Diverse function of aromatase and the N-terminal sequence deleted form

作者：Osawa Y^*; Higashiyama T; Toma Y; Yarborough C

来源：Journal of Steroid Biochemistry and Molecular Biology, 1997, 61(3-6): 117-126.

The diverse function of human placental aromatase including estradiol 6 alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6 alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6 alpha, 7 alpha-H-3(2),4-C-14]estradiol, 7-ethoxycoumarin, and [N-methyl-H-3(3)]cocaine. 6 alpha-Hydroxy[7 alpha-H-3,4-C-14]estradiol was isolated as the metabolite of estradiol and the H-3-water release method based on the 6 alpha-H-3 label was established. The initial rate kinetics of the 6 alpha-hydroxylation gave K-m of 4.3 mu M, V-max of 4.02 nmol min(-1)mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6 alpha-hydroxylation and showed the K-i of 0.15 mu M, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K-m of 200 mu M, V-max of 12.5 nmol min(-1)mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the H-3-release method, giving K-m of 670 mu M, V-max of 4.76 nmol min(-1)mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1)mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

• 出版日期1997-4