The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles

Band 3 protein has been incorporated into lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine or total erythrocyte lipids by means of a Triton X-100 Bio-Beads method, with an additional sonication step prior to the removal of the detergent. This methods results, for both types of band 3 lipid vesicles, in rather homogeneous vesicles with comparable protein content and vesicle trap. Freeze-fracture electron microscopy revealed that band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have considerably more intramembrane particles as compared to the band 3-erythrocyte lipid vesicles. The dimensions of the nonspecific permeation pathways present in the band 3-lipid vesicles were measured using an influx assay procedure for nonelectrolytes of different size, in which the vesicles were sampled and subsequently freed from nonenclosed labeled permeant by means of gel-filtration. The band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have nonspecific permeation pathways (pores), with diameters of up to 60 A. In contrast, the band 3-total erythrocyte lipid vesicles are more homogeneous and show much smaller nonspecific permeation pathways, having a diameter of about 12 A. These results suggest that the nonspecific permeability of the band 3-lipid vesicles is strongly lipid-dependent. Increase in specific anion permeability expected as a consequence of the presence of band 3 in the erythrocyte lipid vesicles was found to be very limited. However, stereospecific, phloretin-inhibitable D-glucose permeability could clearly be demonstrated in these vesicles. The difference of the nonspecific permeability of the band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles and band 3-erythrocyte lipid vesicles, is discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce formation of pores.     

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.     

Transmembrane effects of irreversible inhibitors of anion transport in red blood cells. Evidence for mobile transport sites

Experiments were designed to determine whether band 3, the anion transport protein of the red cell membrane, contains a mobile element that acts as a carrier to move the anions across a permeability barrier. The transport site-specific, nonpenetrating irreversible inhibitor 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) was found to be effective only when applied extracellularly. It was used to sequester transport sites on the extracellular side of the membrane in intact cells. The membranes were then coverted into inside-out vesicles. The number of anion transport sites available on the cytoplasmic side of the vesicle membranes was then estimated by measuring the binding of N-(-4-azido-2-nitrophenyl)-2-aminoethyl-sulfonate (NAP-taurine), a photoreactive probe. Pretreatment with DIDS from the extracullular side substantially reduced the binding of NAP-taurine at the cytoplasmic side. Since NAP-taurine does not appear to penetrate into the intravesicular (normally extracellular) space, a transmembrane effect is apparently involved. About 70% of the DIDS-sensitive NAP-taurine binding sites are located in band 3, with the remainder largely in a lower molecular weight (band 4) region. A similar pattern of reduction in NAP-taurine binding is produced by high concentrations of Cl-, but this anion has little or no effect in vesicles from cells pretreated with DIDS. Thus the DIDS-modulated sites seem to be capable of binding either NAP-taurine or Cl. It is suggested that band 3 contains a mobile transport element that can be recruited to the extracellular surface by DIDS, thus becoming unavailable to NAP-taurine at the cytoplasmic face of the membrane. The results are consistent with a model of carrier-mediated transport in which the movement of the transport site is associated with a local conformational change in band 3 protein.     

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.     

Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange

Treatment of whole erythrocytes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) results in inhibition of ATP and phosphate-dependent Ca2+ transport in subsequently prepared inside-out vesicles (IOV). Accumulation of phosphate into IOV in the presence of Ca2+ is virtually abolished by prior DIDS treatment, consistent with the presumed inhibition of the band III anion-exchange protein by this agent. No inhibition of Ca2+-activatable ATP hydrolysis is observed following DIDS treatment when open membranes are used to prevent development of ion gradients. This indicates that DIDS does not affect the inherent ATPase activity of the calcium pump (Waisman, D. M., Smallwood, J., Lafreniere, D., and Rasmussen, H. (1982) FEBS Lett. 145, 337-340). In IOV prepared from untreated cells, ATP-dependent Ca2+ uptake is stimulated by phosphate, sulfate, or chloride. Rates of Ca2+ uptake into DIDS-IOV are not increased by these anions. Lipid-permeable organic acids such as acetate, however, do promote Ca2+ transport in DIDS-IOV. Lipophilic anions incapable of transporting protons into the vesicle interior (nitrate and thiocyanate) support sustained uptake only when the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone is also added. These results support a model of the (Ca2+-Mg2+)-ATPase as a pump exchanging Ca2+ for protons, not transporting Ca2+ alone. Band III protein appears to promote Ca2+ transport in the presence of phosphate, sulfate, or chloride by exchanging external anion for the accumulating OH- (or HCO3-) produced by the calcium pump.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by stilbene derivatives and the identification of an inhibitor-binding protein

