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Papers In Press, published online ahead of print September 16, 2002
J. Lipid Res., doi:10.1194/jlr.M200260-JLR200
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Submitted on July 3, 2002
Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294
Corresponding Author: charles.falany{at}ccc.uab.edu
Bile acid CoA ligase (BAL) catalyzes the first step in the conjugation of bile acids with amino acids. To investigate the role of BAL, a cDNA encoding this enzyme was isolated, expressed and characterized. Degenerate oligonucleotide primers were synthesized based on the amino acid sequence of peptides obtained from proteolytic and chemical digestion of affinity purified rat liver BAL (rBAL). These primers were used to isolate a partial rBAL cDNA by PCR using rat liver total RNA as a template. The rBAL cDNA fragment was then used to screen a rat liver cDNA library, thereby isolating 3 clones. Sequencing of these putative rBAL cDNA clones led to the identification of a cDNA (rBAL-1) possessing an open reading frame of 2,070 bases translating a protein of 690 aa with a mass of 75,960 Da. Identity of the cDNA was established by the following findings: (1) the rBAL-1 open reading frame encoded the peptides obtained by chemical sequencing of the purified rBAL protein; (2) expressed rBAL-1 generated a protein with a mass similar to purified rBAL; and (3) expressed rBAL-1 had enzymatic properties that were comparable to rBAL isolated from rat liver. The rBAL-1 amino acid sequence displayed homology to mouse and rat very long chain fatty acid CoA ligases (VLACS) and in particular to an unidentified mouse liver VLACS-related protein. rBAL-1 also showed high homology to a human VLACS that possesses bile acid CoA ligase activity. Palmitoleic and oleic acids, but not trans or corresponding saturated fatty acids, showed potent inhibition of rBAL’s chenodeoxycholic acid CoA ligase activity; however, expressed rBAL did not form palmitoleic or oleic acid CoA esters. These results indicate that a rat liver BAL cDNA has been isolated and expression of the rBAL cDNA results in a catalytically active enzyme capable of bile acid CoA ester synthesis.
Revised on August 21, 2002
Accepted on September 5, 2002
Molecular cloning and expression of rat liver bile acid CoA ligase
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