Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes.
In the present study, using the C24 bile acid chenodeoxycholic acid as substrate, rat liver bile acid CoA ligase activity (rBAL) was purified 200-fold from detergent-solubilized microsomes using a combination of Q-Sepharose anion exchange, hydroxyapatite, and CM-Sepharose chromatography. Purified rBAL had a molecular weight of 65 kDa by SDS-PAGE analysis. Gel filtration of purified rBAL indicated that rBAL activity forms a complex with other proteins with an apparent aggregate molecular weight of 243 kDa. A monoclonal antibody raised against the 65-kDa protein and covalently coupled to 6B-Sepharose completely absorbed rBAL activity from a semipurified preparation of rat liver microsomes. Western blot analysis confirmed the elution of the 65-kDa protein from the affinity phase at low pH. Optimum rBAL activity was found at pH 8.5, and activity was dependent on the divalent cation Mg2+. In the presence of 50 microM CoA and 2.5 mM MgCl2, kinetic analysis revealed that the apparent K(m)s of ATP and chenodeoxycholic acid of the purified enzyme were 548 +/- 247 and 18.0 +/- 6.2 microM, respectively, and the apparent Vmax was 9.53 +/- 2.0 nmol min-1 mg protein-1. The formation of chenodeoxycholyl-CoA by rBAL was strongly inhibited by hydrophobic bile acids (the C24 monohydroxy bile acid lithocholic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, the C27 homolog of cholic acid), but only weakly by cholic acid. Chenodeoxycholyl-CoA and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oyl-CoA were confirmed as reaction products of purified rBAL by HPLC-electrospray ionization mass spectrometry.
Wheeler
J B
JB
Department of Pharmacology and Toxicology, University of Alabama at Birmingham 35294, USA.
Shaw
D R
DR
Barnes
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002592:Cetomacrogol; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D002845:Chromatography; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003066:Coenzyme A Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D017886:Durapatite; D005260:Female; D007074:Immunoglobulin G; D007700:Kinetics; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D002635:Chenodeoxycholic Acid; D002592:Cetomacrogol; D017886:Durapatite; D003066:Coenzyme A Ligases; C070656:bile acid-CoA ligase