alpha-chymotrypsin and (7-7-azo-3-12-dihydroxy-5-cholan-24-oyl)-2-aminoethanesulfonic-acid

Reabsorption of bile acids occurs in the terminal ileum by a Na(+)-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6-7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na(+)/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.

Topics: Amino Acid Sequence; Animals; Azo Compounds; Bile Acids and Salts; Binding Sites; Carrier Proteins; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Ileum; In Vitro Techniques; Ligands; Molecular Sequence Data; Organic Anion Transporters, Sodium-Dependent; Photoaffinity Labels; Rabbits; Symporters