Metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes. enzyme properties and kinetic parameters in the formation of estrogens and 17β-hydroxy-1,4-androstadien-3-one

Leon Milewich, Debra J. Bradfield, L. Dean Coe, Bettie Sue S Masters, Paul C. Macdonald

Research output: Contribution to journalArticle

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Abstract

In this investigation the metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes was evaluated. The experiments were conducted with the substrate [3H]-1,4-androstadiene-3,17-dione, which was synthesized using a tritium-hydrogen keto-enol exchange technique. [4-14C]-l,4-Androstadiene-3, 17-dione and [4-14C]-17β-hydroxy-1,4-androstadien-3-one also were synthesized and used, together with [14C]-estrone and [14C]-estradiol-17β, as internal recovery standards for quantification. The radiolabeled metabolites identified from incubation mixtures with placental microsomes were estrone, estradiol-17β and 17β-hydroxy-1,4-androstadien-3-one; two other metabolites, one less polar than the substrate and the other more polar than estradiol-17β, were observed but not investigated further. The enzymatic conversions required either NADPH or NADH as cofactor and there was no apparent tritium loss in the transformation of substrate to products. The rates of formation of metabolites were linear with time for at least 30 min and with the amount of microsomal protein up to 15 mg. Tbe apparent pH optimum for placental microsomal estrogen synthetase was 7.5, and the apparent optimum pH for the 17β-hydroxysteroid oxidoreductase ranged from pH6.0 to pH7.4. The apparent optimal temperature for the estrogen synthetase activity was 37°C and for 17β-hydroxysteroid oxidoreductase it ranged from 37°C to 58°C. The apparent Km of estrogen synthetase for 1,4-androstadiene-3,17-dione was 1.3μM, and the apparent Km of 17β-hydroxysteroid oxidoreductase for 1,4-androstadiene-3, 17-dione was 24 μM. The apparent Km of estrogen synthetase for NADPH and NADH were 0.24 mM and 1.0mM, respectively; the apparent Km of placental microsomal 17β-hydroxysteroid oxidoreductase for NADH was 0.5 mM and that for NADPH was 0.67 mM. Androstenedione was found to be a competitive inhibitor of [3H]-1,4-androstadiene-3,17-dione aromatization (Ki) = 0.75 μM androstenedione) and of 17β-hydroxysteroid oxidoreduction (Ki) = 17μM androstenedione). Fifty per cent inhibition of estrogen synthetase and of 17β-hydroxysteroid oxidoreductase activities were obtained with an androstenedione concentration of 80 μM. The specific activity of NADPH-dependent cytochrome c reductase of placental microsomes was 22 nmol × mg-1 protein × min-1. The dependency of placental microsomal [3H]-1,4-androstadiene-3,17-dione aromatization on cytochrome P-450 and upon NADPH-dependent cytochrome c reductase was demonstrated using both anti-human and anti-porcine hepatic microsomal NADPH-dependent cytochrome c (P-450) reductase antibodies. The estrogen synthetase and NADPH-dependent cytochrome c reductase activities were inhibited greatly by the microsomal specific antibodies, whereas non-immune γ-globulin had little effect on enzyme activity. The 17β-hydroxysteroid oxidoreductase activity was affected only slightly by these antibodies.

Original languageEnglish (US)
Pages (from-to)1115-1125
Number of pages11
JournalJournal of Steroid Biochemistry
Volume14
Issue number11
DOIs
StatePublished - 1981
Externally publishedYes

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Hydroxysteroids
Microsomes
Aromatase
Kinetic parameters
Metabolism
Oxidoreductases
Estrogens
NADPH-Ferrihemoprotein Reductase
NADP
Androstenedione
Enzymes
Cytochromes c
NAD
Estradiol
Tritium
Estrone
Metabolites
Aromatization
Androstadienes
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

Metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes. enzyme properties and kinetic parameters in the formation of estrogens and 17β-hydroxy-1,4-androstadien-3-one. / Milewich, Leon; Bradfield, Debra J.; Coe, L. Dean; Masters, Bettie Sue S; Macdonald, Paul C.

In: Journal of Steroid Biochemistry, Vol. 14, No. 11, 1981, p. 1115-1125.

