Hydroxylations of Bile Acids by Reconstituted Systems from Rat Liver Microsomes

Abstract

Abstract The 7α-hydroxylation of taurodeoxycholic acid and the 6β-hydroxylation of taurochenodeoxycholic acid and lithocholic acid were studied with a reconstituted system from rat liver microsomes consisting of partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, a synthetic phosphatidylcholine and NADPH or an NADPH-generating system. 7α-Hydroxylase activity was observed with cytochrome P-450 from either male or female rats whereas 6β-hydroxylase activity was observed only with cytochrome P-450 from male rats. With male rats, the ratio between 7α-hydroxylase activity and 6β-hydroxylase activity was considerably higher in the reconstituted system than in the original microsomal fraction. The 6β-hydroxylase activity of the reconstituted system was more stable than the 7α-hydroxylase activity during prolonged storage at -20°. The rate of the hydroxylations in the reconstituted system was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. The lipid dependence of the system could be correlated to some extent with the amount of phospholipids in the cytochrome P-450 fraction. Preparations of cytochrome P-450 that had been recentrifuged at 100,000 x g just prior to incubation showed a greater requirement for lipid and had a lower ratio between phospholipid and cytochrome P-450 than before centrifugation. Addition of superoxide dismutase to the reconstituted system did not inhibit 7α or 6β hydroxylation. Treatment with phenobarbital, known to increase 7α- as well as 6β-hydroxylase activity in rat liver microsomes 3- to 4-fold, increased the specific catalytic activity (hydroxylase activity per nanomole of cytochrome P-450) of cytochrome P-450 2- to 5-fold. The catalytic activity of NADPH-cytochrome P-450 reductase per unit of NADPH-cytochrome c reductase activity was about the same regardless of the source of the preparation. The possibility was discussed that different catalytic types of phenobarbitalinducible cytochrome P-450 are involved in 7α- and 6β-hydroxylation of bile acids.