Characterization of matrix-bound Band 3, the anion transport protein from human erythrocyte membranes.
Band 3 (Mr = 95,000), the anion transport protein of human erythrocyte membranes exists primarily as a dimer in solutions of nonionic detergents such as octaethylene glycol mono-n-dodecyl ether (C12E8). The role of the oligomeric structure of Band 3 in the binding of [14C]4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), an inhibitor of anion transport (Ki = 1-2 microM), was studied by characterizing the interaction of BADS with dimers and monomers of Band 3 covalently attached to p-mercuribenzoate-Sepharose 4B. BADS bound to matrix-bound Band 3 dimers with an affinity of approximately 3 microM at a stoichiometry of 1 BADS molecule/Band 3 monomer, in agreement with the BADS binding characteristic of Band 3 in the membrane and in solutions of C12E8. Band 3 dimers could be attached to the matrix via one subunit by limiting the amount of p-chloromercuribenzoate on the Sepharose bead. Matrix-bound monomers were formed by dissociation of the dimers with dodecyl sulfate or guanidine hydrochloride. Complete removal of the denaturants allowed formation of refolded Band 3 monomers since the matrix-bound subunits could not reassociate. These refolded Band 3 monomers were unable to bind BADS. Release of the monomers from the matrix with 2-mercaptoethanol allowed reformation of dimers with recovery of the BADS binding sites. These results suggest that the dimeric structure of Band 3 is required for BADS binding and that the BADS binding sites may be at the interface between the two halves of the Band 3 dimer.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004910:Erythrocyte Membrane / Q000032:analysis*; D006801:Humans; D046911:Macromolecular Substances; D013267:Stilbenes / Q000378:metabolism; D013329:Structure-Activity Relationship
D001457:Anion Exchange Protein 1, Erythrocyte; D046911:Macromolecular Substances; D013267:Stilbenes; C006711:benzoylamido-4'-aminostilbene-2,2'-disulfonate