Structure of guanidinium bicarbonate: a model for the bicarbonate anion binding site of the transferrins

1986 ◽  
Vol 42 (9) ◽  
pp. 1197-1199 ◽  
Author(s):  
D. A. Baldwin ◽  
L. Denner ◽  
T. J. Egan ◽  
A. J. Markwell
Keyword(s):  
Binding Site ◽  
Anion Binding

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.

scholarly journals Mouse Bestrophin-2 Is a Bona fide Cl− Channel

2004 ◽  
Vol 123 (4) ◽  
pp. 327-340 ◽  
Author(s):  
Zhiqiang Qu ◽  
Rodolphe Fischmeister ◽  
Criss Hartzell
Keyword(s):  
Binding Site ◽  
Cell Lines ◽  
Electrostatic Interactions ◽  
Anion Binding ◽  
Wild Type ◽  
Cl Channel ◽  
Mammalian Cell Lines ◽  
Conduction Pathway ◽  
Bona Fide ◽  
Cl Conductance

Bestrophins have recently been proposed to comprise a new family of Cl− channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl− channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca2+-activated Cl− current in all three cell lines (EC50 for Ca2+ = 230 nM). The permeability sequence was SCN−: I−: Br−: Cl−: F− (8.2: 1.9: 1.4: 1: 0.5). Although SCN− was highly permeant, its conductance was ∼10% that of Cl− and SCN− blocked Cl− conductance (IC50 = 12 mM). Therefore, SCN− entered the pore more easily than Cl−, but bound more tightly than Cl−. Mutations in S79 altered the relative permeability and conductance for SCN− as expected if S79 contributed to an anion binding site in the channel. PSCN/PCl = 8.2 ± 1.3 for wild-type and 3.9 ± 0.4 for S79C. GSCN/GCl = 0.14 ± 0.03 for wild-type and 0.94 ± 0.04 for S79C. In the S79 mutants, SCN− did not block Cl− conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN−. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET+ or MTSES− increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl− conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.

scholarly journals Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2781-2786 ◽  
Author(s):  
M Jandrot-Perrus ◽  
KJ Clemetson ◽  
MG Huisse ◽  
MC Guillin
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Protein C ◽  
Anion Binding ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Platelet Receptor ◽  
External Part ◽  
Carboxy Terminal ◽  
Von Willebrand

Abstract We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54–65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.

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