Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance.
We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.
Liu
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D
Department of Biophysics, University of Rochester, New York 14642, USA.
Knauf
P A
PA
Kennedy
S D
SD
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism*; D002713:Chlorine; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013267:Stilbenes / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D013267:Stilbenes; D002713:Chlorine; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C033531:eosin maleimide; D004801:Eosine Yellowish-(YS)