Molecular cloning and expression of rat liver bile acid CoA ligase.
Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.
Falany
Charles N
CN
Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. charles.falany@ccc.uab.edu
Xie
Xiaowei
X
Wheeler
James B
JB
Wang
Jin
J
Smith
Michelle
M
He
Dongning
D
Barnes
Stephen
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary; D003902:Detergents / Q000378:metabolism; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D003902:Detergents; D003066:Coenzyme A Ligases; C070656:bile acid-CoA ligase