TY - JOUR
T1 - Catechol estrogen formation by brain tissue
T2 - A comparison of the release of tritium from [2-3H] estradiol with [6, 7-3H]2-hydroxyestradiol formation from [6, 7-3H]estradiol by rabbit hypothalami in vitro
AU - Hersey, Roscoe M.
AU - Gunsalus, Pamela
AU - Lloyd, Tom
AU - Weisz, Judith
N1 - Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 1981/12
Y1 - 1981/12
N2 - In the course of establishing an assay for estrogen-2-hydroxylase activity, a detailed comparison was made between the formation of tritiated water (3H2O) and [6, 7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6, 7-3H-labeled estradiol, respectively. The amounts of both 3H2O and [6, 7-3H]2-OHE2 formed were stimulated severalfold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S2, 17, 500 × g supernatant, for tritium release; S3, 100, 000 × g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100, 000 × g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 HIM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 μM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/212 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using 3H2O formation from [2-3H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE2 is inhibited, thereby making it practical to quantify enzyme activity on the basis of theamount of catechol estrogen formed. Evidence is also presented that the release of 3H2O from [2-3H]estradiol by hypothalamic tissue, unrelated to 2-OHE2 formation, may be enzymatically mediated.
AB - In the course of establishing an assay for estrogen-2-hydroxylase activity, a detailed comparison was made between the formation of tritiated water (3H2O) and [6, 7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6, 7-3H-labeled estradiol, respectively. The amounts of both 3H2O and [6, 7-3H]2-OHE2 formed were stimulated severalfold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S2, 17, 500 × g supernatant, for tritium release; S3, 100, 000 × g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100, 000 × g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 HIM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 μM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/212 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using 3H2O formation from [2-3H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE2 is inhibited, thereby making it practical to quantify enzyme activity on the basis of theamount of catechol estrogen formed. Evidence is also presented that the release of 3H2O from [2-3H]estradiol by hypothalamic tissue, unrelated to 2-OHE2 formation, may be enzymatically mediated.
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U2 - 10.1210/endo-109-6-1902
DO - 10.1210/endo-109-6-1902
M3 - Article
C2 - 6273121
AN - SCOPUS:0019814646
SN - 0013-7227
VL - 109
SP - 1902
EP - 1911
JO - Endocrinology
JF - Endocrinology
IS - 6
ER -