Interactions of inhibitors on anion transporter of human erythrocyte

1987 ◽  
Vol 252 (2) ◽  
pp. C153-C162 ◽  
Author(s):  
O. Frohlich ◽  
R. B. Gunn
Keyword(s):  
Binding Site ◽  
Human Erythrocyte ◽  
Binding Domain ◽  
Human Erythrocytes ◽  
Anion Binding ◽  
Chloride Binding ◽  
Protein Surface ◽  
Anion Transporter ◽  
Transport Site ◽  
Chloride Concentrations

Chloride tracer efflux was measured from intact human erythrocytes into media containing different chloride concentrations and different concentrations of the inhibitors 4,4'-dinitrostilbene-2-2'-disulfonate (DNDS), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), phloretin, and sulfate. The data were analyzed to test whether these inhibitors were mutually exclusive with each other or whether they could bind at the same time. Under the assumption that mutual exclusiveness is due to steric interference, the data can be used to map out the protein surface near the outward-facing anion binding-transport site. It is concluded that there are separate domains for NAP taurine and phloretin that do not overlap with each other or with the chloride binding site. These two domains do, however, overlap with the binding domain for DNDS that, in addition, excludes the binding of chloride and sulfate.

scholarly journals Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site.

1983 ◽  
Vol 81 (1) ◽  
pp. 127-152 ◽  
Author(s):  
O Fröhlich ◽  
C Leibson ◽  
R B Gunn
Keyword(s):  
Conformational Changes ◽  
Positive Charge ◽  
Chloride Ion ◽  
Anion Binding ◽  
Maximal Inhibition ◽  
Exchange Inhibitor ◽  
Anion Transporter ◽  
Transport Site ◽  
Chloride Concentrations ◽  
Net Positive Charge

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min-1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.

scholarly journals The human erythrocyte anion transport protein, band 3. Characterization of exofacial alkaline titratable groups involved in anion binding/translocation.

1992 ◽  
Vol 100 (2) ◽  
pp. 301-339 ◽  
Author(s):  
P J Bjerrum
Keyword(s):  
Ionic Strength ◽  
Binding Site ◽  
Transport System ◽  
Human Erythrocyte ◽  
Binding Constant ◽  
Anion Transport ◽  
High Ionic Strength ◽  
Anion Binding ◽  
Asymmetry Factor ◽  
Sensitive Group

Chloride self-exchange across the human erythrocyte membrane at alkaline extracellular pH (pHO) and constant neutral intracellular pH (pH(i)) can be described by an exofacial deprotonatable reciprocating anion binding site model. The conversion of the transport system from the neutral to the alkaline state is related to deprotonation of a positively charged ionic strength- and substrate-sensitive group. In the absence of substrate ions ([ClO] = 0) the group has a pK of approximately 9.4 at constant high ionic strength (equivalent to approximately 150 mM KCl) and a pK of approximately 8.7 at approximately zero ionic strength. The alkaline ping-pong system (examined at constant high ionic strength) demonstrates outward recruitment of the binding sites with an asymmetry factor of approximately 0.2, as compared with the inward recruitment of the transport system at neutral pHO with an asymmetry factor of approximately 10. The intrinsic half-saturation constant for chloride binding, with [Cli] = [Clo], increased from approximately 30 mM at neutral to approximately 110 mM at alkaline pHO. The maximal transport rate was a factor of approximately 1.7 higher at alkaline pHO. This increase explains the stimulation of anion transport, the "modifier hump," observed at alkaline pHO. The translocation of anions at alkaline pHO was inhibited by deprotonation of another substrate-sensitive group with an intrinsic pK of approximately 11.3. This group together with the group with a pK of approximately 9.4 appear to form the essential part of the exofacial anion binding site. The effect of extracellular iodide inhibition on chloride transport as a function of pHO could, moreover, be simulated if three extracellular iodide binding constants were included in the model: namely, a competitive intrinsic iodide binding constant of approximately 1 mM in the neutral state, a self-inhibitor binding constant of approximately 120 mM in the neutral state, and a competitive intrinsic binding constant of approximately 38 mM in the alkaline state.

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function

1992 ◽  
Vol 72 (4) ◽  
pp. 1611-1615 ◽  
Author(s):  
R. E. Weber
Keyword(s):  
Binding Sites ◽  
Ionic Composition ◽  
Oxygen Affinity ◽  
Chloride Concentration ◽  
Anion Binding ◽  
Chloride Binding ◽  
Ph Values ◽  
Two Temperatures ◽  
Ethanesulfonic Acid ◽  
Chloride Concentrations

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.

scholarly journals Relative contributions of the slippage and tunneling mechanisms to anion net efflux from human erythrocytes.

