The long term goals of this project are to understand the biochemistry, molecular biology and cell biology in humans of the two enzymes, bile acid CoA synthetase (hBAS) and bile acid CoA: amino acid N-acyltransferase (hBAT), which are responsible for the biosynthesis of the amino acid conjugates of bile acids. The naturally occurring C24 bile acids in human bile are found almost exclusively as their amino acid conjugates. These bile acid conjugates are by far the principal solutes of human bile and their secretion is the major determinant of bile flow. In bile and the small intestine, bile acid amino acid conjugates form mixed micellar complexes with water-insoluble lipid species such as phospholipids, monoacylglycerols, free fatty acids and cholesterol, thereby performing important physiological functions both in normal health and disease. To fully understand how hBAS and hBAT are involved in the synthesis of these conjugates in humans requires knowledge of the chemistry of these enzymes (their primary amino acid sequence), their genetic organization (nucleotide sequence of cDNAs encoding them and the gene(s) from which they were derived and their chromosomal localization), and the regions of each enzyme which are responsible for their catalytic properties.
The specific aims of this application are (1) to characterize the chemical and biochemical properties of purified hBAT by determining whether it has Ping-Pong Bi Bi reaction mechanism, evaluating the importance of its three cysteine residues (cys-235, cys-372 and cys-373) to enzyme function, and examining the rules which govern its substrate specificity; (2) to purify hBAS, characterize its biochemical and chemical properties (as for hBAT) and raise specific polyclonal anti-hBAS antibodies in rabbits; (3) to use the hBAT cDNA to determine the heterogeneity of RNA sequences related to hBAT and the chromosomal localization of the hBAT gene. When these specific aims have been accomplished, it will be possible in future granting periods to clone hBAS and to further analyze which regions of the hBAS and hBAT are essential for enzyme catalytic function and substrate specificity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK046390-01A2
Application #
2145589
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1994-08-15
Project End
1997-07-31
Budget Start
1994-08-15
Budget End
1995-07-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Pharmacology
Type
Schools of Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Barnes, Stephen; Shonsey, Erin M; Eliuk, Shannon M et al. (2008) High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function. Biochem Soc Trans 36:1037-44
Kang, Dae-Joong; Ridlon, Jason M; Moore 2nd, Doyle Ray et al. (2008) Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases. Biochim Biophys Acta 1781:16-25
Styles, Nathan A; Falany, Josie L; Barnes, Stephen et al. (2007) Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver. J Lipid Res 48:1305-15
Shonsey, Erin M; Wheeler, James; Johnson, Michelle et al. (2005) Synthesis of bile acid coenzyme a thioesters in the amino acid conjugation of bile acids. Methods Enzymol 400:360-73
Shonsey, Erin M; Sfakianos, Mindan; Johnson, Michelle et al. (2005) Bile acid coenzyme A: amino acid N-acyltransferase in the amino acid conjugation of bile acids. Methods Enzymol 400:374-94
He, Dongning; Barnes, Stephen; Falany, Charles N (2003) Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization. J Lipid Res 44:2242-9
Sfakianos, Mindan K; Wilson, Landon; Sakalian, Michael et al. (2002) Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase. J Biol Chem 277:47270-5
Falany, Charles N; Xie, Xiaowei; Wheeler, James B et al. (2002) Molecular cloning and expression of rat liver bile acid CoA ligase. J Lipid Res 43:2062-71
King 3rd, L; Barnes, S; Glufke, U et al. (2000) The enzymatic formation of novel bile acid primary amides. Arch Biochem Biophys 374:107-17

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