Enzyme-Kinetic and Immunochemical Characteristics of Mouse cDNA-Expressed, Microsomal, and Purified CYP1A1 and CYP1A2

Abstract

Kinetics of benzo[α]pyrene hydroxylase (AHH), 7-methoxyresorufin o-demethylase (MROD), and 7-ethoxyresorufin o-deethylase (EROD) were estimated in microsomes of Hep G2 cells infected with a recombinant vaccinia virus bearing mouse CYPlAl or CYP1A2 cDNAs. The k cat and K m values obtained were compared with those of liver microsonial and purified mouse CYPlAl and CYP1A2. In the matter of AHH activity, the k cat CYP1A1/CYP1A2 ratios were 21.2, 12.3, and 1.5 for expressed, microsomal, and purified CYPs, respectively. As to MROD activity, the k cat CYP1A2/CYPlAl ratio was 3.0 for both expressed and microsomal CYPs and was 8.0 for purified CYPs. As regards EROD activity, the k cat CYP1A2/CYP1A1 ratios were 1.0, 1.1, and 6.25 for expressed, microsomal, and purified CYPs, respectively. Whereas furafylline displayed an isozyme-specific inhibition of CYP1A2-catalyzing MROD and EROD activities, α-naphthoflavone was an equally strong inhibitor of AHH activity of the CYPlAls and MROD activities of the CYP1A2s. Immunodepleted polyclonal anti-CYP1A1(-A2) and anti-CYP1A2(-A1) showed an isozyme-specific immunoblotting and inhibition of mouse CYP1A1 and CYP1A2 while monoclonal antibody (Mab) 1-7-1 displayed a striking difference between its immunoblotting and inhibitory effects. Western blot/densitometry analysis revealed a 4.8 times lower binding of Mab 1-7-1 to cDNA-expressed CYP1A2 than to CYP1A1. The results demonstrate the reliability of the vaccinia virus expression system for studies on the enzymology of mouse CYP1A1 and CYP1A2.