Human fetal liver estrogen 16α-hydroxylase: precursor specificity, kinetic parameters, and in vitro regulation

L. Milewich, P. C. MacDonald, A. Guerami, W. T. Midgett, W. L. Lassiter, B. R. Carr

Research output: Contribution to journalArticle

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Abstract

The properties of human fetal liver (HFL) estrogen 16α-hydroxylase (16α-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16α-OHase activity based on the enzymatic release of tritium from the C-16α position of stereospecifically labeled 16α-3H-labeled C18-steroids, viz. 17β-[16α-3H]estradiol, 17β-[16α-3H]estradiol 3-sulfate, [16α-3H]estrone, and [16α-3H]estrone sulfate. The percentage of tritium at the C-16α position of 17β-[16α-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16α-hydroxylation of 17β-[16α-3H]estradiol. HFL hepatocyte and microsomal 16α-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7x106 cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent K(m) of 16α-OHase for estrone sulfate (E1S) was greater than that for either 17β-estradiol (E2) or estrone (E1; 2.9-6.4 μM vs 0.70-0.84 μM). The maximum velocity of HFL 16α-OHase also was greater with E1S or 17β-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16α-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17α-3H]estriol from [17α-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16α-OHase activity. 16α-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progresterone did not alter hepatocyte 16α-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.

Original languageEnglish (US)
Pages (from-to)180-191
Number of pages12
JournalJournal of Clinical Endocrinology and Metabolism
Volume63
Issue number1
StatePublished - 1986

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Mixed Function Oxygenases
Kinetic parameters
Liver
Estrogens
Hepatocytes
Estradiol
Liver Microsomes
Hydroxylation
Estriol
Tritium
Estrone
Intercellular Signaling Peptides and Proteins
Diethylstilbestrol
Cholera Toxin
Biosynthesis
Hormones
Xenobiotics
Colforsin
Enzymes
Phenobarbital

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

Human fetal liver estrogen 16α-hydroxylase : precursor specificity, kinetic parameters, and in vitro regulation. / Milewich, L.; MacDonald, P. C.; Guerami, A.; Midgett, W. T.; Lassiter, W. L.; Carr, B. R.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 63, No. 1, 1986, p. 180-191.

Research output: Contribution to journalArticle

Milewich, L. ; MacDonald, P. C. ; Guerami, A. ; Midgett, W. T. ; Lassiter, W. L. ; Carr, B. R. / Human fetal liver estrogen 16α-hydroxylase : precursor specificity, kinetic parameters, and in vitro regulation. In: Journal of Clinical Endocrinology and Metabolism. 1986 ; Vol. 63, No. 1. pp. 180-191.
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abstract = "The properties of human fetal liver (HFL) estrogen 16α-hydroxylase (16α-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16α-OHase activity based on the enzymatic release of tritium from the C-16α position of stereospecifically labeled 16α-3H-labeled C18-steroids, viz. 17β-[16α-3H]estradiol, 17β-[16α-3H]estradiol 3-sulfate, [16α-3H]estrone, and [16α-3H]estrone sulfate. The percentage of tritium at the C-16α position of 17β-[16α-3H]estradiol was 92.5{\%}. There was no kinetic isotope effect in the 16α-hydroxylation of 17β-[16α-3H]estradiol. HFL hepatocyte and microsomal 16α-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7x106 cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent K(m) of 16α-OHase for estrone sulfate (E1S) was greater than that for either 17β-estradiol (E2) or estrone (E1; 2.9-6.4 μM vs 0.70-0.84 μM). The maximum velocity of HFL 16α-OHase also was greater with E1S or 17β-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16α-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17α-3H]estriol from [17α-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16α-OHase activity. 16α-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progresterone did not alter hepatocyte 16α-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.",
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T2 - precursor specificity, kinetic parameters, and in vitro regulation

AU - Milewich, L.

AU - MacDonald, P. C.

AU - Guerami, A.

AU - Midgett, W. T.

AU - Lassiter, W. L.

AU - Carr, B. R.

PY - 1986

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N2 - The properties of human fetal liver (HFL) estrogen 16α-hydroxylase (16α-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16α-OHase activity based on the enzymatic release of tritium from the C-16α position of stereospecifically labeled 16α-3H-labeled C18-steroids, viz. 17β-[16α-3H]estradiol, 17β-[16α-3H]estradiol 3-sulfate, [16α-3H]estrone, and [16α-3H]estrone sulfate. The percentage of tritium at the C-16α position of 17β-[16α-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16α-hydroxylation of 17β-[16α-3H]estradiol. HFL hepatocyte and microsomal 16α-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7x106 cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent K(m) of 16α-OHase for estrone sulfate (E1S) was greater than that for either 17β-estradiol (E2) or estrone (E1; 2.9-6.4 μM vs 0.70-0.84 μM). The maximum velocity of HFL 16α-OHase also was greater with E1S or 17β-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16α-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17α-3H]estriol from [17α-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16α-OHase activity. 16α-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progresterone did not alter hepatocyte 16α-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.

AB - The properties of human fetal liver (HFL) estrogen 16α-hydroxylase (16α-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16α-OHase activity based on the enzymatic release of tritium from the C-16α position of stereospecifically labeled 16α-3H-labeled C18-steroids, viz. 17β-[16α-3H]estradiol, 17β-[16α-3H]estradiol 3-sulfate, [16α-3H]estrone, and [16α-3H]estrone sulfate. The percentage of tritium at the C-16α position of 17β-[16α-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16α-hydroxylation of 17β-[16α-3H]estradiol. HFL hepatocyte and microsomal 16α-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7x106 cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent K(m) of 16α-OHase for estrone sulfate (E1S) was greater than that for either 17β-estradiol (E2) or estrone (E1; 2.9-6.4 μM vs 0.70-0.84 μM). The maximum velocity of HFL 16α-OHase also was greater with E1S or 17β-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16α-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17α-3H]estriol from [17α-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16α-OHase activity. 16α-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progresterone did not alter hepatocyte 16α-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.

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