Purification and characterization of estrogen-2/4-hydroxylase activity from rabbit hypothalami: Peroxidase-mediated catechol estrogen formation

Judith S. Mondschein, Roscoe M. Hersey, Judith Weisz

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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 × g supernatant of hypothalamic tissue was linear with time for at least 10 min and with protein concentration to at least 100 Mg/150 μl. It displayed a pH optimum of 6 and an apparent Michaelis-Menteh constant (Km) of 32 μm 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.

Original languageEnglish (US)
Pages (from-to)1105-1112
Number of pages8
JournalEndocrinology
Volume119
Issue number3
DOIs
StatePublished - Sep 1986

All Science Journal Classification (ASJC) codes

  • Endocrinology

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