Substrate dependency of specific and non-specific estrogen-2/4-hydroxylase activities measured by the radio-enzymatic method in rat brain microsomes.
Putative specific and non-specific estrogen-2/4-hydroxylase activities which might affect the radio-enzymatic assay were characterized in terms of their requirements for NADPH and their substrate dependency. Using rat brain microsomes and partially purified rat liver COMT three main sources of estrogen-2/4-hydroxylase activity could be distinguished; a COMT-related component and NADPH-dependent and NADPH-independent microsomal components. The COMT-related activity required NADPH and showed about equal preferences for estrone and estradiol. The NADPH-dependent component was highly specific for estradiol, the relative activities observed with estrone and estriol being 7 and 1% of that observed with estradiol. The NADPH-independent component exhibited substrate saturation, was heat-labile and could not be inhibited by alpha-naphthoflavone or metyrapone. It showed a preference for estrone over estradiol, with estriol being a very poor substrate. These findings indicate that non-enzymatic factors contribute very little to product formation in the radio-enzymatic assay. The specificity of the major NADPH-dependent microsomal component towards estradiol suggests a stereo-specific requirement for the D-ring configuration of this estrogen. The use of no-cofactor blanks in the radio-enzymatic assay may be very important when different estrogens are compared as substrates for estrogen-2/4-hydroxylases.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002394:Catechol O-Methyltransferase / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1; D065607:Cytochrome P-450 Enzyme Inhibitors; D006454:Hemoglobins / Q000494:pharmacology; D006358:Hot Temperature; D008099:Liver / Q000201:enzymology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D006454:Hemoglobins; D009249:NADP; D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C020120:estrogen 2-hydroxylase; D002394:Catechol O-Methyltransferase