PURIFICATION AND CHARACTERIZATION OF A RAT-LIVER BILE-ACID COENZYME-ALIGASE FROM RAT-LIVER MICROSOMES

In the present study, using the C-24 bile acid chenodeoxycholic acid a s substrate, rat Liver bile acid CoA ligase activity (rBAL) was purifi ed 200-fold from detergent-solubilized microsomes using a combination of Q-Sepharose anion exchange, hydroxyapatite, and CM-Sepharose chroma tography, Purified rBAL had a molecular weight of 65 kDa by SDS-PAGE a nalysis, Gel filtration of purified rBAL indicated that rBAL activity forms a complex with other proteins with an apparent aggregate molecul ar weight of 243 kDa, A monoclonal antibody raised against the 65-kDa protein and covalently coupled to 6B-Sepharose completely absorbed rBA L activity from a semipurified preparation of rat liver microsomes, We stern blot analysis confirmed the elution of the 65-kDa protein from t he 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 prese nce of 50 mu M CoA and 2.5 mM MgCl2, kinetic analysis revealed that th e apparent K(m)s of ATP and chenodeoxycholic acid of the purified enzy me were 548 +/- 247 and 18.0 +/- 6.2 mu M, respectively, and the appar ent V-max was 9.53 +/- 2.0 nmol min(-1) mg protein(-1) The formation o f chenodeoxycholyl-CoA by rBAL was strongly inhibited by hydrophobic b ile acids (the C-24 monohydroxy bile acid lithocholic acid and 3 alpha ,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, the C-27 homolo g of cholic acid), but only weakly by cholic acid, Chenodeoxycholyl-Co A and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oyl-CoA were confirmed as reaction products of purified rBAL by HPLC-electrosp ray ionization mass spectrometry. (C) Academic Press.