Structure of the murine anion exchange protein

A full-length clone encoding the mouse erythrocyte anion exchange protein, band 3, has been isolated from a cDNA library using an antibody against the mature erythrocyte protein. The complete nucleotide sequence has been determined. Substantial homology is evident between the deduced murine amino acid sequence and published sequences of fragments of human band 3 protein. The amino-terminal 420 and the carboxy-terminal 32 residues constitute polar, soluble domains, while the intervening 475 amino acids are likely to be intimately associated with the lipid bilayer. Hydrophobic analysis of this sequence, together with structural studies on the human protein, suggests the possibility of at least 12 membrane spans, predicting that both the amino- and carboxy-termini are intracellular.     

pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.     

pH modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an outwardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistinguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pH_int/pH_ext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at low pH (monovalent F^– predominates) and at least 2 H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pH_ext5.8 (K _m =1.33vs. 1.70 M;V _max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect theK _m for carrier-mediated F transport. These data are consistent with similarK _i ^' s for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H⁺ K _m (or OH^– lC₅₀) corresponding to pH 4.89. External pH effects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺),rather than competitive binding that is mutually exclusive.     

Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum

Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO₃, SO₄ for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO₄OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both¹⁴C-oxalate and³⁵SO₄ were equally sensitive tocis-inhibition by unlabeled SO₄ or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO₄ caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO₃ exchange occur on different carriers because: (i) ClHCO₃ exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO₃ exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO₃ exchanger; (ii) a SO₄OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger.     

Potassium/proton exchange in brush-border membrane of rat ileum

Summary These experiments were designed to determine whether proton-driven⁸⁶Rb uptake was present in apical membrane vesicles prepared from rat ileum. The uptake of⁸⁶Rb was approximately 300 to 350% greater in the presence of a 100-fold H⁺ gradient than in its absence and was greater at 1, 2 and 5 minutes (overshoot) than that at 90 minutes. Proton-driven⁸⁶Rb uptake was decreased by 20% in TMA-nitrate compared to that in TMA-gluconate. 0.3mm amiloride did not significantly inhibit proton-driven⁸⁶Rb uptake; in contrast, proton-driven²²Na uptake was significantly inhibited by 0.3mm amiloride by 34%. Similarly, 25mm KCl inhibited proton-driven⁸⁶Rb uptake more than that of²²Na, while the inhibition of proton-driven²²Na uptake by 25mm NaCl was greater than that of⁸⁶Rb. In additional studies intravesicular acidification measured by acridine orange fluorescence was demonstrated in the presence of an out-wardly directed K gradient. These studies demonstrate that a proton gradient stimulates⁸⁶Rb uptake and a K gradient induces intravesicular acidification; and that these fluxes are mediated by a K/H exchange distinct from Na/H exchange which is also present in this membrane. We conclude that a specific exchange process for K/H is located in ileal apical membrane vesicles.     

Sulfate influx across the rabbit ileal brush border membrane: Sodium and proton dependence,and substrate specificities

Summary In intact ileal mucosa, uptake of SO₄ across the brush border membrane requires the presence of Na and is saturable, withK1/2=1.3mm at 140mm Na (P.L. Smith, S.A. Orellana & M. Field, 1981.J. Membrane Biol. 63:199–206). The present study examines the substrate specificities and transport stoichiometry of the Na-dependent SO₄ uptake process. The effects of variations in medium anion and cation composition on lumen-to-epithelium influx of SO₄ (J _me ^SO4 ) were determined under short-circuit conditions.J _me ^SO4 is inhibited by thiosulfate, but not by phosphate, methylsulfate, vanadate or taurocholate. Cl is weakly inhibitory. Uptake of SO₄ is poorly supported by Li, and is unaffected by K, indicating a specific dependence on Na. At low SO₄ concentration (0.22mm),J _me ^SO4 is a hyperbolic function of medium Na concentration; the corresponding Hill plot is linear with a slope of 1.0, suggesting a transport stoichiometry of 1 Na: 1 SO₄. At high SO₄ concentration (6.7mm), the Na-dependent SO₄ velocity curve is sigmoidal and yields a Hill plot which is again linear but has a slope of 1.56, suggesting transport of more than 1 Na per SO₄. SO₄ uptake in presence of Na exhibits a dependence on medium pH. At 0.22mm SO₄ and 140mm Na,J _me ^SO4 was doubled by lowering pH from 7.4 to 6.8. However, at 6.7mm SO₄ and 140mm Na, changing pH had no effect onJ _me ^SO4 over the range 6.8 to 8.5. The pH dependence ofJ _me ^SO4 at 6.7mm SO₄ was restored when medium Na was lowered to 3mm, suggesting that pH sensitivity is a function of the concentration of preformed NaSO ₄ ^– ion pair. The results suggest that SO₄ influx across the ileal brush border occurs by electroneutral Na⁺/NaSO ₄ ^– or Na⁺/H⁺/SO ₄ ^2– cotransport, the former being favored by high concentrations of Na and SO₄.     