The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H₂DIDS bound to the vesicles was determined by using [³H]-H₂DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 μmol/g protein. SDS-polyacrylamide gel electrophoresis of the vesicles treated with [³H]H₂DIDS showed that about 10% of the total bound H₂DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H₂DIDS-binding protein was about 0.5 μmol/g protein. These results suggest that the H₂DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.     

人红细胞膜带3蛋白重组人脂质体及其~(35)SO_4~(2-)运输活性

用Triton X-100选择性抽提法,部分提纯了人红细胞膜带3蛋白。在除去去污剂后,带3蛋白被插入由磷脂酰胆碱/胆固醇组成的脂质体中。研究表明,用0.05％Triton抽提膜能除去大部分唾液酸糖蛋白,不会损失太多的带3蛋白。再用0.5％Triton抽提,可增溶出大量带3蛋白和少量其它膜蛋白。把它重组入脂质体后,在最后超离心步骤中得到了小的单层囊泡和大的多层囊泡。本文研究了单层重组囊泡输入~(35)SO_4~(2-)的功能,并和那些由人红细胞“发芽”得到的天然囊泡输入~(35)SO_肋~(2-)的功能作了比较。     

Intermembrane protein transfer. Band 3, the erythrocyte anion transporter, transfers in native orientation from human red blood cells into the bilayer of phospholipid vesicles

Band 3, the erythrocyte anion transporter, has been shown to transfer between human erythrocytes and sonicated vesicles (Newton, A. C., Cook, S. L., and Huestis, W. H. (1983) Biochemistry 22, 6110-6117). Functional band 3 becomes associated with dimyristoylphosphatidylcholine vesicles incubated with human red blood cells. Proteolytic degradation patterns reveal that the transporter is transferred to the vesicles in native orientation. In erythrocytes, native band 3 is degraded on the exoplasmic membrane face by chymotrypsin and on the cytoplasmic surface by trypsin (Cabantchik, Z. I., and Rothstein, A. (1974) J. Membr. Biol. 15, 227-248; Jennings, M. L., Anderson, M. P., and Monaghan, R. (1986) J. Biol. Chem. 261, 9002-9010). Band 3 in intact protein-vesicle complexes is degraded by exogenous chymotrypsin but not by trypsin. In contrast, trypsin entrapped in the lumen of the vesicles proteolyses the vesicle-bound band 3 quantitatively. Band 3 remaining in the membranes of vesicle-treated cells and in cell fragments is not degraded detectably by vesicle-entrapped trypsin. These observations indicate that band 3 is unlikely to transfer between cell and vesicle membranes via a water-soluble form or to adhere nonspecifically to the vesicle surface; the aqueous contents of vesicles and cells (or membrane fragments) are not pooled during cell-vesicle incubations, hence no cell-vesicle fusion occurs; and the band 3 associated with the sonicated vesicle fraction is inserted in the vesicle bilayer in native orientation, with its cytoplasmic segment contacting the aqueous contents of the vesicle lumen.     

The turnover number for band 3-mediated sulfate transport in phosphatidylcholine bilayers

The anion transport system of the human erythrocyte membrane was reconstituted in unilamellar phosphatidylcholine vesicles, and a vesicle subpopulation of a narrow size distribution was isolated from the sample by gel filtration. In this subpopulation, the turnover number of the transport protein (the band 3 protein) for sulfate transport was determined. It was found that, in the reconstituted system, the protein transports sulfate 5-10 times faster than in the human erythrocyte membrane.     

