Catechol Estrogen-Forming Enzyme of Brain: Demonstration of a Cytochrome P450 Monooxygenase

Abstract

A catechol estrogen-forming enzyme was demonstrated in rat brain microsomes using a sensitive radioenzymatic assay. This method is based on conversion of the relatively labile catechol estrogens to their stable O-methylated derivatives. By employing a methyl donor of high specific activity (S-adenosyl-L-[methyl-3H]methionine), a partially purified preparation of catechol-O-methyltransferase (COMT), and selective solvent extraction this method was extremely sensitive. The specificity of the assay was established by subjecting the O-methylated product to TLC in several solvent systems and further confirmed by mass spectral analysis of the chromatographed product. The enzymatic activity of rat brain is optimal using reduced NADP and is inhibited by well known inhibitors of P450-dependent mixed function oxidases, CO and SKF-525A [.beta.-diethylaminoethyl diphenylpropyl acetate hydrochloride]. The subcellular distribution, cofactor requirements, and the effects of various inhibitors strongly suggest that the enzymatic activity of brain is similar if not identical to cytochrome P450-dependent monooxygenases found in peripheral tissues. The microsomal enzyme was found to form catechols from naturally occurring estrogens and synthetic estrogenic compounds such as diethylstilbestrol and 17.alpha.-ethynylestradiol, but not from non-aromatic steroids such as testosterone, progesterone and cholesterol. The normal occurrence of catechol estrogens and the relatively high enzymatic activity in brain suggests a physiological role for the catechol estrogen-forming enzyme.