Inhibition of inorganic anion transport across the human red blood cell membrane by chloride-dependent association of dipyridamole with a stilbene disulfonate binding site on the band 3 protein.
The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding.
Legrum
B
B
Max-Planck-Institut für Biophysik, Frankfurt am Main, F.R.G.
Passow
H
H
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D000495:Allosteric Site; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000838:Anions; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002712:Chlorides / Q000097:blood / Q000494:pharmacology*; D004176:Dipyridamole / Q000097:blood* / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D010710:Phosphates / Q000097:blood*; D013267:Stilbenes / Q000378:metabolism*; D013431:Sulfates / Q000097:blood*
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D002712:Chlorides; D010710:Phosphates; D013267:Stilbenes; D013431:Sulfates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D004176:Dipyridamole; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid