Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3 |
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Authors: | James A. Dix A. S. Verkman A. K. Solomon |
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Affiliation: | (1) Biophysical Laboratory, Department of Physiology and Biophysics, Harvard Medical School, 02115 Boston, Massachusetts;(2) Department of Chemistry, State University of New York, 13901 Binghamton, New York;(3) Present address: Division of Nephrology, University of California, 94143 San Francisco, California |
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Abstract: | Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK1=0.67±0.16 m,k2=1.6±0.7 sec–1,k–2=0.17±0.09 sec–1,K1=6.3±1.7 m,k2=9±4 sec–1 andk–2=7±3 sec–1. The apparent dissociation constants of chloride from band 3,KCl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3. |
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Keywords: | red cell anion transport disulfonic stilbene band 3 |
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