cDNA-directed expression of rat P450s IIA1 and IIA2. Catalytic activities toward steroids and xenobiotics and comparison with the enzymes purified from liver

T Aoyama, K Korzekwa, T Matsunaga, K Nagata, J Gillette, HV Gelboin and FJ Gonzalez

Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

Cytochromes P-450IIA1 and IIA2 are steroid hydroxylases that are expressed in rat liver. The cDNAs for these enzymes were recently sequenced and compared. To study and compare the catalytic activities of IIA1 and IIA2, their cDNAs were inserted into a vaccinia virus expression vector and expressed in human hepatoma Hep G2 cells. IIA2 was able to efficiently catalyze ethoxycoumarin O-deethylation and propoxycoumarin O-depropylation, while IIA1 was inactive toward these substrates. Neither enzyme could catalyze ethoxy- and pentoxyresorufin dealkylation reactions. Both cDNA-expressed IIA1 and IIA2 metabolize testosterone and these activities were quantitatively and qualitatively similar to those obtained with the purified enzymes. IIA1 produced 7 alpha-hydroxy, 6 alpha-hydroxy, and delta 6-testosterone at ratios of 9:0.5:0.5 while IIA2 formed 15 alpha-hydroxytestosterone, an unknown metabolite and four minor metabolites. Progesterone metabolism was also studied. IIA1 yielded a 9.5:0.5 ratio of 7 alpha-hydroxy and 6 alpha- hydroxyprogesterone, while IIA2 produced at least six metabolites. These studies establish the conditions and verify the reliability and accuracy of the vaccinia virus expression system for studies on the enzymology of IIA1 and IIA2.

Volume 18, Issue 3, pp. 378-382, 05/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics