scholarly journals Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system.

1984 ◽  
Vol 83 (5) ◽  
pp. 703-725 ◽  
Author(s):  
P A Knauf ◽  
N A Mann
Keyword(s):  
Anion Exchange ◽  
Dissociation Constants ◽  
Energy Difference ◽  
Protein Band ◽  
Band 3 ◽  
Niflumic Acid ◽  
Inhibitory Potency ◽  
The Asymmetry ◽  
High Concentrations ◽  
Transport Site

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl-, the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride-loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.

NIP- and NAP-taurine bind to external modifier site of AE1 (band 3), at which iodide inhibits anion exchange

1995 ◽  
Vol 269 (2) ◽  
pp. C410-C416 ◽  
Author(s):  
P. A. Knauf ◽  
L. J. Spinelli
Keyword(s):  
Anion Exchange ◽  
Blood Cells ◽  
Inhibitory Effect ◽  
Band 3 ◽  
Reaction Site ◽  
Inhibitory Potency ◽  
Human Red Blood Cells ◽  
Inhibitory Site ◽  
Probable Reaction ◽  
Transport Site

External iodide (I-o) inhibits AE1 (band 3)-mediated anion exchange in human red blood cells by binding to a noncompetitive inhibitory site, the external halide modifier site. External N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) and N-(4-isothiocyano-2-nitrophenyl)-2-aminoethyl sulfonate (NIP-taurine) also inhibit Cl- exchange noncompetitively. Increasing I-o decreases the inhibitory potency of NIP-taurine in a competitive fashion; this effect is not due to I- binding to the transport site, which has little effect on the NIP-taurine affinity. Bis(sulfosuccinimidyl)-suberate (BSSS) abolishes the noncompetitive inhibitory effect of I-o and greatly reduces the inhibitory effect of NAP-taurine. Together with previous work, these data suggest that external halides, such as I-, Br-, and probably also Cl-, bind to the same noncompetitive inhibitory site as do NAP- and NIP-taurine and that these reagents can be used to label the halide modifier site. Lys-539, a probable reaction site of BSSS, lies within the same segment of AE1 that is labeled by NAP-taurine and thus may be part of the modifier site.

scholarly journals Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein.

1984 ◽  
Vol 83 (5) ◽  
pp. 657-681 ◽  
Author(s):  
W Furuya ◽  
T Tarshis ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Competitive Inhibitor ◽  
Transport Protein ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Anion Exchange Protein ◽  
Ping Pong ◽  
Chloride Exchange ◽  
Transport Site

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in two different conformations, one in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular chloride should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS is a competitive inhibitor of chloride exchange that does not cross the membrane, it must bind only to the Eo form. Thus, an increase in Eo should cause an increase in H2DIDS inhibition. When intracellular chloride was increased at constant extracellular chloride, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the chloride gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. These data provide support for the ping-pong model and also demonstrate that chloride gradients can be used to change the orientation of the transport protein.

Anion exchange protein (band 3) in human erythrocytes characterized by different abnormalities

1993 ◽  
Vol 220 (2) ◽  
pp. 211-217 ◽  
Author(s):  
A. Giuliani ◽  
S. Marini ◽  
L. Ferroni ◽  
S.G. Condo' ◽  
M.T. Ramacci ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Band 3 ◽  
Human Erythrocytes ◽  
Anion Exchange Protein ◽  
Exchange Protein

scholarly journals Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry.

1984 ◽  
Vol 83 (5) ◽  
pp. 683-701 ◽  
Author(s):  
P A Knauf ◽  
F Y Law ◽  
T Tarshis ◽  
W Furuya
Keyword(s):  
Anion Exchange ◽  
Chloride Transport ◽  
Chloride Concentration ◽  
Competitive Inhibitor ◽  
Disulfonic Acid ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Chloride Exchange ◽  
A Site ◽  
Transport Site

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.

scholarly journals Flexibility of the Cytoplasmic Domain of the Anion Exchange Protein, Band 3, in Human Erythrocytes

2001 ◽  
Vol 81 (6) ◽  
pp. 3363-3376 ◽  
Author(s):  
Scott M. Blackman ◽  
Eric J. Hustedt ◽  
Charles E. Cobb ◽  
Albert H. Beth
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Human Erythrocytes ◽  
Anion Exchange Protein ◽  
Exchange Protein

Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation

1993 ◽  
Vol 264 (5) ◽  
pp. C1144-C1154 ◽  
Author(s):  
P. A. Knauf ◽  
N. M. Strong ◽  
J. Penikas ◽  
R. B. Wheeler ◽  
S. Q. Liu
Keyword(s):  
Inhibitory Concentration ◽  
Potent Inhibitor ◽  
Chloride Concentration ◽  
Competitive Inhibitor ◽  
Band 3 ◽  
Reversible Inhibitor ◽  
Red Cell ◽  
Inhibitory Potency ◽  
A Site ◽  
Transport Site

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.

