Identification of a ligand-binding site in the Na+/bile acid cotransporting protein from rabbit ileum.
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.
Kramer
W
W
Aventis Pharma Deutschland GmbH, D-65926 Frankfurt am Main, Germany. Werner.Kramer@aventis.com
Girbig
F
F
Glombik
H
H
Corsiero
D
D
Stengelin
S
S
Weyland
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001391:Azo Compounds / Q000737:chemistry; D001647:Bile Acids and Salts / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007082:Ileum / Q000378:metabolism*; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent; D019920:Photoaffinity Labels; D011817:Rabbits; D027981:Symporters
D001391:Azo Compounds; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D008024:Ligands; D029363:Organic Anion Transporters, Sodium-Dependent; D019920:Photoaffinity Labels; D027981:Symporters; C071908:sodium-bile acid cotransporter; C037641:(7,7-azo-3,12-dihydroxy-5-cholan-24-oyl)-2-aminoethanesulfonic acid; D002918:Chymotrypsin