Enzymic Studies of Bile Acid Metabolism

Abstract

Abstract 6β-Hydroxylation of chenodeoxycholic acid-24-14C and taurochenodeoxycholic acid-24-14C was demonstrated in vitro by cell-free preparations of rat liver. The enzymic activity, requiring O2 and NADPH, was located in the microsomes prepared in 0.1 m phosphate buffer and could be stimulated by addition of the 105,000 x g supernatant fluid. The microsomal fraction prepared in 0.01 m phosphate buffer had full activity and was not further stimulated by the 105,000 x g supernatant fluid. A clear extract made by suspending this microsomal pellet in 1.0 m phosphate and subsequently centrifuging it at 105,000 x g contained the complete 6β-hydroxylase system. The activity of the 1.0 m phosphate extract was inhibited by sulfhydryl reagents, Cu++, cytochrome c, and CO. The inhibition by CO could be reversed by light. The presence of cytochrome b5 and the CO-binding pigment, P-450, in the extract was detected by differential spectrophotometry.