Barbara 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.

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.

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.

Anreicherung und Charakterisierung einer „Transhydrogenase-Aktivität“ und der 17 β-Hydroxysteroid-Oxidoreduktase aus der Cytoplasma-Fraktion der Leber von weiblichen Ratten / The Enrichment and Characterisation of Cytoplasmatic “Transhydrogenase-activity” and 17 β-hydroxysteroid-oxidoreductase from Rat Liver

The cytoplasmatic fraction of rat liver contains both 17 β-hydroxysteroid-oxidoreductase and a “transhydrogenase-activity”, which catalyses the transfer of hydrogen from the 17 β-position of estradiol-17 β to the 17-position of 4-androstene-3,17-dione. The 17 β-hydroxysteroid-oxidoreductase was purified 718-fold and the “transhydrogenase-activity” 264-fold by ammonium sulphate precipitation, gel chromatography with Sephadex G-200, column chromatography on DEAE-Sephadex and isoelectric focusing. The two enzymic activities could not be separated. The characteristics of the two enzymatic activities give some evidence that the “transhydrogenase-activity” is identical with the already known 17 β-hydroxysteroid-oxidoreductase. Isoelectric focusing of the chromatographycally enriched 17 β-enzyme gave an isoelectric point at 5,2. The 17 β-enzyme has a molecular weight of 62 — 65 000 as determined by mobility on Sephadex G-200 superfine. The kinetic constants for both the 17 β-enzyme and the “transhydrogenase-activity” were determined.

Charakterisierung und Kinetik des in der Rattenplacenta vorkommenden mikrosomalen Enzyms, das den Wasserstoff-Transfer von Oestradiol auf Androstendion katalysiert / Characterization and Kinetics of the Microsomal Enzyme of Rat Placenta which Functions as a “Transhydrogenase” between Estradiol-17β and Androstenedione

The microsomal fraction of rat placenta contains a 17β-hydroxysteroid-oxidoreductase which transfers hydrogen from position 17 of estradiol to androstenedione. This hydrogen transfer is dependent on NAD, NADP as cofactor is without effect. The optimum pH is at 6,9. In the presence of NAD the Michaelis constant for estradiol is 4,17 · 10-5м at pH 7,4. In the presence of androstenedione in the incubation medium the Km-value for estradiol is decreased, which indicates an increased affinity for the enzyme. The temperature optimum of the enzyme is 38 °C. Addition of SH-blocking agents inhibited the enzyme activity. Zinc and magnesium ions had an inhibitory effect on the “transhydrogenase” and B-NADPT specifically labelled from [1-T]-glucose showed that the non-effect of NADP on transhydrogenation from estradiol to androstenedione resulting in reduction of position 17 is not due to different stereospecifity. The results show a close relation between the oxidative metabolism of estradiol and the reduction of androstenedione, indicating that estradiol-17β, as the preferred hydrogen-donating substrate, is an essential component of the androstenedione-hydrogenating system in the microsomal fraction of rat placenta.