Vesicle-to-cell protein transfer: insertion of band 3, the erythrocyte anion transporter, into lymphoid cells

Band 3, the erythrocyte anion transporter, transfers spontaneously between human red cells and model membranes. During incubation of intact erythrocytes with sonicated dimyristoylphosphatidylcholine vesicles, the transporter inserts in functional form and native orientation into the liposome bilayer, with the cytoplasmic segment of the protein contacting the lumen of the vesicle [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117; Huestis, W. H., & Newton, A. C. (1986) J. Biol. Chem. 261, 16274-16278]. When band 3-vesicle complexes are incubated with erythrocytes whose native band 3 has been inhibited irreversibly, reverse transfer of the protein restores anion transport capacity to the cells [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117]. Here we report the vesicle-mediated transfer of band 3 to human peripheral blood lymphocytes and to cultured murine lymphoma cells (BL/VL3). Subsequent to incubation with protein-vesicle complexes, both lymphoid cell types exhibit a 2-4-fold increase in the rate of chloride uptake. This enhanced permeability is inhibited greater than or equal to 98% by the exofacial band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, consistent with right-side-out insertion of functional band 3 into the lymphoid cell membrane.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

Anion transport in normal erythrocytes,sickle red cells,and ghosts in relation to hemoglobins and magnesium

"Band 3," an integral membrane protein of red blood cells, plays a relevant role in anionic transport. The C- and N-terminal portions of band 3 are cytoplasmatics, and the last is the link site for different glycolitic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, and hemoglobin. All or some of these interactions on the CDB3 protein could allow a subtle modulation of anion flux. The interaction among HbA, Mg(2+), and membrane proteins has been sufficiently investigated, but not the effect of Mg(2+) on pathological hemoglobin in relation to the influx of the SO(4)(2-). The aim of this study was to evaluate the involvement of hemoglobin S in sulfate transport. This has been measured with native and increased concentrations of Mg(2+), using normal erythrocytes containing HbA, sickle red cells containing HbS, or ghosts obtained from both erythrocytes and normal erythrocytes ghosts with HbS added. The magnitude of the SO(4)(2-) rate constant measured in normal red blood cells increased markedly when measured in the presence of varied Mg(2+) concentrations. The results show that a low increase of intracellular Mg(2+) concentrations exercises a different HbA modulation on band 3 protein and consequently higher anion transport activity. The same experiments carried out in sickle red cells showed that the SO(4)(2-) rate constant measured in the presence of native concentrations of Mg(2+) was normal, compared to normal red cells, and was not affected by any increase of intracellular Mg(2+). Our suppositions with regard to the importance exercised by the hemoglobin and the Mg(2+) on the SO(4)(2-) influx were confirmed by comparison of the data obtained through measuring SO(4)(2-) influx with native and increased concentrations of Mg(2+) in both normal and sickle red cell ghosts. Both revealed the same sensitivity to Mg(2+) due to withdrawal of hemoglobins. The incorporation of HbS in normal as well as in sickle red cell ghosts reduced the Mg(2+) response to sulfate influx in both the reconstituted ghosts. Our research demonstrated that the different effects exercised on the rate constants of SO(4)(2-) influx in normal (HbA) and sickle red cells (HbS) by the increased intracellular Mg(2+) could be ascribed to the physical-chemical influence exercised either on the hemoglobins or on the intracellular contents of erythrocytes.     