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

Summary The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate substites are required for the preferred substrates. Cl^– and HCO ₃ ^– , to account for tight binding in the transition state (K _diss1m). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unsual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and tunneling, which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.     

Release of vasoactive intestinal polypeptide by electrical field stimulation of rabbit ileum

The release of vasoactive intestinal polypeptide (VIP) induced by electrical field stimulation (EFS) of rabbit ileum was studied in vitro. EFS parallel to the muscularis propria caused a significant increase in VIP concentration in the buffer bathing the serosal surface of full-thickness ileum. This effect was blocked by 10^?7 M tetrodotoxin. When circular and longitudinal muscle was removed, the amount of measurable VIP in the tissue decreased to about one-half that of full-thickness ileum, and EFS no longer caused release of VIP into the serosal or mucosal buffers. Our data indicate that EFS of rabbit ileum causes release of VIP, presumably from VIP-containing nerves present in the tissue. These results support the idea that VIP may be a physiological neuroregulator of intestinal function.     

Sodium dependence of neutral amino acid uptake into rabbit ileum

Alanine uses two mediated pathways to enter the rabbit ileal mucosa. Present results suggest that one of them (K_m = 4.1 mM) is fully dependent on sodium in the mucosal medium, while the other (K_m = 91 mM) is sodium-independent. Similar results are obtained for methionine and serine. Reinterpretation of previous alanine/sodium coupling coefficients suggests that two sodium ions per alanine molecule are transported via the high affinity system.     

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K_p value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425–434, 2017     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Sulfate transport in brush border membrane vesicles prepared from human placental syncytiotrophoblast

Isolated brush-border membrane vesicles prepared from human placenta are known to transport amino acids via a Na⁺-dependent mechanism akin to that found in gut and kidney vesicle preparations. We studied sulfate transport in placental vesicles and failed to identify any Na⁺-dependent uptake mechanism. Rather, uptake is a non-electrogenic process that is trans-stimulated by outwardly directed anion flux which is independent of cation. If anion exchange is tightly coupled $\text{in}\text{vivo}$, the net transfer of sulfate from mother to the growing fetus may be driven by the continuous flux of bicarbonate in the opposite direction.     

Transport of neutral and cationic amino acids across the brush-border membrane of the rabbit ileum

Summary The transport of sugars and amino acids across the brush-border membrane of the distal rabbit ileum has been studied. The kinetics of the transport of glucose demonstrated that the data obtained with the present technique are less distorted by unstirred layers than those obtained with the same technique adapted to the use of magnetic stirring. The role of depolarization of the electrical potential difference across the brush-border membrane in mutual inhibition between different classes of amino acids was estimated by measurements of the effects of high concentrations of alanine and lysine on the transport of galactose. It was found that this role would be insignificant in the present study. By measurements of the transport of alanine, leucine and lysine and the inhibitory interactions between these amino acids the function of three transport systems has been delineated. The transport of lysine is resolved in a high- and a low-affinity contribution. At 140mm sodium these transport systems may also function as respectively high- and low-affinity contributors to the transport of neutral amino acids. At 0mm sodium the high-affinity system remains a high-affinity system for cationic and neutral amino acids with reduced capacity especially for the neutral amino acids. At 0mm sodium the low-affinity system's affinity for lysine is reduced and it is inaccessible to neutral amino acids. In addition to the two systems for lysine transport the existence of a lysine-resistant, sodium-dependent, high-affinity system for the transport of neutral amino acids has been confirmed. It seems unlikely that the distal ileum is equipped with a low-affinity, sodium-independent system for the transport of neutral amino acids.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Alterations in band 3 protein and anion exchange in red blood cells of renal failure patients

The precise nature of band 3 protein and its involvement in oxalate exchange in the red blood cells (RBCs) of renal failure patients has not been studied in detail. Therefore, here we studied the oxalate exchange and binding by band 3 protein in RBCs of humans with conditions of acute and chronic renal failure (ARF and CRF). The RBCs of ARF and CRF patients exhibited abnormal red cell morphology and an increased resistance to osmotic hemolysis. Further, an increase in the cholesterol content and decrease in the activities of Na⁺-K⁺-, Ca²⁺-, and Mg²⁺-ATPases of membranes were observed in the RBCs of ARF and CRF patients. A decrease in the oxalate flux was observed in the RBCs of ARF and CRF patients. The oxalate-binding activities of the RBC membranes were significantly lower in ARF (20 pmoles/mg protein) and CRF (5.3 pmoles/mg protein) patients as compared to that in the normal subjects (36 pmoles/mg protein). DEAE-cellulose and Sephadex G-200 column chromatography purification profiles revealed a distinctive shift in oxalate-binding activity of band 3 protein of RBCs of ARF and CRF patients as compared to that of the normal subjects. It was also observed from the binding studies with a fluorescent dye, eosin-5-maleimide, which specifically binds to band 3 protein, that the RBCs of ARF and CRF patients exhibited only 53 and 32% of abundance of band 3 protein, respectively, as compared to that in the RBCs of the normal subjects, thus revealing a decrease in the band 3 protein content in ARF and CRF patients. These results for the first time showed a decrease in the oxalate exchange in RBCs of patients with ARF and CRF, which was also concomitant with the low levels of abundance of band 3 protein.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Denaturation of a membrane transport protein by urea: The erythrocyte anion exchanger

