion transport that is highly localized in an abundant intrinsic protein known as band 3, the probe is largely recovered in the 15,000 dalton peptide. The part of band 3 from which it is derived is a previously reported 17,000 transmembrane segment (Steck, T.L., Ramos, R., Strapazon, E., 1976,Biochemistry 15:1154). The 9,000-dalton peptide is present in the vesicles in a one-to-one mole ratio with the 15,000-dalton peptide, suggesting that both are derived from the same protein. This conclusion is supported by the finding that the 35,000-dalton C-terminal end of band 3, derived by chymotrypsin treatment of cells, is further proteolysed if the cells are converted to ghosts and its disappearance coincides with the appearance of the 9,000-dalton fragment. Evidence is presented that the 9,000-dalton fragment crosses the bilayer and that it is closely associated with the 15,000-dalton peptide.This paper is dedicated to the memory of Walther Wilbrandt.     

Reactive sulfhydryl groups of the band 3 polypeptide from human erythroycte membranes. Location in the primary structure.

Human erythrocyte membranes contain a major transmembrane protein, known as Band 3, that is involved in anion transport. This protein contains a total of five reactive sulfhydryl groups, which can be assigned to either of two classes on the basis of their susceptibility to release from the membrane by trypsin. Two of the groups are located in the region COOH-terminal to the extracellular chymotrypsin-sensitive site of the protein and remain with a membrane-bound 55,000-dalton fragment generated by trypsin treatment. The three sulfhydryl groups NH2-terminal to the extracellular chymotrypsin site are released from the cytoplasmic surface of the membrane by trypsin. All three groups are present in a 20,000-dalton tryptic fragment of Band 3. Two of these groups are located very close to the sites of trypsin cleavage that generate the 20,000-dalton fragment. The third reactve group is probably located about 15,000-daltons from the most NH2-terminal sulfhydryl group. Two other well defined fragments of the protein do not contain reactive sulfhydryl groups. They are a 23,000-dalton fragment derived from the NH2-terminal end that is also released by trypsin from the cytoplasmic surface of the membrane and a 19,000-dalton membrane-bound region of the protein that is produced by treatment with chymotrypsin in ghosts. The 20,000-dalton tryptic fragment may, therefore, constitute a sulfhydryl-containing domain of the Band 3 protein.     

The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

Structural stability of the erythrocyte anion transporter, band 3, in different lipid environments. A differential scanning calorimetric study

In order to understand how subtle variations in lipid structure can influence the stability of an integral membrane protein, the purified, delipidated anion transport domain of human erythrocyte band 3 was reconstituted into a series of well-defined lipids and examined by differential scanning calorimetry. From the calorimetric scans, plots of denaturation temperature (Tm), enthalpy (delta Hd), and heat capacity (delta Cdp) as a function of phospholipid chain length, degree of unsaturation, headgroup type, and cholesterol content were constructed. The data show that the stability of the 55,000-dalton membrane-spanning domain of band 3 is exquisitely sensitive to the acyl chain length of its phospholipid environment, increasing almost linearly from a Tm of 47 degrees C in dimyristoleylphosphatidylcholine (C14:1) to 66 degrees C in dinervonylphosphatidylcholine (C24:1). The integral domain was also found to be significantly stabilized by increasing the degree of saturation of the fatty acyl chains and by elevating the cholesterol content of the membrane. Although band 3 was native in all reconstituted lipid systems, the transport protein's stability was clearly much greater in zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) than anionic lipids (phosphatidylserine and phosphatidylglycerol). Enthalpy and delta Cdp values were generally within the ranges expected of globular proteins in the various reconstituted systems, except the values for the anionic and polyunsaturated phospholipids were anomalously low. Much of the data can be accounted for by the hypothesis that band 3 has a long hydrophobic cross-section and that a close match between the hydrophobic zone of the membrane-spanning protein and the nonpolar region of the bilayer is necessary for maximum protein stability. Because the integral domain of band 3 may be structurally representative of a larger group of transport proteins, the data should be useful in interpreting structural observations on protein-lipid interactions in other membrane systems.     

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled byN-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites are preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.     

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

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.     

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.     

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.     

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.     

Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane.

Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.     

Cloning and characterization of a murine band 3-related cDNA from kidney and from a lymphoid cell line

Anion exchange is a nearly ubiquitous cellular transport function which contributes to the regulation of cell pH and of cell volume. However, the only plasma membrane anion exchanger of known identity and sequence is erythroid band 3. Both hybridization and immunologic data support the presence of band 3-related mRNAs and proteins in nonerythroid tissues. We have used low stringency hybridization with the murine band 3 cDNA to clone a band 3-related cDNA from murine kidney and from 70Z/3 pre-B cells. The cDNA encodes a band 3-related protein (B3RP) of 1237 amino acids, with a predicted mass of 137 kDa. The carboxyl-terminal hydrophobic domain of B3RP has an amino acid sequence 67% identical to that of band 3, with a very similar predicted secondary structure. The amino-terminal hydrophilic domain of B3RP has two sections. The section adjacent to the putative membrane-associated segment is 33% identical in amino acid sequence to the amino-terminal, cytoplasmic domain of band 3. The other, far amino-terminal section of B3RP has no correspondent in the band 3 sequence. B3RP mRNA is present in a variety of epithelial and other tissues and probably encodes an anion exchange protein of wide distribution.     

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.     

Isolation of a 5,300-dalton peptide containing a pyridoxal phosphate binding site from the 38,000-dalton domain of band 3 of human erythrocyte membranes

A hydrophobic 5,300-dalton peptide was isolated from the 38,000-dalton domain of Band 3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The peptide was affinity labeled with pyridoxal phosphate and sodium [3H]borohydride when erythrocytes were incubated in vitro. The peptide was not labeled with these agents when cells were incubated in the presence of a specific inhibitor of anion transport, suggesting that the peptide contains at least a part of the active center for the anion transport system in the cell membrane. The peptide was eluted from a reversed-phase high-performance liquid chromatography column with a high concentration of acetonitrile (more than 65%), although the elution pattern of the hydrophobic peptide was not as sharp as that of the soluble peptides. However, a satisfactory separation was achieved when this procedure was employed in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Fine structure of the band 3 protein in human red cell membranes: Freeze-fracture studies

The major red cell membrane protein, band 3, is a glycoprotein which extends across the membrane from the extracellular space into the cytoplasmic compartment. It is widely held that band 3 is a component of the intramembrane particles (IMP) which can be demonstrated by freeze-fracture electron microscopy. In this study, we find that the outer surface poles of the IMP can be seen by freeze-etching after they are unmasked by proteolysis under conditions which excise the surrounding sialopeptides from the membrane. The poles appear as distinctive projections, 30–50 Å in diameter, the “ES particles.” The ES particles remain associated with the outer surface of the membrane following cleavage of the band 3 polypeptide by chymotrypsin or pronase. This is consistent with previous biochemical studies which have shown that the 38,000-dalton outer surface segment of band 3 is intercalated in the lipid bilayer. A granulofibrillar component at the inner surface of the membrane is provisonally identified as the 40,000-dalton inner-surface domain of band 3.     

Amino acid sequence of the N alpha-terminal 201 residues of human erythrocyte membrane band 3

We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane. The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation. The sequence had these notable features. 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase. The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group. 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues. 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character. The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites.     

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.     

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.     

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.