1984 ◽  
Vol 84 (6) ◽  
pp. 877-893 ◽  
Author(s):  
O Fröhlich
Keyword(s):  
Membrane Potential ◽  
Conformational Change ◽  
Chemical Nature ◽  
Human Erythrocytes ◽  
Alternative Process ◽  
Anion Transporter ◽  
Transport Site ◽  
Cl Conductance ◽  
Channel Type

The rates of anion net efflux from gramicidin-treated erythrocytes in the presence of a K gradient were measured at 25 degrees C, pH 7.8, as rates of loss of Ki. The experiments served to estimate the relative contributions of two hypothetical mechanisms to Cl net efflux at low extracellular Cl concentrations. Cl, Br, and NO3 net effluxes were measured into media of different Cl, Br, or NO3 concentrations, respectively, to determine and compare the relative rates of the extracellular anion-inhibitable components. They were 48, 160, and 230 mmol/(kg Hb X min), respectively, at a membrane potential of about -90 mV. This indicates that the anion-inhibitable efflux is not due solely to the return translocation of the empty transport site ("slippage") because slippage should be independent of the chemical nature of the anion. Cl net efflux was also measured as a function of the intracellular Cl concentration into media containing either 0 or 50 mM Cl. Under both conditions, net efflux was linearly dependent on Cli between 30 and 300 mM Cli and was 0 when back-extrapolated to 0 Cli. This observation is not compatible with the slippage process, which under these conditions would have been expected to be independent of Cli above 15 mM Cli. It was concluded that slippage contributes negligibly to Cl net efflux even at low extracellular anion concentrations and that the alternative process of "tunneling"--that is, movement of the anion through the anion transporter without a conformational change in a channel-type behavior--is the major, if not the sole, mechanism underlying Cl conductance.

Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3

1999 ◽  
Vol 277 (4) ◽  
pp. C791-C799 ◽  
Author(s):  
Sonya Bahar ◽  
Christopher T. Gunter ◽  
Cheryl Wu ◽  
Scott D. Kennedy ◽  
Philip A. Knauf
Keyword(s):  
Binding Site ◽  
External Surface ◽  
Band 3 ◽  
Low Ph ◽  
Anion Binding ◽  
Band 3 Protein ◽  
Proton Binding ◽  
Monovalent Anion ◽  
Woodward’S Reagent K ◽  
Transport Site

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward’s reagent K ( N-ethyl-5-phenylisoxazolium-3′-sulfonate; WRK). Measurements of Cl− binding by35Cl-NMR show that external Cl− binds to band 3 even when Cl− transport is inhibited ∼95% by WRK modification of Glu-681. This indicates that the external Cl− binding site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl− binding even when WRK is bound to Glu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facing Cl−transport site are located nearer to the external surface of the membrane than Glu-681.

Elemental analysis of human erythrocytes

1989 ◽  
Vol 47 ◽  
pp. 242-243
Author(s):  
S. A. Livesey ◽  
A. A. del Campo ◽  
E. S. Griffey ◽  
D. Ohlmer ◽  
T. Schifani ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Elemental Analysis ◽  
Human Erythrocyte ◽  
Spectroscopic Analysis ◽  
Model System ◽  
Human Erythrocytes ◽  
List Type ◽  
Chemical Fixation ◽  
Ethanol Dehydration ◽  
Lowicryl K4m

The aim of this study is to compare methods of sample preparation for elemental analysis. The model system which is used is the human erythrocyte. Energy dispersive spectroscopic analysis has been previously reported for cryofixed and cryosectioned erythrocytes. Such work represents the reference point for this study. The use of plastic embedded samples for elemental analysis has also been documented. The work which is presented here is based on human erythrocytes which have been either chemically fixed and embedded or cryofixed and subsequently processed by a variety of techniques which culminated in plastic embedded samples.Heparinized and washed erythrocytes were prepared by the following methods for this study :(1). Chemical fixation in 4% paraformaldehyde/0.25% glutaraldehyde/0.2 M sucrose in 0.1 M Na cacodylate, pH 7.3 for 30 min, followed by ethanol dehydration, infiltration and embedding in Lowicryl K4M at -20° C.

scholarly journals Mapping the ankyrin-binding site of the human erythrocyte anion exchanger

1989 ◽  
Vol 264 (16) ◽  
pp. 9665-9672 ◽  
Author(s):  
L Davis ◽  
S E Lux ◽  
V Bennett
Keyword(s):  
Binding Site ◽  
Anion Exchanger ◽  
Human Erythrocyte

Phosphate Binding to Isolated Chloroplast Coupling Factor (CF1)

1988 ◽  
Vol 43 (3-4) ◽  
pp. 213-218 ◽  
Author(s):  
Bernhard Huchzermeyer
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Inorganic Phosphate ◽  
Coupling Factor ◽  
Binding Domain ◽  
Atp Binding ◽  
Phosphate Binding ◽  
Chloroplast Coupling Factor ◽  
Single Binding Site ◽  
Site Binding

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a Kd of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ-32P]ATP the amount of 32P bound per CF1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

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