Activation of purified cytoplasmic 17 beta-hydroxysteroid dehydrogenase from human placenta by human albumin and globulin

The cytoplasmic 17 beta-hydroxysteroid dehydrogenase of human placenta, purified more than 2500-fold, was activated by small amounts of human albumin and globulin. This activation was dependent on substrate concentration. At 20 microM estradiol (10 X KM) and two different concentrations of enzyme (0.01 and 2 micrograms/ml), the activation was greatest at albumin or globulin concentrations between 0 and 30 micrograms/ml. At "low" concentrations of estradiol (20 nM = 10(-2) X KM) and enzyme (0.01 microgram/ml), maximal activity occurred at approximately 10 micrograms/ml. Higher concentrations of albumin and globulin led to a decline in activity.     

Immunological measurement of human 17 beta-hydroxysteroid dehydrogenase

Human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) was purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on Red-Agarose and DEAE-Sepharose columns. Electrophoresis on polyacrylamide gels under denaturing conditions and using silver staining showed a single protein with an apparent molecular weight of 37,800. Antibodies to the purified protein were raised in rabbits and were found by immunoblotting to be specific to 17-HSD. A sensitive radioimmunoassay was established using 125I-labeled 17-HSD as a tracer, an appropriate dilution of the antibody, and a kaolin-coupled double antibody for separating the antibody-bound and free fractions. The detection limit of the assay was approximately 150 pg/tube (1.5 micrograms/l). The cytosol fraction (105,000 g) of term placental tissue contained approximately 0.7 mg of 17-HSD per gram of protein, and the concentrations of 17-HSD measured by immunoassay and enzymatic activity proved to be strictly parallel in different partly purified placental preparations. The supernatants from centrifugations of human endometrial homogenates at 800 g and 105,000 g (after detergent treatment) displayed cross-reactivity with the antibody. The mean concentration of the cross-reacting substance in the radioimmunoassay was 14.1 micrograms/g protein (range 2-62.3) in specimens taken on different days in the cycle. These concentrations showed a significant correlation with the 17-HSD activities measured in the endometrial specimens (r = 0.722, P less than 0.001, n = 21). Mean concentrations of substance were 8.3 micrograms/g protein in endometrial specimens taken during the follicular phase (days 4-12, n = 8) and 22.9 micrograms/g protein during the luteal phase (days 16-22, n = 6) were obtained using the radioimmunoassay. There was excellent parallelism between the competition curves for [125I]iodo-17-HSD with purified 17-HSD standards and placental and endometrial homogenate dilutions. These data strongly suggest that the substance measured in the endometrial specimens was 17-HSD.     

Multiple forms of formaldehyde dehydrogenase from human red blood cells

Red cell hemolysates from nonrelated Finns were analyzed by electrofocusing on polyacrylamide gel, and formaldehyde dehydrogenase (EC 1.2.1.1) was located by an activity-staining method. Three forms of the enzyme were constantly found for all the individuals studied but no variants were observed in this population (n = 217). Human liver also had three formaldehyde dehydrogenase forms with locations identical to those of the red cell formaldehyde dehydrogenase. Population genetic studies of formaldehyde dehydrogenase can easily be performed with red cell hemolysates with the techniques described here, and there is no need to use liver biopsy samples.     

Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta.

Human placental 17 beta-hydroxysteroid dehydrogenase has been purified with a new rapid procedure based on fast protein liquid chromatography, yielding quantitatively a homogeneous preparation with high specific activity catalyzing the oxidation of 7.2 mumol of estradiol/min/mg of enzyme protein at 23 degrees C, pH 9.2. This preparation was shown to have a subunit mass of 34.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while having a molecular mass of 68 kDa by both Superose-12 gel-filtration and native pore gradient gel electrophoresis. When 17 beta-hydroxysteroid dehydrogenase was expressed in HeLa cells or overproduced in insect cells using the baculovirus expression system, both from its cDNA encoding a protein of 34 kDa, the enzyme had the same migration in native and sodium dodecyl sulfate-gel electrophoresis as the purified one from human placenta and eluted from the Superose-12 column at the same elution volume. Moreover, all the above forms of this enzyme have similar specific activity. These results clearly demonstrate the identity of the three enzyme forms. The enzyme produced from the cDNA is expressed as a dimer, and its two subunits are identical. 17 beta-Hydroxysteroid dehydrogenase subunit identity is thus proved. The NH2-terminal analysis revealed a unique sequence of Ala-Arg-Thr-Val-Val-Leu-Ile for the purified enzyme from placenta, further confirming the above conclusion.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

Localization of 17 beta-hydroxysteroid dehydrogenase throughout gestation in human placenta

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) is the enzyme responsible for the formation of all sex steroids in gonadal as well as extragonadal tissues. To obtain more information about the age-specific expression of 17 beta-HSD in the human placenta, we have localized this enzyme by immunocytochemistry at the light microscopic level at different periods of gestation. In the 7- and 9-week-old placenta, immunostaining was detected exclusively in the cytoplasm of the syncytiotrophoblast. Between the tenth and thirteenth weeks of gestation, immunolabeling was also observed in the cytoplasm of the cytotrophoblastic cells, suggesting that these cells could be transiently involved in the biosynthesis of sex steroids. Interestingly, between the fourteenth and twenty-fifth weeks of gestation, 17 beta-HSD was observed in both the cytoplasm and nucleus of the syncytiotrophoblast. The reaction product was much more intense in nuclei than in cytoplasm. During the last trimester of gestation, strong immunocytochemical staining was observed in all the nuclei of the syncytiotrophoblast, the cytoplasm being unstained. The meaning of this nuclear staining for 17 beta-HSD is still unclear and remains to be extensively investigated.     

