The influence of two anion-transport inhibitors, 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate, on the self-association of erythrocyte band 3 protein.
4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate potently inhibit the erythrocyte anion transporter. These inhibitors act by binding, with a 1:1 stoichiometry, to the band 3 transport protein. We have studied, by sedimentation equilibrium analysis in an analytical ultracentrifuge, the effect of the two closely related stilbenedisulfonates on the state of association of band 3 in the nonionic detergent nonaethyleneglycol lauryl ether. It was found that covalent binding of 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate to band 3 did not significantly disturb the monomer/dimer/tetramer association equilibrium shown by the unliganded protein. An entirely different result was obtained after addition of 4,4'-dibenzoylstilbene-2,2'-disulfonate to the protein, at both low and high chloride concentrations. The amount of band 3 dimer in the samples increased with increasing inhibitor concentration c1, and for c1 > or = 15 microM virtually all of the protein was present as dimer. After removal of the inhibitor (by gel filtration or dialysis), the original monomer/dimer/tetramer distribution of the band 3 protein was restored. Our data show that the (noncovalent) binding of 4,4'-dibenzoylstilbene-2,2'-disulfonate drastically changes the coupling between band 3 protomers. In addition, a reversible change in the state of association of band 3 induced by ligand binding is demonstrated.
Schuck
P
P
Institut für Biophysik, J. W. Goethe-Universität, Frankfurt am Main, Germany.
Legrum
B
B
Passow
H
H
Schubert
D
D
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000187:drug effects* / Q000378:metabolism; D000838:Anions; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D006454:Hemoglobins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid