CLONING, EXPRESSION, AND CHROMOSOMAL LOCALIZATION OF MOUSE-LIVER BILE-ACID COA-AMINO ACID N-ACYLTRANSFERASE

A mouse liver lambda Zap XR cDNA library was screened using the coding region of human bile acid CoA:amino acid N-acyltransferase (BAT) cDNA as a probe. Ten positive clones were isolated and purified, two of wh ich apparently possessed complete open reading frames for BAT based on sequence analysis of the ends of;he cDNAs. One clone (mBAT#9) was sel ected for sequence analysis and characterization. mBAT#9 is 1869 basep airs in length and the full-length cDNA possesses a 189 basepair 5'-no ntranslated region, an open-reading frame of 1260 basepairs, and a 404 basepair 3'-nontranslated region followed by a poly(A) tail. The open -reading frame codes for a 420 amino acid protein with a calculated mo lecular mass of 46,525 daltons. The structural gene for mBAT was mappe d to mouse Chromosome 4. The amino acid sequence of mBAT is 69% identi cal and 84% similar to that of hBAT, and 86% identical and 95% similar to that of kan-1, a putative rat liver BAT. Enzymatically active mBAT was expressed in E. coli using the bacterial expression vector pKK233 -2. Immunoblot analysis of expressed mBAT with rabbit anti-human BAT p olyclonal antibodies detected a single protein with a molecular mass o f approximately 45,000 daltons. Cytosol from cells transformed with mB AT#9/pKK233-2 possessed significant amounts of BAT-catalyzed conjugati ng activity with taurine as substrate but the expressed enzyme did not use glycine or fluoro-beta-alanine as substrates. The K-m value for t aurine was 1.9 mM +/- 0.1 mM in reactions with cholyl CoA as a cosubst rate. The specificity of mBAT for taurine as a substrate was confirmed by the demonstration, using HPLC-electrospray ionization mass spectro metry: that mouse gallbladder bile contained only taurine conjugates o f bile acids. The identification of the types of amino acid conjugates of bile acids present in mouse bile had not been previously reported. These results indicate that a taurine-specific form of BAT has been c loned and expressed from mouse liver.