Specific interaction of the water transport inhibitor, pCMBS, with band 3 in red blood cell membranes.
The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4'-dibenzoamido-2,2'-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D001834:Body Water / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D008433:Mathematics; D010663:Phenylmercury Compounds / Q000494:pharmacology*; D013997:Time Factors
D001457:Anion Exchange Protein 1, Erythrocyte; D010663:Phenylmercury Compounds; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D002731:4-Chloromercuribenzenesulfonate; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D005033:Ethylmaleimide