Kunhard Pollow

Comparison of the in vitro conversion of estradiol-17β to estrone of normal and neoplastic human breast tissue

Specific activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) was measured in 48 tissue specimens of human female breast cancer and, in addition, 48 nonmalignant tissue specimens obtained in each case from the same cancer-bearing breast. In all cases the nonmalignant tissue showed greater conversion of estradiol-17 beta into estrone than the neoplastic tissues. In normal human breast tissue of premenopausal women specific enzyme activity depended on the phase of the MENSTRUAL CYCLE: the highest values of 17 beta-HSD activity were found in the early secretory phase. To determine the intracellular distribution of the 17 beta-HSD, purified microsomes, mitochondria, peroxysomes, lysosomes, nuclei and cytosol fractions were prepared. The purity of each fraction was monitored by marker enzymes. It was found that the 17 beta-HSD was mainly located in mitochondria and microsomes. Furthermore it could be demonstrated that the microsomal enzyme was bound tightly to the membranes of the endoplasmic reticulum, while the mitochondrial 17 beta-HSD was mainly associated with the outer membranes of the organelle. Kinetic parameters (Km-values, coenzyme requirements and maximal velocities) of a cytoplasmic, nuclear, mitochondrial and microsomal 17 beta-HSD of normal and neoplastic human mammary tissue were compared. Maximal velocity was highest in enzyme preparations of normal mammary tissue obtained from premenopausal women in the early secretory phase. Km-values wrere nearly identical in normal and neoplastic mammary tissue preparations (approx. 1 X 10(-6) M). NAD was more efficient than NADP as a cofactor. For the conversion of estradiol to estrone the optimum temperature was approximately 40 degrees C and the optimum pH 9.5. For the reduction of estrone the optimum pH was 6.5. Sulphydryl groups were shown to be essential for catalysis.

Sex steroid levels in serum, myometrium, and fibromyomata in correlation with cytoplasmic receptors and 17β-HSD activity in different age-groups and phases of the menstrual cycle

ZusammenfassungDer Einfluß der Gewebekonzentrationen von Östradiol, Östron und Progesteron auf den Gehalt cytoplasmatischer Östradiol- und Progesteron-bindender Rezeptoren sowie der Enzymaktivität der 17β-Hydroxysteroiddehydrogenase (17β-HSD) im normalen Myometrium und Myomen während verschiedener reproduktiver Phasen der Frau wurde untersucht. Die gemessenen Parameter waren in ihren Konzentrationen von der endokrinen Situation abhängig.Die Gewebespiegel von Östradiol und Progesteron verhielten sich im normalen Myometrium während des mensuellen Cyclus ähnlich wie die Serumkonzentrationen. Die Östronspiegel des normalen Myometrium hingegen waren — im Vergleich zum Serum — in der lutealen Phase höher als in der follikulären. Die Östradiol-bindenden Rezeptoren wiesen ihre höchsten Konzentrationen im Cytoplasma in der proliferativen Cyclushälfte auf und fielen post ovulationem stark ab. Der Gehalt an cytoplasmatischen Progesteron-Rezeptoren war mittcyclisch am höchsten. Die enzymatische Aktivität der 17β-HSD stieg in der frühen Sekretionsphase etwa um das Zehnfache und korrelierte mit der Zunahme der Östron-Gewebespiegel.Die auffälligsten Unterschiede zwischen normalem Myometrium und Myom waren: niedrige Östron-Gewebespiegel und nur ein geringer Anstieg der 17β-HSD-Aktivität in der zweiten Cyclushälfte. Die cytoplasmatischen Rezeptorkonzentrationen für Östradiol und Progesteron waren während des Cyclus stets niedrig und zeigten keinerlei signifikante Schwankungen.Nach der Menopause entsprachen die Konzentrationen des cytoplasmatischen Östradiolrezeptor denen der follikulären Cyclusphase. Die Progesteron bindenden Rezeptorproteine sowie die 17β-HSD-Aktivität zeigten hingegen sehr niedrige Werte. Im Myometrium von Frauen, die hormonelle Kontrazeptiva nahmen, entsprachen die cytoplasmatischen Rezeptorkonzentrationen mittcyclischen Werten. Die 17β-HSD-Aktivität war trotz niedriger Progesteron-Gewebespiegel hoch.SummaryA study has been made on the influence of estradiol, estrone, and progesterone on cytoplasmic estradiol and progesterone receptors and the 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in normal myometrium and in fibromyomata during various phases of the menstrual cycle.The concentrations of estradiol, estrone, and progesterone in normal myometrium during the menstrual cycle were similar to those in serum except for higher levels of tissue estrone in the luteal phase. The number of estradiol-binding sites was highest during the proliferative phase and decreased after ovulation. The highest progesterone receptor content was found at midcycle. The 17β-HSD activity increased approximately 10-fold during the early secretory phase and correlated with the increase of tissue estrone levels.In fibromyomata there were low tissue estrone concentrations and only a moderate increase of 17β-HSD activity during the secretory phase. Furthermore, the number of cytoplasmic estradiol and progesterone receptor binding sites was low and did not change during the menstrual cycle, thus indicating in fibromyomata a defect in formation of cytoplasmic receptors, especially for progesterone.After the menopause the number of estradiol-binding sites was high, as in the proliferative phase of the cycle, whereas the number of progesterone-binding sites and the amount of 17β-HSD activity were extremely low. Myometrium from women on oral contraceptives showed an intermediate number of cytoplasmic receptors and a high 17β-HSD activity despite low tissue progesterone concentrations.

