The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide.
Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.
Werner
P K
PK
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Reithmeier
R A
RA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000502:physiology*; D000838:Anions / Q000097:blood*; D002234:Carbodiimides / Q000494:pharmacology*; D002951:Citrates / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects*; D013267:Stilbenes / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D002234:Carbodiimides; D002951:Citrates; D007473:Ion Channels; D013267:Stilbenes; C006711:benzoylamido-4'-aminostilbene-2,2'-disulfonate; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D005022:Ethyldimethylaminopropyl Carbodiimide