Flufenamic acid senses conformation and asymmetry of human erythrocyte band 3 anion transport protein

1989 ◽  
Vol 257 (2) ◽  
pp. C277-C289 ◽  
Author(s):  
P. A. Knauf ◽  
L. J. Spinelli ◽  
N. A. Mann
Keyword(s):  
Conformational Changes ◽  
External Medium ◽  
Band 3 ◽  
Anion Transport ◽  
Flufenamic Acid ◽  
Band 3 Protein ◽  
Inhibitory Potency ◽  
External Site ◽  
Transport Molecules ◽  
Transport Site

With Cl as substrate, the human red blood cell anion transport (band 3) protein can exist in four conformations: Ei, with the transport site facing the cytoplasm; Eo, with the transport site facing the external medium; and ECli and EClo, the corresponding forms loaded with Cl. Flufenamic acid (FA), an inhibitor that binds to an external site different from the transport site, binds to Eo with a dissociation constant of 0.0826 +/- 0.0049 (SE) microM. Binding of iodide or sulfate to the external-facing transport site reduces the affinity by 1.66 or 14.3-fold, respectively. Changing from Eo to Ei lowers the affinity by 3.7-fold, and binding of cytoplasmic iodide to Ei further decreases the affinity by 5.5-fold. Thus changes in orientation of the transport site and substrate binding, even at the opposite side of the membrane, cause sufficient conformational changes in band 3 to affect FA binding substantially. If the possible effects of Cl binding to the transport site on FA affinity are estimated from the iodide data, the dependence of FA inhibitory potency on Cl concentrations inside and outside the cell suggests that there are at least 6.5 times as many inward-facing as outward-facing Cl-loaded transport sites. This information can be used to calculate the distribution of capnophorin among the various conformations under different circumstances and to devise conditions for recruiting the transport molecules toward a particular conformation.

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

1979 ◽  
Vol 73 (4) ◽  
pp. 493-514 ◽  
Author(s):  
S Grinstein ◽  
L McCulloch ◽  
A Rothstein
Keyword(s):  
Mobile Element ◽  
Band 3 ◽  
Anion Transport ◽  
Permeability Barrier ◽  
Irreversible Inhibitor ◽  
Red Cell Membrane ◽  
Intact Cells ◽  
High Concentrations ◽  
Cytoplasmic Side ◽  
Transport Site

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.

Identification of a 185-kDa band 3-related polypeptide in oxyntic cells

1989 ◽  
Vol 257 (3) ◽  
pp. C537-C544 ◽  
Author(s):  
H. A. Thomas ◽  
T. E. Machen ◽  
A. Smolka ◽  
R. Baron ◽  
R. R. Kopito
Keyword(s):  
Anion Exchange ◽  
Anion Exchanger ◽  
Polyclonal Antibodies ◽  
Basolateral Membrane ◽  
Gastric Gland ◽  
Protein Band ◽  
Band 3 ◽  
Gastric Glands ◽  
Mouse Erythrocyte ◽  
Exchange Activity

Polyclonal antibodies to the purified mouse erythrocyte anion exchange protein (band 3) and to a conserved COOH-terminal peptide of mouse band 3 (alpha-Ct) recognized a single major 185-kDa polypeptide in immunoblots of a membrane fraction prepared from rabbit gastric glands. Competition studies revealed that the epitopes shared between the rabbit gastric 185-kDa antigen and the approximately 100-kDa mouse erythrocyte band 3 protein are restricted to the COOH-terminal domain of band 3, which is known to contain the catalytic site for anion exchange activity. Immunofluorescence microscopy was used to demonstrate that this band 3-related polypeptide is associated with the plasma membrane in a subpopulation of gastric gland cells composed exclusively of oxyntic cells, as judged by the coincidence of immunofluorescence with alpha-Ct and with a monoclonal antibody to the gastric H+-K+-ATPase. This alpha-Ct-reactive antigen was further localized to the cytoplasmic face of the basolateral membrane of oxyntic cells, which correlates well with the physiologically determined site of anion exchange activity. These data demonstrate the presence in gastric oxyntic cells of a novel member of the family of proteins related to the erythrocyte anion exchanger. The possibility that the 185-kDa polypeptide is an anion exchanger is discussed.

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