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  • Title: Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3.
    Author: Macara IG, Kuo S, Cantley LC.
    Journal: J Biol Chem; 1983 Feb 10; 258(3):1785-92. PubMed ID: 6185490.
    Abstract:
    The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.
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