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Human Fetal Liver Estrogen 16-Hydroxylase:Precursor Specificity, Kinetic Parameters, and in Vitro Regulation^*

Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics and Gynecology and Biochemistry, The University of Texas Southwestern Medical School Dallas, Texas 75235

Address all correspondence and requests for reprints to: Dr. Leon Milewich, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235.

The properties of human fetal liver (HFL) estrogen 16-hydroxylase (16-0Hase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16- OHase activity based on the enzymatic release of tritium from the C-16 position of stereospecifically labeled 16-³H-labeled C¹⁸-steroids, viz. 17β-[16-³H]estradiol, 17βl6-³H]estradiol3- sulfate, [16-³H]estrone, and [16-³H]estrone sulfate. The percentage of tritium at the C-16 position of 17β[16-³H]estradiol was 92.5%. There was no kinetic isotope effect in the 16hydroxylation of 17β[16-³H]estradiol. HFL hepatocyte and microsomal 16-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7 x 10⁶ cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent K_m of 16-OHase for estrone sulfate (E1S) was greater than that for either 17β-estradiol (E2) or estrone (El; 2.9–6.4 µM us. 0.70–0.84 µM). The maximum velocity of HFL 16-OHase also was greater with ElS or 17β-estradiol 3-sulfate than with either El or E2, and ElS was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0.16-Hydroxylation of ElS by HFL microsomes was inhibited noncompetitively by El. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to El as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17-³H]estriol from [17-³H] 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 16a-OHase activity. 16-OHase activity was stimulated by dexamethasone, forskolin, (Bu)₂cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progesterone did not alter hepatocyte 16-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.

^* This work was supported in part by USPHS Grants 5-P50-HD-11149 and HD-17814.

Received December 10, 1985.

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