On the mitochondrial 17β-hydroxysteroid dehydrogenase from human endometrium and endometrial carcinoma: Characterization and intramitochondrial distribution

Abstract To determine intramitochondrial location of an endometrial 17β-hydroxysteroid dehydrogenase (17β-HSD), purified mitochondria from secretory endometrium were separated into inner and outer membranes, matrix and an intermembrane fraction. The purity of each fraction was monitored by marker enzymes. A radiochemical assay was used for the determination of 17β-HSD activities. It was found that the 17β-HSD was mainly located in the outer membranes of the mitochondria. Kinetic parameters and substrate specificity of the enzyme were determined. The conversion of estradiol (E 2 ) to estrone (E 1 ) by purified mitochondria was linear with time and protein concentration. The optimum temperature was approximately 40°C and the optimum pH 9·5. For the reduction of E 1 the optimum pH was 6·0. With NAD E 2 was oxidized approximately 15 times more rapidly than with NADP. The apparent K m -values for E 2 (in the presence of NAD) were 13 × 10 −6 M and 4 × 10 −6 M in proliferative and secretory endometrium, respectively. The maximal velocity was highest in secretory endometrium. Testosterone and androstenedione could also serve as substrates. In normal endometrium they were interconverted at approximately 50% of the rate of E 2 and E 1 . In endometrial carcinoma androstenedione was reduced twice as rapidly as estrone. Sulfhydryl groups were shown to be essential for catalysis. The mitochondrial 17β-HSD was similar in character to the cytoplasmic, microsomal and nuclear enzymes described previously.

Subcellular distribution and properties of 17β-hydroxysteroid dehydrogenase in pregnant rabbit uterus

Abstract To determine the intracellular distribution of the 17β-hydroxysteroid dehydrogenase, purified microsomes, mitochondria, peroxysomes, lysosomes, nuclei and cytosol were prepared. The purity of each fraction was monitored by marker enzymes. It was found that the 17β-hydroxysteroid dehydrogenase was mainly located in mitochondrial, microsomal and cytoplasmic fractions. Furthermore, it could be demonstrated that the microsomal enzyme was bound tightly to the membranes of the endoplasmic reticulum, while the mitochondrial 17β-hydroxysteroid dehydrogenase was mainly associated with the outer membranes of the organelle. Kinetic parameters of the mitochondrial and microsomal 17β-hydroxysteroid dehydrogenase were compared. K m -values were nearly identical in both enzyme preparations. NADP was more efficient than NAD as cofactor. For the conversion of estradiol to estrone the optimum temperature was approximately 42°C and the optimum pH 8.0. For the reduction of estrone the optimum pH was 6.0. Sulfhydryl groups were shown to be essential for catalysis.

Partial purification and evidence for heterogeneity of the cytoplasmic 17β-hydroxysteroid dehydrogenase (17β-HSD) from normal human endometrium and endometrial carcinoma

Abstract This study was designed primarily to purify the 17β-HSD from human endometrium. The cytoplasmic 17β-HSD of secretory endometrium was partially purified by ammonium-sulphate precipitation, gel filtration, ion-exchange chromatography and isoelectric focusing. During purification procedures 17β-HSD activity was determined by a radiochemical method. Activity patterns of the enzyme(s) from normal and neoplastic endometrium were obtained by isoelectric focusing in polyacrylamide gels and subsequent staining of the gels for enzyme activity. Estradiol (E2) and testosterone (T) served as substrates, NAD and NADP as cosubstrates. All solutions and gels contained glycerol. A 40-fold purification of the 17β-HSD from secretory endometrium was achieved. The overall recovery was 10%. Isoelectric focusing in sucrose of the 17β-HSD from secretory endometrium yielded three enzymatically active bands with both E2 and T as substrates (focused around pH 5). In contrast, the activity patterns from endometrial carcinoma gave 2, 4 or 7 bands depending on the substrate and the cofactor. These findings may indicate that in normal and neoplastic endometrium the 17β-HSD exists in different molecular forms.

17β-Carboxamide Steroids: Highly Effective Inhibitors of the Phytohaemagglutinin Mediated Blastogenesis of Normal Human Peripheral Lymphocytes

: Several novel 17 beta-carboxamide analogues of dexamethasone were synthesized. The common precursor, 9-fluoro-16 alpha-methyl-11 beta,17-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxylic acid, did not bind to the glucocorticoid receptors of rat liver and human spleen tumours. In addition, no inhibition of the mitogen-induced blastogenesis of cultured human peripheral lymphocytes was observed. The 17 beta-carboxamide analogues, however, bound with similar affinities to the glucocorticoid receptors of both tissues. They inhibited the mitogen-induced blastogenesis of peripheral lymphocytes, showing the same potency and same order of binding affinity as the natural glucocorticoids.

A new high sensitive analytical micro-scale procedure for chromosomal proteins

Utilizing male rat liver cells we describe a method for isolating and fractionating chromosomal proteins. About 99%of chromosomal proteins was dissociated using a three step dissociation procedure. DNA was removed by sedimentation and the histone fractions were separated from the non-histone chromosomal proteins by Bio Rex 70 chromatography. The nonhistone chromosomal proteins were fractionated by micro-gradient electrophoresis on SDS-polyacrylamide gels, which proved to be superior to the electrophoretic procedures currently in use. The histones were further separated on polyacrylamide-SDS slab gels using a micro-two-dimensional electrophoretic system. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.

Comparison of nonhistone chromosomal proteins from neuronal and glial chromatin by isoelectric focussing and microdisc electrophoresis

Abstract The present study describes optimal conditions for preparative fractionation of nonhistone chromosomal proteins from neuronal and glial nuclei. We have detected about 1200 nonhistone chromosomal proteins. Microgels of glial origin contained 20% more protein bands than those of neuronal origin. However, neuronal bands prevailed clearly in the acidic respectively in the high-molecular range. Amino acid analysis confirmed the observed heterogeneity.

Purification and properties of the soluble 17 beta-hydroxysteroid dehydrogenase of rabbit uterus.

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromatography yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

Kinetische Untersuchungen und Charakterisierung der solubilisierten Oestradiol-bzw. Testosteron-"sensitiven" 17 β-HSD-Aktivitäten / Microsome-Associated 17β-Hydroxysteroid Dehydrogenases of Human Placenta, II Kinetic Studies and Characterization of the Solubilized Estradioland Testosterone-“Sensitive” 17β-HSD-Activities

Abstract Detailed enzyme kinetic parameters of the reactions catalyzed by the two 17β-hydroxysteroid dehydrogenases (17β-HSD), which were solubilized from the microsomes of human placenta by treatment with phospholipase A, followed by enrichment and separation were determined. Both enzymes are strictly substrate specific. The most active substrate of one of the 17β-HSD (fraction A) is estradiol-17β, the other 17β-HSD (fraction B) is sensitive to testosterone. Both NAD and NADP can serve as hydrogen transferring coenzymes, the latter giving about one-third of the initial rate of the former. With respect to the influence of temperature, different buffers and pH values, Michaelis constants [Km] with estradiol-17β and testosterone as substrates, the solubilized and separated microsomal 17β-HSD behave like those isolated from the cytoplasmic fraction. The two 17β-HSD, after solubilization from the microsomal fraction of human placenta, enrichment and separation from each other, show only a little activity for the transfer of hydrogen between C17 of estradiol-17β and C17 of androstenedione. On the other hand, intact microsomes and an integrated system prepared by recombination of the 17β-enzymes by preincubation in phosphate buffer are able to catalyse very actively the transfer of hydrogen between estradiol-17β and androstenedione. The effect of temperature and time on the recombination of the two enriched and separated microsomal enzyme activities and the determination of the pH-optimum of the hydrogen transfer reaction are described. Finally it is proposed that the hydrogen transfer between steroid hormones represents an aspect of the true reaction mechanism of steroid hormones: Steroid hormones function as hydrogen transferring coenzymes by forming part of a chain of hydrogen carriers.