Research output: Contribution to journalArticle

Milewich, Leon ; Bradfield, Debra J. ; Coe, L. Dean ; Masters, Bettie Sue S ; Macdonald, Paul C. / Metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes. enzyme properties and kinetic parameters in the formation of estrogens and 17β-hydroxy-1,4-androstadien-3-one. In: Journal of Steroid Biochemistry. 1981 ; Vol. 14, No. 11. pp. 1115-1125.
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abstract = "In this investigation the metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes was evaluated. The experiments were conducted with the substrate [3H]-1,4-androstadiene-3,17-dione, which was synthesized using a tritium-hydrogen keto-enol exchange technique. [4-14C]-l,4-Androstadiene-3, 17-dione and [4-14C]-17β-hydroxy-1,4-androstadien-3-one also were synthesized and used, together with [14C]-estrone and [14C]-estradiol-17β, as internal recovery standards for quantification. The radiolabeled metabolites identified from incubation mixtures with placental microsomes were estrone, estradiol-17β and 17β-hydroxy-1,4-androstadien-3-one; two other metabolites, one less polar than the substrate and the other more polar than estradiol-17β, were observed but not investigated further. The enzymatic conversions required either NADPH or NADH as cofactor and there was no apparent tritium loss in the transformation of substrate to products. The rates of formation of metabolites were linear with time for at least 30 min and with the amount of microsomal protein up to 15 mg. Tbe apparent pH optimum for placental microsomal estrogen synthetase was 7.5, and the apparent optimum pH for the 17β-hydroxysteroid oxidoreductase ranged from pH6.0 to pH7.4. The apparent optimal temperature for the estrogen synthetase activity was 37°C and for 17β-hydroxysteroid oxidoreductase it ranged from 37°C to 58°C. The apparent Km of estrogen synthetase for 1,4-androstadiene-3,17-dione was 1.3μM, and the apparent Km of 17β-hydroxysteroid oxidoreductase for 1,4-androstadiene-3, 17-dione was 24 μM. The apparent Km of estrogen synthetase for NADPH and NADH were 0.24 mM and 1.0mM, respectively; the apparent Km of placental microsomal 17β-hydroxysteroid oxidoreductase for NADH was 0.5 mM and that for NADPH was 0.67 mM. Androstenedione was found to be a competitive inhibitor of [3H]-1,4-androstadiene-3,17-dione aromatization (Ki) = 0.75 μM androstenedione) and of 17β-hydroxysteroid oxidoreduction (Ki) = 17μM androstenedione). Fifty per cent inhibition of estrogen synthetase and of 17β-hydroxysteroid oxidoreductase activities were obtained with an androstenedione concentration of 80 μM. The specific activity of NADPH-dependent cytochrome c reductase of placental microsomes was 22 nmol × mg-1 protein × min-1. The dependency of placental microsomal [3H]-1,4-androstadiene-3,17-dione aromatization on cytochrome P-450 and upon NADPH-dependent cytochrome c reductase was demonstrated using both anti-human and anti-porcine hepatic microsomal NADPH-dependent cytochrome c (P-450) reductase antibodies. The estrogen synthetase and NADPH-dependent cytochrome c reductase activities were inhibited greatly by the microsomal specific antibodies, whereas non-immune γ-globulin had little effect on enzyme activity. The 17β-hydroxysteroid oxidoreductase activity was affected only slightly by these antibodies.",
author = "Leon Milewich and Bradfield, {Debra J.} and Coe, {L. Dean} and Masters, {Bettie Sue S} and Macdonald, {Paul C.}",
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TY - JOUR

T1 - Metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes. enzyme properties and kinetic parameters in the formation of estrogens and 17β-hydroxy-1,4-androstadien-3-one

AU - Milewich, Leon

AU - Bradfield, Debra J.

AU - Coe, L. Dean

AU - Masters, Bettie Sue S

AU - Macdonald, Paul C.