Pepsin cleavage of band 3 produces its membrane-crossing domains

After prolonged treatment of red-cell ghosts with pepsin followed by SDS-urea-acrylamide gel electrophoresis of the membrane peptide fraction, a heavily stained band representing peptides of about 4 kDa (with traces of higher molecular weights) was found. If the cells were first labelled with the disulfonic stilbene, DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) or with N-ethylmaleimide, probes that react with specific sites in Band 3 the anion transport protein, both agents were largely located in the 4 kDA band. With less intensive pepsin treatment, Stained bands of about 17, 12 and 8 kDa were also visible, and DIDS labelling was associated with these higher molecular weight peptides. The 4 kDa band apparently contains at least five or six different peptides. A single peptide containing the DIDS-binding site was separated from others in the band by ion-exchange chromatography. The location of the DIDS-peptide in the primary structure of Band 3 was determined by matching the known location of DIDS and of a methionine residue cleavable by cyanogen bromide. It is concluded that two additional 4 kDA peptides are labelled with N-ethylmaleimide. Because the location of the N-ethylmaleimide-binding sites are known, these two peptides could also be mapped in the primary structure of Band 3. The findings are consistent with the suggestion that pepsin can digest those portions of Band 3 (and probably of other intrinsic peptides) that are exposed on either side of the membrane, leaving only those domains that cross the bilayer. For Band 3, the data are consistent with a structure containing five crossing strands per monomer, each crossing strand being about 4 kDa.     

External anions regulate stilbene-sensitive proton transport in placental brush border vesicles

The mechanism for HCO3-(-)independent proton permeability in microvillus membrane vesicles (MVV) isolated from human placenta was examined by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Proton fluxes (JH) across MVV were determined in response to induced pH and anion gradients from the time course of 6CF fluorescence, the MVV buffer capacity, and the 6CF vs. pH calibration. In the absence of anions, JH was 12 +/- 2 nequiv s-1 (mg of protein)-1 (pHin 7.4, pHout 6.0, MVV voltage-clamped with K+/valinomycin, 23 degrees C), corresponding to a proton permeability coefficient of 0.02 cm/s, with an activation energy of 9.1 +/- 0.3 kcal/mol. JH was inhibited 20% by dihydro-4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (H2DIDS) with KI = 8 microM [( Cl-]out = 0 mM). For a 0.5-unit pH gradient JH increased from 1.5 to 4.6 nequiv s-1 (mg of protein)-1 as the internal MVV pH was increased (5.5-7.5). External Cl-, Br-, and I- (but not SO4(2-) and PO4-) increased JH 1.3-2.5-fold for both inwardly and outwardly directed pH gradients with KD = 1.0 +/- 0.4 mM (Br-) and greater than 100 mM (Cl-). This increase was blocked by 100 microM H2DIDS but not by amiloride or furosemide. Internal Cl- did not alter JH induced by pH gradients nor were proton fluxes induced by anion gradients in the absence of a pH gradient. Experiments in which JH was driven by membrane potentials (induced by valinomycin and K+ gradients) indicated that proton transport was voltage-sensitive. These experiments demonstrate a stilbene-sensitive electrogenic proton transport mechanism in MVV that is regulated allosterically by anions at an external binding site.     

Identification of an anion channel protein from electric organ of Narke japonica

The anion permeability of membrane vesicles prepared from the electric organ of $\text{Narke japonica}$ was inhibited by the addition of 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS). The permeability was measured by measuring changes in the scattered-light intensity caused by the osmotic volume change of vesicles; and also by the efflux measurement of ions from the vesicles using radioisotopes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane vesicles treated with dihydro analog of DIDS ([³H]H₂DIDS) showed that the H₂DIDS binding protein has a molecular weight of 180,000, and exists in membrane vesicles as a dimer formed by a disulfide bond between monomers of molecular weight 90,000.     

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.     

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.     

The effect of the presence of integral membrane protein (human band 3) on the membrane lytic properties of melittin in reconstituted systems

Abstract Reconstitution of the anion exchange protein from human erythrocytes (band 3) into phospholipid vesicles was shown to have a protective effect on melittin lysis of the vesicles when compared to pure lipid vesicles. Low salt buffer was found to cause an inhibition of lysis in both proteoliposomes and pure lipid vesicles compared to salt buffer. High phosphate concentration did not seem to cause inhibition of lysis in the reconstituted system. However, an inhibition is observed in pure lipid vesicle control, which is contradictory to previous reports.     

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.     

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.