Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum

Kramer, Werner; Girbig, Frank; Gutjahr, Ulrike; Kowalewski, Simone

doi:10.1042/bj3060241

Cited by 32 publications

(29 citation statements)

References 49 publications

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“…The Na-dependent uptake involves the ileal apical bile acid transporter ASBT, the most important transporter in humans and mice (26,27). This transporter has different affinities to different bile acid species and operates as a homotetramer that interacts with bile acid transport protein (28). The Na-independent uptake involves the Oatp3 transporter, also with different affinities to different bile acid species, which localizes to the apical membrane of jejunal enterocytes (29).…”

Section: Discussionmentioning

confidence: 99%

Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprit

Sehayek

Hagey

Fung

et al. 2006

Journal of Lipid Research

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An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower b-muricholic acid (bMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %bMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38-73 cM (9KK) and 68-73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %bMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %bMC was decreased. Sterol 12a-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or highlow biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68-73 cM) major determinant in males, and a more proximal (38-68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible. In humans and mice, biliary bile acid composition affects the enterohepatic metabolism of lipids, including the synthesis and transport of bile acids across hepatocytes, the excretion of biliary lipids, and the absorption of sterols from the intestines (1-6). In humans, the two major primary bile acids are chenodeoxycholic acid (CDCA), a dihydroxy bile acid, and cholic acid (CA), a trihydroxy bile acid. The difference between them is hydroxylation of the 12 carbon of the sterol ring, which is carried out by the enzyme sterol 12a-hydroxylase (CYP8B1). As a result, CDCA is more hydrophobic and CA is more hydrophilic. In mice, CA is one of the major primary bile acids; it is formed by CYP8B1-mediated 12 hydroxylation. Muricholic acids comprise the other major murine bile acids, and they are formed from CDCA by 6 hydroxylation and 7 epimerization to generate a-and b-muricholic acid (bMC), with bMC predominating. bMC is even more hydrophilic than CA. Although the mouse does not have CDCA as a major species, it is possible to use this model to identify genes that influence the 12 hydroxylation pathway and uptake of bile acids from the intestines by studying the proportions of CA and bMC in bile. Identifying the genes that influence 12 hydroxylation and bile acid uptake from the intestines is particularly important because they determine the proportions of CA and CDCA and hence the hydrophobicity of the bile acid pool in humans.In humans, the proportions of CA and CDCA vary between individuals (7, 8), and there is some evidence for genetic control. For example, studies in monozygotic and dizygotic twins found a significant pair-...

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Section: Discussionmentioning

confidence: 99%

Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprit

Sehayek

Hagey

Fung

et al. 2006

Journal of Lipid Research

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“…For an unequivocal localization of the ligand binding a covalent cross-linking of the ligand to the peptide backbone and subsequent determination of the cross-linking position is necessary. The ileal Na ϩ /bile acid cotransport system in intact ileal tissue is a heteromeric protein complex of 451 kDa composed of several transporter units and associated ileal lipidbinding proteins (3,6). Because the structure and composition of the ileal Na ϩ /bile acid cotransport system in recombinant cell lines is not known and because functional differences of transporters found in intact tissues and recombinant cell lines were reported (22), we decided to photolabel the ileal Na ϩ /bile acid cotransporter in situ using freshly isolated rabbit ileal brush border membrane vesicles with subsequent determination of the attachment site.…”

Section: Discussionmentioning

confidence: 99%

“…Materials-Photoaffinity labeling was carried out with the photoreactive bile acid analogue 2-(7,7-azo-3␣,12␣-dihydroxy-5␤-[3␤- 3 H]cholan-24-oylamino)-ethane-sulfonic acid (specific radioactivity, 20.25 Ci/mmol) synthesized as described elsewhere (16,17). Acrylamide, N,NЈ-methylene bisacrylamide and Serva Blue R-250 were from Serva (Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…The functional ileal and hepatic Na ϩ /bile acid cotransport systems are protein complexes composed of several transporter protein subunits (3,4). The ileal Na ϩ /bile acid cotransport system with a molecular mass of 451 Ϯ 35 kDa contains additionally several subunits of the cytoplasmically attached ileal lipid-binding protein (ILBP) 1 (3,5,6). The mammalian Na ϩ /bile acid cotransport proteins have 347-362 amino acids with 35-37% identity and 46 -48% similarity between the ileal and the hepatic transporter (Refs.…”

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confidence: 99%

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Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Kramer

Girbig

Glombik

et al. 2001

Journal of Biological Chemistry

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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.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 Na ϩ -dependent transport systems localized in the apical membrane of ileocytes and renal proximal tubule cells and the basolateral membrane of hepatocytes (1, 2). The functional ileal and hepatic Na ϩ /bile acid cotransport systems are protein complexes composed of several transporter protein subunits (3, 4). The ileal Na ϩ /bile acid cotransport system with a molecular mass of 451 Ϯ 35 kDa contains additionally several subunits of the cytoplasmically attached ileal lipid-binding protein (ILBP) 1 (3, 5, 6). The mammalian Na ϩ /bile acid cotransport proteins have 347-362 amino acids with 35-37% identity and 46 -48% similarity between the ileal and the hepatic transporter (Refs. 7-12 and GenBank TM accession numbers 254357 and AJ131361). Originally a topology model with seven transmembrane domains has been suggested (2), but novel investigations with in vitro translation approaches favor a topology model with nine transmembrane domains (13). Investigations of the substrate specificity of the ileal and hepatic Na ϩ /bile acid cotransporters using recombinant cell lines and brush border membrane vesicles (14) enabled us to generate the first predictive three-dimensional quantitative structure activity relationships pharmacophore model for mammalian Na ϩ /bile acid cotransporting proteins (15). As a next step to the understanding of intestinal bile acid absorption we describe in the present manuscript the identification of a bile acid-binding site of the rabbit ileal Na ϩ /cotransporter localized to the C-term...

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“…Functional analysis of the Na ϩ /bile acid cotransport systems in ileocytes and hepatocytes by target size analysis revealed that these transport systems are in their functional states protein complexes composed of several subunits (29 -31). The rabbit ileal Na ϩ /bile acid cotransport system revealed a functional molecular mass of 451 Ϯ 35 kDa (31) being probably composed of four integral membrane proteins of 93 kDa (identified as dimers of the Na ϩ /bile acid cotransporting protein (19)) and four cytoplasmically attached 14-kDa peripheral membrane proteins (19,22,31). The 14-kDa protein, the ileal lipid-binding protein (ILBP), 1 has originally been identified as a predominant bile acid binder in the cytosol of ileocytes (21,(32)(33)(34)(35)(36)(37)(38)(39).…”

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confidence: 99%

Identification of the Bile Acid-binding Site of the Ileal Lipid-binding Protein by Photoaffinity Labeling, Matrix-assisted Laser Desorption Ionization-Mass Spectrometry, and NMR Structure

Kramer¹,

Sauber²,

Baringhaus³

et al. 2001

Journal of Biological Chemistry

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Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum

Cited by 32 publications

References 49 publications

Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprit

Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprit

Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Identification of the Bile Acid-binding Site of the Ileal Lipid-binding Protein by Photoaffinity Labeling, Matrix-assisted Laser Desorption Ionization-Mass Spectrometry, and NMR Structure

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