Interactions between transport inhibitors at the anion binding sites of the band 3 dimer

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Abstract
Evidence is presented that the binding of aromatic disulfonates to the external transport sites of the red cell anion-exchange protein (band 3) can exhibit negative cooperativity. Fluorescence resonance energy transfer was used to compare the affinities of an aromatic disulfonate 4,4''-bis-(4-nitro-2,1,3-benzoxadiazolyl)dihydrostilbene-2,2''-disulfonate [H2(NBD)2DS] for empty band 3 dimers (in which neither external transport site is occupied) and for half-filled dimers (in which 1 site/dimer is occupied by a covalently attached fluorescent stilbenedisulfonate). H2(NBD)2DS apparently binds to the external anion transport site since it is a potent inhibitor of [35S]sulfate influx into red cells (Ki = 20-50 nM), binds reversibly to .apprx. 1 site/band 3 monomer (1.6 .times. 106 sites/cell), and is displaced by covalent labeling with a disulfonic stilbene. The affinity of H2(NB-D)2DS for membranes in which 80% of the transport sites are occupied by covalently attached 4-benzamido-4''-isothio-cyanostilbene-2,2''-disulfonate (BIDS) was .apprx. 1 order of magnitude lower than that for unmodified membranes. When a similar proportion of the transport sites on red cells was blocked by reaction with BIDS, [35S]sulfate was taken up with a lower Vmax but with a Km identical with that observed for unmodified cells, suggesting that no subunit interactions are necessary for transport. To test whether the observed negative cooperativity of aromatic disulfonate binding could be ascribed simply to steric hindrance, the distance between transport sites was measured by fluorescence resonance energy transfer. H2(NBD)2DS and eosin maleimide were used as acceptors, with BIDS as donor. Transfer efficiencies were determined by donor fluorescence quenching, by acceptor fluorescence enhancement, and from donor lifetime changes. Uncertainties in the distance were estimated from measured depolarization factors. The donor-acceptor distance was only 28-52 .ANG.. Since the probes are large molecules, they could be very close together. The observed negative cooperativity might be explained by overlapping sites. The subunits of a band 3 dimer apparently transport anions independently. Access to the transport sites may be provided by a cavity between the subunits.

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