Summary: The occurrence and characteristics of macromolecular components of normal human myometrium and leiomyoma which bind [ 3 H]estradiol and [ 3 H]progesterone were investigated, employing dextran coated charcoal, density gradient centrifugation and gel filtration techniques.On sucrose density gradient centrigugation, [ 3 H]progesterone was bound by macromolecules with sedimentation rates of about 4 S and 8 S.The major [ 3 H]progesterone binding component had a sedimentation coefficient of about 4 S, which contained specific and nonspecific binding sites.Sedimentation patterns as well as elution profiles from agarose gel revealed a striking similarity between biochemical properties of the progesterone receptors from normal myometrium and leiomyomas of the same organ.Both progesterone and estradiql receptor change in concentration during the normal menstrual cycle. During the early proliferative phase the number of estradiol receptor binding sites was highest; after ovulation, a rapid decrease of estradiol receptor level was seen. On the other hand, using [ 3 H]progesterone as the ligand, the highest receptor concentration was found at midcycle.The leiomyoma steroid hormone receptor levels were compared with those in normal myometrium. Whereas leiomyoma exhibited higher estradiol binding capacity, the concentration of progesterone receptors was low in fibroid tumors. In normalem Gewebe variieren die Prqgesterqn-sowie Östradiol-Rezeptorkonzentrationen in der Cytoplasmafraktion. Die höchsten Östradiol-Rezeptorbindungsaktivitäten wurden während der frühen Proliferationsphase beobachtet; nach Oviüation kommt es zu einem rapiden Abfall der Östradipl-Rezeptorkohzentration. Die höchsten ProgesteronRezeptorkonzentratipnen zeichneten sich ab in der Mitte des Menstruationszyklus zum Zeitpunkt der Ovulation. Myome waren charakterisiert durch hohe Östradiql-Bindungskäpazität und eine niedrigere Progesteron-Rezeptorkonzentration. Östrogen-und Progesterön-Rezeptören in normalem Human-Myometrium und Myomen
Sedimentation coefficients of cytoplasmic estradiol and progesterone receptors of human proliferative endometrium and endometrial carcinoma were determined by sucrose gradient centrifugation. In the absence of KCl, receptors from proliferative endometrium sedimented as single bands in the 8 S region and in the presence of 0.3M KCl in the 4 S region of the gradients. Receptors from endometrial carcinoma sedimented in several bands (between 3 and 9 S). When chromatographed on agarose gel comumns, the receptors (from both normal and neoplastic tissue) showed different molecular weights in the presence and absence of KCl (approximately 40,000 and 120,000, respectively). Elution profiles from agarose gel and ion exchange columns, as well as electrophoretic patterns from isoelectric focusing, revealed a similarity between biochemical properties of the receptors from endometrial carcinoma and proliferative endometrium. While the concentration of binding sites for estradiol and progesterone in normal endometrium depended on the day of the cycle, in endometrial carcinoma it depended on the degree of differentiation of the tumor. The binding of estradiol was highest at the beginning of the proliferative phase and declined continuously towards the 14th day of the cycle. In contrast, the concentration of progesterone binding sites was relatively low throughout the proliferative phase. In endometrial carcinoma low binding of estradiol was obtained in well differentiated tumors and high binding (as high as in proliferative endometrium) in undifferentiated tumors. For progesterone the contrary was the case. There was no difference in pH sensitivity between cytoplasmic receptors from normal and neoplastic tissue, optimal binding occurring at pH 7. Dissociation constants (Kd) for estradiol and progesterone depended on the degree of tumor differentiation. Kd values increased for E2 and decreased for P with increasing differentiation of the tumor. Competition studies with various unlabeled steroids revealed no significant difference between the specificity of the receptors from proliverative and neoplastic endometrium.
