Nobutaka Yoshida

Steroid specificity of human placental 5-ene-3β-hydroxysteroid oxidoreductase

The properties of 5-ene-3 beta hydroxysteroid oxidoreductase (3 beta-HSD) from human placental homogenates were studied in vitro. The apparent Michaelis constants for 3 beta-HSD with the substrates pregnenolone (delta 5P) and dehydroepiandrosterone (DHA) were 170 nM and nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for the pregnenolone was 20 microM and for DHA, 17 microM. The activity of 3 beta-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17 beta (Ki=46 nM), cholesterol (Ki=0.68 microM) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOHDHA) (Ki=2.2 microM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17 beta (Ki=69 nM), pregnenolone (Ki=91 nM), cholesterol (Ki=1.3 microM) and 16 alphaOHDHA (Ki=1.9 microM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (delta 4P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.

Distribution and cyclic change of aromatase cytochrome P-450 activity in human uteri

Activities of aromatase cytochrome P-450 in the columnar epithelial region (CE), squamous epithelial region (SE) and connective tissue (CT) of uterine cervix, and endometrium (EM) during the menstrual cycle were determined using [4-14C] and [1β-3H]androstenedione. Aromatase activities in the proliferative phase (n=8) were 15.0±7.9, 10.9±10.3, 9.4±10.6 and 8.0±7.3 (mean±SD) fmol/h/mg protein in CE, SE, CT and EM, respectively, and aromatase activities in the secretory phase (n=6) were 31.5±7.6, 19.1±7.1, 5.6±4.6 and 6.3±1.5 fmol/h/mg protein, respectively. The results show that the aromatase activities in these regions in the proliferative phase were not significantly different from each other. On the other hand, the aromatase activity in the secretory phase was significantly higher in CE than in any other regions (P<0.05), and significantly higher in SE than in CT or EM (P<0.05). There was no significant difference in aromatase activity between CT and EM. By comparison of aromatase activity between these two phases, the activity in CE was significantly higher in the secretory phase than in the proliferative phase (P<0.05), but no significant difference was observed in other regions.

Steroid 16α-Hydroxylase from Human Fetal Liver; Inhibition by Steroids

The 16α‐hydroxylase system in fetal liver which used dehydroepiandrosterone (DHA) or pregnenolone as substrate, was apparently inhibited by various endogenous and synthetic steroids: DHA, pregnenolone, their sulfates, androstenediol, androstenetriol, estrone, estradiol‐17β, ethynylestradiol and chlormadinone‐acetate. The inhibition constants (Ki) towards DHA were as follows; pregnenolone 22 μM, DHA‐sulfate 13 μM, pregnenolone‐sulfate 21 μM, androstenediol 16 μM, androstenetriol 53 μM, estrone 32 μM, estradiol‐17β 75 μM, ethynylestradiol 22 μM and chlormadinone‐acetate 27 μM. The Ki values towards pregnenolone were DHA 6.3 μM, DHA‐sulfate 8.3 μM, pregnenolone‐sulfate 3.9 μM, androstenediol 8.7 μM, androstenetriol 14.7 μM, estradiol‐17β 15.4 μM and ethynylestradiol 16.0 μM, respectively. The reaction products, 16αOH‐DHA and 16αOH‐pregnenolone, showed little inhibitory effect upon the 16α‐hydroxylase.

Rebuttal to the 1α,2β-hydrogen elimination mechanism of estrogen biosynthesis by reconstituted aromatase [Poster 03]

Abstract The stereochemistry of 1,2-hydrogen elimination during androstenedione aromatization was determined to be stereospecific 1s, 2s for reconstituted and cross-reconstituted aromatase preparations as well as for intact and solubilized forms of the human placental enzyme. The [3H]water method for assaying aromatase based on the 1s-3H elimination is thus valid, irrespective of the stage of purification such as particulate, soluble, or reconstituted forms, or of coupling with reductase of different species and tissues.