Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes.

Academic Article

Abstract

  • 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.
  • Keywords

  • Animals, Antibodies, Monoclonal, Cattle, Cetomacrogol, Chenodeoxycholic Acid, Chromatography, Chromatography, Affinity, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Coenzyme A Ligases, Durapatite, Female, Immunoglobulin G, Kinetics, Mass Spectrometry, Microsomes, Liver, Molecular Weight, Rats, Rats, Sprague-Dawley, Substrate Specificity
  • Digital Object Identifier (doi)

    Pubmed Id

  • 23083583
  • Author List

  • Wheeler JB; Shaw DR; Barnes S
  • Start Page

  • 15
  • End Page

  • 24
  • Volume

  • 348
  • Issue

  • 1