title: Purification and characterization of estrogen-2/4-hydroxylase activity from rabbit hypothalami: peroxidase-mediated catechol estrogen formation.
abstract: Estrogen-2/4-hydroxylase (E-2/4-H) activity of rabbit hypothalamic tissue was previously found to be localized in the soluble subcellular fraction. In the present study, the enzymatic activity responsible for catechol estrogen formation in this subcellular fraction of the rabbit hypothalamus was purified by ammonium sulfate fractionation, ion exchange chromatography, and chromatofocusing. E-2/4-H activity was found to be associated with a group of hemoproteins with peroxidase activity. The characteristics of this hypothalamic E-2/4-H activity were reestablished in light of a peroxidatic mechanism for catechol estrogen formation. Organic hydroperoxides stimulated E-2/4-H activity, presumably by serving as oxidizing cosubstrate required for peroxidase-mediated reactions. E-2/4-H activity in a 17,500 X g supernatant of hypothalamic tissue was linear with time for at least 10 min and with protein concentration to at least 100 micrograms/150 microliter. It displayed a pH optimum of 6 and an apparent Michaelis-Menten constant (Km) of 32 microM with respect to estradiol. The amounts of 4-hydroxyestradiol formed were comparable to those of 2-hydroxyestradiol. The characteristics established in this study for the peroxidase-type E-2/4-H were distinct from those of the particulate, NADPH-dependent 2-hydroxylases found in rat liver and in porcine blastocyst and ovary. These differences provide a basis for differentiating between the two types of enzymatic activity that can lead to catechol estrogen formation in vitro.
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001555:Benzene Derivatives / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D002393:Estrogens, Catechol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007031:Hypothalamus / Q000201:enzymology*; D007525:Isoelectric Focusing; D011136:Polysorbates; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000302:isolation & purification*; D013997:Time Factors
chemical_list: D001555:Benzene Derivatives; D002393:Estrogens, Catechol; D011136:Polysorbates; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585499:Cyp1b1 protein, rat; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; C020120:estrogen 2-hydroxylase; C007164:cumene hydroperoxide