Bile acids are conjugated with the amino acids glycine and taurine by the liver, a metabolic step catalyzed by two enzymes, bile acid CoA ligase (BAL) and bile acid CoA: amino acid N-acyltransferase (BAT). The long term goal of this project is to use molecular biology approaches to establish the physiological importance of amidation of bile acids, and hence to determine the role of bile acid amidation in diseases of the gastrointestinal and hepatobiliary systems. In the present application: (1) Since a deficiency or either BAL or BAT will create a defect in bile acid N-acyl amidation, cDNAs encoding BAL will be isolated from a rat liver cDNA library, characterized, sequenced and expressed in a suitable expression system. cDNAs encoding rat BAL will be used to isolate the corresponding cDNAs in human and mouse liver cDNA libraries. (2) The amino acid residues in BAT that characterize its choice of amino acid substrates (either taurine alone, or glycine/ taurine) will be investigated. To identify critical amino acid residues for BAT activity, a putative rat liver BAT cDNA (Kan-1) will be expressed to determine its substrate specificity- if it is a taurine- specific BAT, other rBAT cDNAs will be cloned, sequenced and expressed. Substrate protection experiments will be carried out on BATs to determine which cysteine is the site of covalent attachment of the bile acid CoA substrate. HPLC-MS and MALDI-time-of-flight mass spectrometry will identify the cysteine-containing tryptic peptide that is protected by addition of cholyl CoA from alkylation by N-ethylmaleimide. Other amino acid residues associated with the catalytic properties of BAT will be identified by chemical modification, and by DNA site-specific metagenesis experiments. A potential new pathway of metabolism of glycine-conjugated bile acids will be explored. (3) A knockout model of BAT deficiency will be developed so as to provide an experimental system for testing the physiological effects of loss of BAT activity. The organization of the Baat gene will be determined in order to transform pluripotent embryonic stem cells from the 129 mice with a Baat positive/negative selection vector and identify cells containing the BAT knockout.
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