A high spin form of cytochrome P-448 highly purified from PCB-treated rats--II. Characteristic requirement of cytochrome b5 for maximum activity.

Abstract A high spin form of cytochrome P-448 (PCB P-448-H), highly purified from microsomes of PCB-treated rats, catalyzed oxidations of several compounds and required cytochrome b 5 for its full activities in all oxidations examined. PCB P-448-H catalyzed the hydroxylation of aniline and O -dealkylations of p -alkoxy derivatives of aniline and nitrobenzene and 7-alkoxy derivatives of coumarin. Among the activities measured, hydroxylation of aniline and O -dealkylation of p -alkoxy derivatives of aniline were catalyzed by PCB P-448-H more efficiently than by PCB P-448-L, which was a low spin form of cytochrome P-448 purified from liver microsomes of PCB-treated rats. In all reactions, PCB P-448-H required cytochrome b 5 for maximum activity. Slight requirements were also seen with PCB P-448-L but varied equivocally depending on the substrates. Cytochrome b 5 showed its maximum effects on p -propoxyaniline O -depropylation activity at a molar ratio of cytochrome b 5 to PCB P-448-H of 1:2. The enhancement by cytochrome b 5 was more pronounced when lower concentrations of either the substrate or NADPH-cytochrome P-450 reductase were added to the reconstituted system. Based on these results, we confirm that PCB P-448-H is a unique form of cytochrome P-448 with respect to the requirements for cytochrome b 5 and is a good probe to study the mechanisms involved in the enhancement of drug oxidations by cytochrome b 5 .