Summary Chloride equilibrium exchange was measured in the presence of intracellular and extracellular urea, several different alkylureas and thiourea. Urea half-inhibited Cl exchange at about 2.5m, but the other, less polar analogs had significantly higher potencies; e.g., butylurea half-inhibited at about 60mm. Onset and reversal of inhibition occurred within less than 2 sec. The inhibition exhibited no obvious sigmoidal dependence on urea concentration, and at low concentrations dimethylurea was a noncompetitive inhibitor of Cl exchange. However, at higher concentrations the Dixon plots were curved upward and a Hill analysis of the dimethylurea data yielded a Hill coefficient of at least 1.5. When present on only one side of the membrane, the slowly penetrating thiourea inhibited Cl exchange with a higher potency from the outside of the cell. Cl/Br exchange was inhibited less under conditions of self-inhibition of anion exchange than in the absence of self-inhibition. These data indicate that the ureas inactivate the anion transporter by a reversible denaturation process, and that the function of the anion transport mechanism may be more sensitive to small perturbations of protein structure than are spectroscopically derived structural parameters.     

Effect of zinc on L-threonine transport across the jejunum of rabbit

Zinc is an essential trace element for life. Many metalloenzymes involved in the metabolism of carbohydrates, lipids, protein, and nucleic acids require zinc for their functions. The aim of this study was to characterize how zinc acts on the intestinal amino acid absorption in rabbit. Results obtained show that zinc inhibits both L-threonine accumulation in the jejunum tissue, and mucosal-to-serosal transepithelial flux of this amino acid in a dose-dependent way. The inhibition does not increase by a 10-min previous intestinal exposure of the mucosa to the heavy metal, and is not reversed by washing the intestinal tissue with saline solution or 10mM EDTA, but is appreciably reversed with 10mM dithioerythritol. Zinc seems not to modify amino acid diffusion across the intestinal epithelium. The inhibition of intestinal amino acid transport by zinc seems to be of a competitive type, and appears to be a result of impairment of the active transport that is altered by its binding to proteins (prevailing to thiol groups) of the brush-border membrane of enterocytes.     

Characterization of the Band 3 substrate site in human red cell ghosts by NDS-TEMPO,a disulfonatostilbene spin probe: The function of protons in NDS-TEMPO and substrate-anion binding in relation to anion transport

Summary NDS-TEMPO is a specific disulfonatostilbene spin label for the Band 3 substrate site (K. F. Schnell, W. Elbe, J. Käsbauer & E. Kaufmann,Biochim. Biophys. Acta 732:266–275, 1983). The pH dependence of NDS-TEMPO binding and of chloride and sulfate binding was studied in resealed human erythrocyte ghosts. pH was varied from 6.0 to 9.0. The ESR spectra from NDS-TEMPO-labeled red cell ghosts exhibited a strong immobilization of membrane-bound NDS-TEMPO. Changes of pH had no effect upon the mobility of membrane-bound NDS-TEMPO. A mutual competition between NDS-TEMPO binding and the binding of the substrate-anions, chloride and sulfate, was observed throughout the entire pH range. The maximal number of NDS-TEMPO binding sites per cell was in the range of 9.0×10⁵ to 1.10×10⁶ and was found to be insusceptible to changes of pH. The NDS-TEMPO/substrate-site and the chloride/substratesite dissociation constants amounted to 1.25 m and to 17mm and were independent of pH from pH 6.0 to 8.0, while the sulfate/substrate-site dissociation constant displayed a strong pH dependency with a maximum of 50mm at about pH 7.0. The NDS-TEMPO inhibition constants from the chloride and the sulfate flux experiments were 0.5 m (0°C) and 1.8 m (25°C), respectively, and are in close accordance with the NDS-TEMPO/substrate-site dissociation constants. Our studies provide strong evidence for the assumption that NDS-TEMPO binds in fact to the substrate site of Band 3. They show that the strong pH dependence of the chloride and of the sulfate transport cannot result from the pH dependency of substrate-anion binding, but point to the participation of ionizable regulator sites in transport catalysis. These regulator sites seem to be positioned outside the substrate site of the Band 3 transport domain.