Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.
A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.
Taylor
A M
AM
Biomembrane Structure Unit, Department of Biochemistry, Oxford University, U.K.
Gröbner
G
G
Williamson
P T
PT
Watts
A
A
D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017920:Antiporters / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry; D001665:Binding Sites; D002250:Carbon Radioisotopes; D027963:Chloride-Bicarbonate Antiporters; D003903:Deuterium; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010758:Phosphorus; D011208:Powders; D011489:Protein Denaturation; D013267:Stilbenes / Q000097:blood* / Q000737:chemistry; D013431:Sulfates / Q000037:antagonists & inhibitors / Q000097:blood; D013451:Sulfonic Acids / Q000097:blood* / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
C121274:4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid; D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D002250:Carbon Radioisotopes; D027963:Chloride-Bicarbonate Antiporters; D011208:Powders; D013267:Stilbenes; D013431:Sulfates; D013451:Sulfonic Acids; D010758:Phosphorus; D003903:Deuterium