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

ABSTRACT: 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.

SUBMITTER: Mawby WJ

PROVIDER: S-EPMC1158509 | biostudies-other | 1982 Sep

REPOSITORIES: biostudies-other

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Project description:The N-terminal 72 residues of an integral membrane fragment, P5, of the human erythrocyte anion-transport protein, which is known to be directly involved in the anion-exchange process, was shown to have the following amino acid sequence: Met-Val-Pro-Lys-Pro-Gln-Gly-Pro-Leu-Pro-Asn-Thr-Ala-Leu-Leu-Ser-Leu-Val-Leu-Met -Ala-Gly-Thr-Phe-Phe-Phe-Ala-Met-Met-Leu-Arg-Lys-Phe-Lys-Asn-Ser-Ser-Tyr-Phe-Pro-Gly-Lys-Leu-Arg-Arg-Val-Ile-Gly-Asp-Phe-Gly-Val-Pro-Ile-Ser-Ile-Leu-Ile-Met-Val-Leu-Val-Asp-Phe-Phe-Ile-Gln-Asp-Thr-Tyr-Thr-Gln- The structure of this fragment was analysed, with account being taken of the constraints that apply to the folding of integral membrane proteins and the topographical locations of various sites in the sequence. It was concluded that this sequence forms two transmembrane alpha-helices. These are probably part of a cluster of amphipathic transmembrane alpha-helices, which could comprise that part of the protein responsible for transport activity. The presently available evidence relating to the anion-exchange process was considered with the structural features noted in this study and a possible molecular mechanism is proposed. In this model the rearrangement of a network of intramembranous charged pairs mediates the translocation of an anion between anion-binding regions at each surface of the membrane, which are composed of clusters of positively charged amino acids. This model imposes a sequential exchange mechanism on the system. Supplementary material, including Tables and Figures describing the compositions of peptides determined by amino acid analysis and sequence studies, quantitative and qualitative data that provide a residue-by-residue justification for the sequence assignment and a description of modifications to and use of the solid-phase sequencer has been deposited as Supplementary Publication SUP 50123 (12 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained as indicated in Biochem. J. (1983) 209, 5.

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

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