Characterization and partial sequence of di-iodosulphophenyl isothiocyanate-binding peptide from human erythrocyte anion-transport protein

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Abstract
The presumed anion-binding domain of the anion-transport protein from human erythrocyte membranes, using 2,6-di-iodo-4-sulfophenyl isothiocyanate, an inhibitor of anion transport. The 125I-labeled reagent binds covalently to the protein with a half-maximal inhibitory concentration of 86 .mu.M. Treatment of unsealed erythrocyte ghosts with chymotrypsin yielded a membrane-bound fragment (MW 14,500 .+-. 1000) that contained all the protein-bound radioactivity. The binding of the inhibitor to this peptide gave a pattern very similar to that obtained for the effect of the compound on phosphate transport into erythrocytes. The peptide is therefore presumed to be intimately involved in the mediation of anion exchange. Cleavage of the 14,500-MW transmembrane fragment with CNBr resulted in the production of 2 peptides with apparent MW of 8800 and 4700. The 4700-MW peptide is the N-terminal portion of the 14,500-MW peptide. The attachment site for 2,6-di-iodo-4-sulfophenyl isothiocyanate is situated near the C-terminal of the 8800-MW peptide. This locates the inhibitor-binding site near the chymotrypsin cleavage point at the extracellular surface of the membrane. A partial sequence (residues 1-38) of the 8800-MW peptide was obtained.