Target molecular weights for red cell band 3 stilbene and mercurial binding sites.
Radiation inactivation was used to measure the target sizes for binding of disulfonic stilbene anion transport inhibitor 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) and mercurial water transport inhibitor p-chloromercuribenzene sulfonate (pCMBS) to human erythrocytes. The measured target size for erythrocyte ghost acetylcholinesterase was 78 +/- 3 kDa. DBDS binding to ghost membranes was measured by a fluorescence enhancement technique. Radiation (0-26 Mrad) had no effect on total membrane protein and DBDS binding affinity, whereas DBDS binding stoichiometry decreased exponentially with radiation dose, giving a target size of 59 +/- 4 kDa. H2-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2-DIDS, 5 microM) blocked greater than 95% of DBDS binding at all radiation doses. pCMBS binding was measured from the time course of tryptophan fluorescence quenching in ghosts treated with the sulfhydryl reagent N-ethylmaleimide (NEM). Radiation did not affect the kinetics of tryptophan quenching, whereas the total amplitude of the fluorescence signal inactivated with radiation with a target size of 31 +/- 6 kDa. These results support the notion that DBDS and pCMBS bind to the transmembrane domain of erythrocyte band 3 in NEM-treated ghosts and demonstrate that radiation inactivation may probe a target significantly smaller than a covalently linked protein subunit. The small target size for the band 3 stilbene binding site may correspond to the intramembrane domain of the band 3 monomer (52 kDa), which is physically distinct from the cytoplasmic domain (42 kDa).
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D002731:4-Chloromercuribenzenesulfonate / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000838:Anions; D001665:Binding Sites / Q000528:radiation effects; D001692:Biological Transport / Q000528:radiation effects; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D010663:Phenylmercury Compounds / Q000378:metabolism*; D013267:Stilbenes / Q000378:metabolism*; D014508:Urea / Q000378:metabolism; D014867:Water / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D010663:Phenylmercury Compounds; D013267:Stilbenes; D014867:Water; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D002731:4-Chloromercuribenzenesulfonate; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D014508:Urea; D005033:Ethylmaleimide