Characteristic properties of a retinoic acid synthetic cytochrome P-450 purified from liver microsomes of 3-methylcholanthrene-induced rats.

An inducible cytochrome P-450 (P-450) catalyzing retinoic acid synthesis was purified from liver microsomes of 3-methylcholanthrene (3-MC)-treated rats, based on the activity of all-trans-retinoic acid formation from all-trans-retinal. We previously reported that the retinoic acid synthesis by microsomes was catalyzed by a cytochrome P-450-linked monooxygenase system (Tomita et al. (1993) Int. J. Biochem. 25, 1775-1784). This microsomal retinoic acid synthesis in rat liver was induced more than 8-fold by 3-MC. The purified P-450 electophoretically gave a single protein band and its minimum molecular weight was estimated to be 57.2 kDa on SDS-PAGE. The optical spectrum of the oxidized P-450 without retinal revealed it was the low-spin form, and the CO-complex exhibited a maximum peak at 447 nm. The specific activity of the reconstituted P-450-linked monooxygenase system was 29.5 nmol/min per nmol P-450 at pH 7.6 and 37 degrees C. The K(m) and Vmax values for all-trans-retinal were 11.6 microM and 38.5 nmol/min per nmol P-450, respectively. The amino-acid sequence of the N-terminal region of the P-450 was identical to that of rat P-450 1A1 (CYP 1A1). Xenobiotic activities, such as 7-ethoxycoumarin O-deethylase (7-ECOD) and 7-ethoxyresorufin O-deethylase (7-EROD) activities, of the P-450-linked monooxygenase system were specific to the P-450 1A1. The retinoic acid formation in the reconstituted monooxygenase system was specifically inhibited by alpha-naphthoflavone (alpha-NF), which is a P-450 1A1-specific inhibitor, citral, which is a retinoid analogue structurally, and an anti-rat P-450 1A1 antibody. These results further support that the purified P-450 is P-450 1A1. This paper describes that P-450 1A1 was purified and characterized as a retinoic acid synthetic P-450.