Structure of two in tandem human 17 beta-hydroxysteroid dehydrogenase genes

Two human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes (h17 beta-HSDI and h17 beta-HSDII) included in tandem within an approximately 13 kilobase pair fragment were isolated from a genomic lambda EMBL3 DNA library using cDNA encoding human 17 beta-HSD (hpE2DH216) as probe. We have determined the complete exon and intron sequences of the two genes as well as their 5' and 3'-flanking regions. Human 17 beta-HSDII contains six exons and five short introns for a total length of 3250 base pairs. The exon sequence of h17 beta-HSDII is identical to the previously reported hpE2DH216 cDNA while the overlapping nucleotide sequences of the corresponding exons and introns of h17 beta-HSDI and h17 beta-HSDII show 89% homology. In addition, we have used the hpE2DH216 cDNA to demonstrate the widespread expression of 17 beta-HSD mRNAs in steroidogenic and peripheral target tissues. These new findings provide the basis for a better understanding of the molecular mechanisms involved in 17 beta-HSD deficiency and peripheral sex steroid metabolism.     

Analysis and characteristics of multiple types of human 17beta-hydroxysteroid dehydrogenase

Androgens and estrogens are not only synthesized in the gonads but also in peripheral target tissues. Accordingly, recent molecular cloning has allowed us to identify multiple types of 17β-hydroxysteroid dehydrogenases (17β-HSD), the key and exclusive enzymes involved in the formation and inactivation of sex steroids. However, only one form, namely, type 3 17β-HSD, is responsible for pseudohermaphroditism in deficient boys. To date, seven human 17β-HSDs have been isolated and characterized. Although they catalyze substrates having a similar structure, 17β-HSDs have very low homology. In intact cells in culture, these enzymes catalyze the reaction in a unidirectional way — types 1, 3, 5 and 7 catalyze the reductive reaction, while types 2, 4 and 8 catalyze the oxidative reaction. It is noteworthy that rat type 6 17β-HSD also catalyzes the reaction in the oxidative direction. In this report, we analyze the different characteristics of the multiple types of human 17β-HSD.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Characterization and localization of human type10 17beta-hydroxysteroid dehydrogenase.

The tissue distribution, subcellular localization, and metabolic functions of human 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase have been investigated. Human liver and gonads are abundant in this enzyme, but it is present in only negligible amounts in skeletal muscle. Its N-terminal sequence is a mitochondrial targeting sequence, but is not required for directing this protein to mitochondria. Immunocytochemical studies demonstrate that this protein, which has been referred to as ER-associated amyloid beta-binding protein (ERAB), is not detectable in the ER of normal tissues. We have established that protocols employed to investigate the subcellular distribution of ERAB yield ER fractions rich in mitochondria. Mitochondria-associated membrane fractions believed to be ER fractions were employed in ERAB/Abeta-binding alcohol dehydrogenase studies. The present studies establish that in normal tissues this protein is located in mitochondria. This feature distinguishes it from all known 17beta-hydroxysteroid dehydrogenases, and endows mitochondria with the capability of modulating intracellular levels of the active forms of sex steroids.     

Ontogenesis of oxidative reaction of 17beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase in rat Leydig cells,a histochemical study

The enzyme 17beta-hydroxysteroid dehydrogenase is required for the synthesis and 11beta-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1-90. Using NAD or NADP as the cofactor, 17beta-hydroxysteroid dehydrogenase (substrate: 5-androstene-3beta,17beta-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17beta-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11beta-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17beta-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11beta-hydroxysteroid dehydrogenase in addition to types 1 and 2.     

17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.     

Studies on the activity of 17 beta-hydroxysteroid dehydrogenase in human adipose tissue

The activity of 17 beta-hydroxysteroid dehydrogenase has been investigated in human subcutaneous adipose tissue. Using oestrone as substrate, oestradiol formation was linear with time and the concentration of protein in the tissue homogenate. The optimum pH was 8.0 and the Km for oestrone was 2.5 x 10(-6) M. With NADH, the production of oestradiol was about 30% of that with NADPH. Oestradiol was also a substrate for the enzyme although under the experimental conditions used reduction of oestrone appeared to be favoured in adipose tissue. In the presence of progesterone (31.8 x 10(-6) M) the Km for oestrone was increased fivefold.     

人17β羟类固醇脱氢酶（17β—HSD）基因在家蚕系统中的表达（简报）

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a crucial role in the synthesis of estrogens, but it also produces negative action of estrogens in promoting the growth of hormone-sensitive cancers, especially in breast and prostate. The high specific activity can be taken as an important signal for the diagnosis of cancers. Recombinant rAcBm-NPV/17 beta-HSD virus which contains the human 17 beta-HSD cDNA under the control of polyhedron gene promoter is generated by cotransfection of the BmN cells with the transfer plasmid pVL/17 beta-HSD and wild BmNPV genomic DNA. 17 beta-HSD is maximally expressed 72 h and 120 h post infection in BmN cells and the 5th instar silkworm larvae respectively. At those time interval, intracellular and hemolymphic enzymatic activity reach 0.12 U/mg and 0.15 U/mg of protein which produced total activity of 0.97 U/1.5 x 10(6) cells and 4.7 U/larva. The expressed quantities in female larvae are a little higher than that in male larvae. The present data shows that Silkworm/BmNPV expression system can express 3-5 times higher than that of the richest human placenta. It also indicates that there is an apparent band with a molecular mass of 35 kDa using SDS-PAGE method, the size of which is similar to that of the crude enzyme from placenta.     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.