Abstract
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.
Highlights
Enterohepatic circulation of bile acids with a specific and highly efficient extraction of bile acids from the intestinal lumen, portal blood, and the primary filtrate in the kidney occurs by Naϩ-dependent transport systems localized in the apical membrane of ileocytes and renal proximal tubule cells and the basolateral membrane of hepatocytes [1, 2]
Strategy to Localize the Bile Acid-binding Site of the Ileal Bile Acid Transporter Protein ileal bile acid transporter (IBAT)—The three-dimensional quantitative structure activity relationship pharmacophore model generated for mammalian Naϩ/bile acid cotransporters revealed the following specific interactions of a bile acid molecule with the transporter ligand-binding site [15]: (i) the methyl groups 18 and 21 and the five-membered ring D of the steroid nucleus occupy two of the three hydrophobic binding sites; (ii) the negatively charged side chain maps the hydrogen bond acceptor site; (iii) the ␣-oriented hydroxy groups at position 7 or 12 act as hydrogen bond donors and (iv) the 3␣hydroxy groups of cis-configurated ring A does not show a specific interaction with any of the five specific binding features of the Naϩ/bile acid cotransporter
The following strategy was used to localize bile acid-binding sites of the ileal Naϩ/bile acid cotransporter: (i) photoaffinity labeling of the intact functional Naϩ/bile acid transport system in rabbit ileal brush border membrane vesicles with 2-(7,7-azo-3␣,12␣dihydroxy-5-[3-3H]cholan-24 oylamino)-ethanesulfonic acid; (ii) enrichment of the radiolabeled monomeric 46-kDa and dimeric 93-kDa forms of IBAT protein by preparative SDS gel electrophoresis; (iii) enzymatic fragmentation of the enriched IBAT protein; (iv) separation of peptide fragments by electrophoresis; and (v) identification of radiolabeled peptides with epitope-specific antibodies raised against the C terminus
Summary
Materials—Photoaffinity labeling was carried out with the photoreactive bile acid analogue 2-(7,7-azo-3␣,12␣-dihydroxy-5-[3-3H]cholan24-oylamino)-ethane-sulfonic acid (specific radioactivity, 20.25 Ci/mmol) synthesized as described elsewhere [16, 17]. 10-l aliquots were removed for determination of the distribution of radioactivity, and 10 l of each fraction were precipitated [19] for analysis of protein composition by SDS-PAGE on 9% precasted gels followed by Serva Blue R-250 staining or blotting and detection of IBAT proteins with anti-IBAT antibodies. The protein in 70 l of those fractions immunoreactive for the monomeric 46 kDa or the dimeric 93 kDa IBAT protein was precipitated by chloroform/methanol, and after SDSPAGE on 9% gels the distribution of radioactivity was determined by slicing of the gels into 2-mm pieces and subsequent liquid scintillation counting after digestion of proteins with Biolute S (Zinsser Analytic GmbH, Frankfurt, Germany). Enzymatic Fragmentation—Brush border membrane vesicles or fractions from preparative SDS gel electrophoresis containing enriched IBAT protein were delipidated with chloroform/methanol [19] and redissolved in 3 l of Tris/HCl buffer (pH 6.8), 0.2% SDS, 0.5% 2-mercaptoethanol/0.005% bromphenol blue. After the addition of 4 ml of Scintillator Quickszint 501 radioactivity was measured by liquid scintillation counting
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