Characterization and partial sequence of di-iodosulphophenyl isothiocyanate-binding peptide from human erythrocyte anion-transport protein.
We investigated the presumed anion-binding domain of the anion-transport protein from human erythrocyte membranes, using 2,6-di-iodo-4-sulphophenyl isothiocyanate, an inhibitor of anion transport. The 125I-labelled reagent binds covalently to the protein with a half-maximal inhibitory concentration of 86 microM. Treatment of unsealed erythrocyte 'ghosts' with chymotrypsin yielded a membrane-bound fragment (mol.wt. 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-mol.wt. transmembrane fragment with CNBr resulted in the production of two peptides with apparent molecular weights of 8800 and 4700. The 4700-mol.wt. peptide is the N-terminal portion of the 14 500-mol.wt. peptide. The attachment site for 2,6-di-iodo-4-sulphophenyl isothiocyanate is situated near the C-terminal of the 8800-mol.wt. 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-mol.wt. peptide was obtained.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D027321:Anion Transport Proteins; D001557:Benzenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D017879:Isothiocyanates; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*
D000345:Affinity Labels; D027321:Anion Transport Proteins; D001557:Benzenesulfonates; D002352:Carrier Proteins; D017879:Isothiocyanates; D010446:Peptide Fragments; D010455:Peptides; C022932:diiodosulfophenylisothiocyanate