Chloride binding to the anion transport binding sites of band 3. A 35Cl NMR study.
Band 3 is an integral membrane protein that exchanges anions across the red cell membrane. Due to the abundance and the high turnover rate of the band 3 transport unit, the band 3 system is the most heavily used ion-transport system in a typical vertebrate organism. Here we show that 35Cl NMR enables direct and specific observation of substrate Cl- binding to band 3 transport sites, which are identified by a variety of criteria: (a) the sites are inhibited by 4,4'- dinitrostilbene -2,2'-disulfonate, which is known to inhibit competitively Cl- binding to band 3 transport sites; (b) the sites have affinities for 4,4'- dinitrostilbene -2,2'-disulfonate and Cl- that are quantitatively similar to the known affinities of band 3 transport sites for these anions; and (c) the sites have relative affinities for Cl-, HCO-3, F-, and I- that are quantitatively similar to the known relative affinities of band 3 transport sites for these anions. The 35Cl NMR assay also reveals a class of low affinity Cl- binding sites (KD much greater than 0.5 M) that are not affected by 4,4'- dinitrostilbene -2,2'-disulfonate. These low affinity sites may be responsible for the inhibition of band 3 catalyzed anion exchange that has been previously observed at high [Cl-]. In the following paper the 35Cl NMR assay is used to resolve the band 3 transport sites on opposite sides of the membrane, thereby enabling direct observation of the transmembrane recruitment of transport sites.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000838:Anions; D001665:Binding Sites; D001693:Biological Transport, Active; D002712:Chlorides / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008954:Models, Biological; D011485:Protein Binding; D013267:Stilbenes
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D002712:Chlorides; D013267:Stilbenes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid