Hydroxylations of bile acids by reconstituted systems from rat liver microsomes.

The 7a-hydroxylation of taurodeoxycholic acid and the 6&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. 7a-Hydroxylase activity was observed with cytochrome P-450 from either male or female rats whereas 6/3-hydroxylase activity was observed only with cytochrome P-450 from male rats. With male rats, the ratio between 7cY-hydroxylase activity and 6fi-hydroxylase activity was considerably higher in the reconstituted system than in the original microsomal fraction. The 6/3-hydroxylase activity of the reconstituted system was more stable than the SLYhydroxylase 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 7a or 6fi hydroxylation. Treatment with phenobarbital, known to increase 7cuas well as 6P-hydroxylase activity in rat liver microsomes 3to 4-fold, increased the specific catalytic activity (hydroxylase activity per nanomole of cytoc@ome P-450) of cytochrome P-450 2to S-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 7aand 6@-hydroxylation of bile acids.