Year-end Sale % OFF 
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.
Highlights
Since both Cs’ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these resultsindicate that afterbinding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface
Since we investigated by titration of the tryptophanemission of red cell already haveshown the IOVs to be impermeant to Cs', these membranes against eosin maleimide and a novel nonfluorescent stilbenedisulfonate derivative, HzDBDS
We have examined the susceptibility of membrane tryptophan and eosin 5-maleimidefluorescence toquenching by
Summary
Fluorescence lifetimes were measured on an SLM 4800 phase shift spectrofluorometer, using 505 nm excitation modulated at 6, 18, and. Emission was monitored through a Corning 3-67 filter Both phase shifts and changes in modulation were used to determine lifetimes, as described previously [7]. The data are well fit by a single exponential decay (7,). Titrating the eosin emission of the membrane-bound fragments and of the supernatant fragments against CsCl showed that only 2.1% of the total quenching by Cs’ is attributable to the nonspecifically bound eosin present on chymotrypsin-released protein fragments. The eosin maleimide was found to be attached exclusively to the membrane-bound fragment of Band 3 (Fig. 1). Neither Cs’ nor eosin maleimide appears able to penetrate the red cell membrane, and at least 85% of the maleimide is bound at (or near) the external transport site of Band 3. The results support the hypothesis that reaction of eosin maleimide at the external transport site of Band 3 leads to the exposure of the fluorophore to the opposite (cytoplasmic) side of the membrane
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
