Kinetic studies of inhibition of estradiol 2-and 16α-hydroxylases in rat liver microsomes with various cytochrome P-450 inhibitors

Abstract

Kinetic studies of inhibition of estradiol 2- and 16α-hydroxylase activities in male rat liver microsomes with cytochrome P-450 inhibitors, α-naphthoflavone, DL-aminoglutethimide, SKF-525A and metyrapone, were extensively carried out. All of the inhibitors competitively blocked the two enzyme activities. The former three inhibitors preferentially inhibited the 16α-hydroxylase activity while the reverse result was obtained in the case of metyrapone, and SKF-525A was the most potent inhibitors for the two enzyme among the four inhibitors. The kinetic data, the apparent K i 's for the four inhibitors and K m 's for the substrate estradiol in the assays, along with the inhibition results with carbon monooxide suggest that different forms of cytochrome P-450 may be involved in the two hydroxylations. Kinetic parameters of the two hydroxylase activities in female rat liver microsomes were then determined to be an apparent K m of 23.0 and 158 μM and V max of 99.0 and 5.65 pmol/min/mg protein for the 2-hydroxylation and the 16α-hydroxylation, respectively. The kinetic data show that the 2-hydroxylation may be quantitatively an exclusive hydroxylative pathway in estrogen metabolism in female.