PY - 1981

Y1 - 1981

N2 - In this investigation the metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes was evaluated. The experiments were conducted with the substrate [3H]-1,4-androstadiene-3,17-dione, which was synthesized using a tritium-hydrogen keto-enol exchange technique. [4-14C]-l,4-Androstadiene-3, 17-dione and [4-14C]-17β-hydroxy-1,4-androstadien-3-one also were synthesized and used, together with [14C]-estrone and [14C]-estradiol-17β, as internal recovery standards for quantification. The radiolabeled metabolites identified from incubation mixtures with placental microsomes were estrone, estradiol-17β and 17β-hydroxy-1,4-androstadien-3-one; two other metabolites, one less polar than the substrate and the other more polar than estradiol-17β, were observed but not investigated further. The enzymatic conversions required either NADPH or NADH as cofactor and there was no apparent tritium loss in the transformation of substrate to products. The rates of formation of metabolites were linear with time for at least 30 min and with the amount of microsomal protein up to 15 mg. Tbe apparent pH optimum for placental microsomal estrogen synthetase was 7.5, and the apparent optimum pH for the 17β-hydroxysteroid oxidoreductase ranged from pH6.0 to pH7.4. The apparent optimal temperature for the estrogen synthetase activity was 37°C and for 17β-hydroxysteroid oxidoreductase it ranged from 37°C to 58°C. The apparent Km of estrogen synthetase for 1,4-androstadiene-3,17-dione was 1.3μM, and the apparent Km of 17β-hydroxysteroid oxidoreductase for 1,4-androstadiene-3, 17-dione was 24 μM. The apparent Km of estrogen synthetase for NADPH and NADH were 0.24 mM and 1.0mM, respectively; the apparent Km of placental microsomal 17β-hydroxysteroid oxidoreductase for NADH was 0.5 mM and that for NADPH was 0.67 mM. Androstenedione was found to be a competitive inhibitor of [3H]-1,4-androstadiene-3,17-dione aromatization (Ki) = 0.75 μM androstenedione) and of 17β-hydroxysteroid oxidoreduction (Ki) = 17μM androstenedione). Fifty per cent inhibition of estrogen synthetase and of 17β-hydroxysteroid oxidoreductase activities were obtained with an androstenedione concentration of 80 μM. The specific activity of NADPH-dependent cytochrome c reductase of placental microsomes was 22 nmol × mg-1 protein × min-1. The dependency of placental microsomal [3H]-1,4-androstadiene-3,17-dione aromatization on cytochrome P-450 and upon NADPH-dependent cytochrome c reductase was demonstrated using both anti-human and anti-porcine hepatic microsomal NADPH-dependent cytochrome c (P-450) reductase antibodies. The estrogen synthetase and NADPH-dependent cytochrome c reductase activities were inhibited greatly by the microsomal specific antibodies, whereas non-immune γ-globulin had little effect on enzyme activity. The 17β-hydroxysteroid oxidoreductase activity was affected only slightly by these antibodies.

AB - In this investigation the metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes was evaluated. The experiments were conducted with the substrate [3H]-1,4-androstadiene-3,17-dione, which was synthesized using a tritium-hydrogen keto-enol exchange technique. [4-14C]-l,4-Androstadiene-3, 17-dione and [4-14C]-17β-hydroxy-1,4-androstadien-3-one also were synthesized and used, together with [14C]-estrone and [14C]-estradiol-17β, as internal recovery standards for quantification. The radiolabeled metabolites identified from incubation mixtures with placental microsomes were estrone, estradiol-17β and 17β-hydroxy-1,4-androstadien-3-one; two other metabolites, one less polar than the substrate and the other more polar than estradiol-17β, were observed but not investigated further. The enzymatic conversions required either NADPH or NADH as cofactor and there was no apparent tritium loss in the transformation of substrate to products. The rates of formation of metabolites were linear with time for at least 30 min and with the amount of microsomal protein up to 15 mg. Tbe apparent pH optimum for placental microsomal estrogen synthetase was 7.5, and the apparent optimum pH for the 17β-hydroxysteroid oxidoreductase ranged from pH6.0 to pH7.4. The apparent optimal temperature for the estrogen synthetase activity was 37°C and for 17β-hydroxysteroid oxidoreductase it ranged from 37°C to 58°C. The apparent Km of estrogen synthetase for 1,4-androstadiene-3,17-dione was 1.3μM, and the apparent Km of 17β-hydroxysteroid oxidoreductase for 1,4-androstadiene-3, 17-dione was 24 μM. The apparent Km of estrogen synthetase for NADPH and NADH were 0.24 mM and 1.0mM, respectively; the apparent Km of placental microsomal 17β-hydroxysteroid oxidoreductase for NADH was 0.5 mM and that for NADPH was 0.67 mM. Androstenedione was found to be a competitive inhibitor of [3H]-1,4-androstadiene-3,17-dione aromatization (Ki) = 0.75 μM androstenedione) and of 17β-hydroxysteroid oxidoreduction (Ki) = 17μM androstenedione). Fifty per cent inhibition of estrogen synthetase and of 17β-hydroxysteroid oxidoreductase activities were obtained with an androstenedione concentration of 80 μM. The specific activity of NADPH-dependent cytochrome c reductase of placental microsomes was 22 nmol × mg-1 protein × min-1. The dependency of placental microsomal [3H]-1,4-androstadiene-3,17-dione aromatization on cytochrome P-450 and upon NADPH-dependent cytochrome c reductase was demonstrated using both anti-human and anti-porcine hepatic microsomal NADPH-dependent cytochrome c (P-450) reductase antibodies. The estrogen synthetase and NADPH-dependent cytochrome c reductase activities were inhibited greatly by the microsomal specific antibodies, whereas non-immune γ-globulin had little effect on enzyme activity. The 17β-hydroxysteroid oxidoreductase activity was affected only slightly by these antibodies.

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