Enzyme-kinetic and immunochemical characteristics of mouse cDNA-expressed, microsomal, and purified CYP1A1 and CYP1A2

Abstract

Kinetics of benzo[alpha]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 CYP1A1 or CYP1A2 cDNAs. The kcat and Km values obtained were compared with those of liver microsomal and purified mouse CYP1A1 and CYP1A2. In the matter of AHH activity, the kcat CYP1A1/CYP1A2 ratios were 21.2, 12.3, and 1.5 for expressed, microsomal, and purified CYPs, respectively. As to MROD activity, the kcat CYP1A2/CYP1A1 ratio was 3.0 for both expressed and microsomal CYPs and was 8.0 for purified CYPs. As regards EROD activity, the kcat 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, alpha-naphthoflavone was an equally strong inhibitor of AHH activity of the CYP1A1s 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.