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.
Tomita
S
S
Department of Biochemistry, Kagawa Medical School, Japan. stomita@mailbox.kms.ac.jp
Okuyama
E
E
Ohnishi
T
T
Ichikawa
Y
Y
D000080462:Acyclic Monoterpenes; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001571:Benzoflavones / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015786:Cytochromes b5 / Q000032:analysis; D004790:Enzyme Induction; D007074:Immunoglobulin G / Q000494:pharmacology; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology*; D039821:Monoterpenes; D051381:Rats; D017207:Rats, Sprague-Dawley; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D017421:Sequence Analysis; D013729:Terpenes / Q000494:pharmacology; D014212:Tretinoin / Q000378:metabolism*; D015262:Xenobiotics / Q000378:metabolism
D000080462:Acyclic Monoterpenes; D000906:Antibodies; D001571:Benzoflavones; D007074:Immunoglobulin G; D007527:Isoenzymes; D039821:Monoterpenes; D013729:Terpenes; D015262:Xenobiotics; D008748:Methylcholanthrene; D014212:Tretinoin; C011512:alpha-naphthoflavone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D012172:Retinaldehyde; C007076:citral