Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein.
Flufenamate, non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6 . 10(-7) M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2 . 10(-7) M) to 8.5 . 10(5) sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10(-4) M) to a second set of sites (4.6 . 10(7) per cell). Pretreatment of cells with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport of [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D001798:Blood Proteins / Q000378:metabolism*; D002712:Chlorides / Q000097:blood; D002918:Chymotrypsin / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005439:Flufenamic Acid / Q000097:blood*; D006801:Humans; D007700:Kinetics; D010206:Papain / Q000494:pharmacology; D011402:Pronase / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D001798:Blood Proteins; D002712:Chlorides; D005439:Flufenamic Acid; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain; D011402:Pronase