nap-taurine and 4-4--dinitro-2-2--stilbenedisulfonic-acid

Chloride tracer efflux was measured from intact human erythrocytes into media containing different chloride concentrations and different concentrations of the inhibitors 4,4'-dinitrostilbene-2-2'-disulfonate (DNDS), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), phloretin, and sulfate. The data were analyzed to test whether these inhibitors were mutually exclusive with each other or whether they could bind at the same time. Under the assumption that mutual exclusiveness is due to steric interference, the data can be used to map out the protein surface near the outward-facing anion binding-transport site. It is concluded that there are separate domains for NAP taurine and phloretin that do not overlap with each other or with the chloride binding site. These two domains do, however, overlap with the binding domain for DNDS that, in addition, excludes the binding of chloride and sulfate.

Topics: Anions; Binding Sites; Binding, Competitive; Biological Transport; Chlorides; Erythrocytes; Humans; Phloretin; Stilbenes; Sulfates; Taurine

Phosphate entry into chloride-loaded human erythrocytes is inhibited by treatment of cells with the water-soluble carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the absence of added nucleophile. EAC does not penetrate the erythrocyte membrane or lead to significant intermolecular cross-linking of membrane proteins. At neutral extracellular pH in chloride-free medium, only about 50% of transport is rapidly and irreversibly inhibited, but at alkaline pH, inhibition is more rapid and complete. Inhibition by EAC was reversible in the presence of extracellular NaCl. Modification of membrane sulfhydryl groups does not prevent inhibition of phosphate transport by EAC but almost complete protection is afforded by 4,4-dinitrostilbene-2,2-disulfonic acid, a reversible competitive inhibitor of anion transport. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate, a reversible noncompetitive inhibitor of anion transport did not protect against EAC inhibition of transport but prevented reversal of inhibition in saline medium. Transport inhibition by [3H]EAC did not lead to specific incorporation of radioactivity into Band 3, the anion transport protein. These results suggest that inhibition of anion transport by EAC is due to modification of a carboxylic acid residue in or near the transport site accessible from the external face of the membrane. The subsequent fate of the modified carboxyl residue appears to be sensitive to the orientation of the anion transport site.

Topics: 4-Chloromercuribenzenesulfonate; Anions; Biological Transport; Carbodiimides; Cell Membrane Permeability; Chlorides; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; Humans; Hydrogen-Ion Concentration; Phosphates; Stilbenes; Taurine