Different subcellular fractions (purity checked by electron microscopy and respective marker enzymes) were incubated with 0.1 muCi 14C-progesterone (10 muM) in 0.15 M phosphate buffer at pH 7.4 and 37 C under air for varying periods of time in the presence of NAD(P)H (500 muM). By the preparation of chromic acid oxidation products and acetates, thin-layer chromatography, and crystallisation to constant specific activity, the following metabolites were identified: 20alpha-hydroxypregn-4-en-3-one, 20alpha-hydroxy-5alpha-pregnan-3-one, 20alpha-hydroxy-5beta-pregnan-3-one, 5alpha-pregnane-3,20-dione, and 5beta-pregnane-3,20-dione, indicating the presence of a 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and 5alpha- and 5beta-reductases. Most of the 20alpha-HSD activity was located in mitochondria (associated mainly with outer membranes) and microsomes. Purified nuclei and cytosol contained 1/6 to 1/18 of the activity of mitochondria and microsomes, respectively. SUBFRACTIONS OF ENDOMETRIAL CELLS ONLY CONTAINED EITHER 5ALPHA- OR 5BETA-REDUCTASE ACTIVITY. 5alpha-reductase activity was mainly associated with microsomes, 5beta-reductase activity was found only in the cytosol. While in normal endometrium specific enzyme activities in subcellular fractions depended on the phase of the cycle, in endometrial carcinoma it depended on the degree of tumour differentiation. The highest values of 5alpha-reductase activity were found in the early proliferative phase. 20alpha-HSD activity was highest in the middle of the secretory phase. The specific activity of the 5alpha-reductase increased with decreasing differentiation of the tumour while the specific activity of the 20alpha-HSD decreased. Kinetic parameters (Km-values, coenzyme requirements and maximum velocities) were determined. The Km-value for progesterone of the 20alpha-HSD in proliferative endometrium was significantly higher than in secretory endometrium, while the Km-values of the 5alpha- and 5beta-reductases were considerably lower during the proliferative than secretory phase.
Summary:The specific activity of 170-hydroxysteroid dehydrogenase was measured in normal human myometrium, and in leiomyoma specimens obtained from the same tumor-bearing uterus. In all cases the normal tissue showed greater conversion of estradiol-17/3 into estrone than the neoplastic tissues. In normal myometrium of fertile women, the specific enzyme activity depended on the phase of the menstrual cycle, the highest values of 170-hydroxysteroid dehydrogenase activity being found in the early secretory phase.To determine the intracellular distribution of the 17/3-hydroxysteroid dehydrogenase, purified microsomes, mitochondria, nuclei and cytosol fractions were prepared. The purity of each fraction was monitored by marker enzymes. It was found that the enzyme was mainly located in mitochondria and microsomes. Furthermore it was demonstrated that the microsomal enzyme was bound tightly to the membranes of the endoplasmic reticulum, while the mitochondrial 17j34iydroxysteroid dehydrogenase was mainly associated with the outer membranes of the organelle.Kinetic parameters (K m -values, coenzyme requirements, temperature and pH-optima) of the cytoplasmic, nuclear, mitochondrial and microsomal enzyme of normal and neoplastic tissue were compared. In vitrO'Umwandlung von Östradiol-17ß in Östron in normalem Human-Myometrium und LeiomyomenZusammenfassung: Die spezifische Aktivität der 17|8-Hydroxysteroiddehydrogenase wurde in normalem HumanMyometrium und in Gewebeproben von Leiomyomen vergleichend gemessen. Die Gewebsproben wurden aus dem gleichen Tumor-tragenden Uterus gewonnen. Normalgewebe zeigte einen größeren Umsatz von Östradiol-170 zu Östron als Tumorgewebe. In Normal-Myometrium geschlechtsreifer Frauen variierte die spezifische Enzymaktivität in Abhängigkeit vom Menstruationszyklus: die höchsten Werte wurden in der frühen Sekretionsphase beobachtet.Um die intrazellulare Verteilung der 17|8-Hydroxysteroiddehydrogenase-Aktivität zu untersuchen, wurden hochgereinigte Mikrosomen, Mitochpndrien, Kerne und Cytoplasma präpariert. Der Reinheitsgrad der Fraktion wurde durch Leitenzyme charakterisiert. Die höchsten spezifischen Enzymaktivitäten wurden in Mitochondrien und Mikrosomen lokalisiert. Darüberhinaus wurde beobachtet, daß das mikrosomale Enzym fest mit der Membran des endoplasmatischen Retikulums verbunden ist, während die mitochondriale 17j3-Hydroxysteroiddehydrogenase mit der äußeren Membran der Mitochondrien assoziiert ist.Kinetische Parameter (K m -Werte, Coenzym-Abhängigkeit, Temperatur-und pH-Optima) des cytoplasmatischen, nuklearen, mitochondrialen und mikrosonialen Enzyms aus normalem und neoplastischem Gewebe wurden vergleichend untersucht.
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