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Query: UNIPROT:P06889 (Mol)
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Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes, but not by the adrenals. Inhibition of the key enzyme for androgen biosynthesis, cytochrome P450(17) alpha, could prevent androgen production from both sources. The in vivo effects of 17-(3-pyridyl)androsta-5,16-dien-3 beta-ol (CB7598) and 17-(3-pyridyl)androsta-5,16-dien-3-one (CB7627), novel potent steroidal inhibitors of this enzyme, on WHT mice were compared with those of castration and two clinically active compounds, ketoconazole and flutamide. Flutamide and surgical castration caused significant reductions in the weights of the ventral prostate and seminal vesicles. CB7598, in its 3 beta-O-acetate form (CB7630), and CB7627 caused significant reductions in the weights of the ventral prostate, seminal vesicles, kidneys and testes when administered once daily for 2 weeks. Ketoconazole, given on the same schedule, caused no reductions. Plasma testosterone was reduced to < or = 0.1 nM by CB7630, despite a 3- to 4-fold increase in the plasma level of luteinizing hormone. Adrenal weights were unchanged following treatment with CB7630 or CB7627 but were markedly increased following ketoconazole, indicating no inhibition of corticosterone production by these steroidal compounds. These results indicate that CB7598, CB7630 or CB7627 may be useful in the treatment of hormone-dependent prostatic cancers.
J Steroid Biochem Mol Biol 1994 Sep
PMID:Pharmacology of novel steroidal inhibitors of cytochrome P450(17) alpha (17 alpha-hydroxylase/C17-20 lyase). 791 12

The structures of cDNA clones encoding four members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family were characterized. The rat type I, type II and the novel type IV are genuine NAD+/H-dependent 3 beta-HSD isoenzymes. On the other hand, the liver-specific type III protein is a specific 3-keto-reductase (3-KSR) that catalyzes the conversion of 5 alpha-androstane-3-one-17 beta-ol (DHT) and 5 alpha-androstane 3,17-dione (A-dione) into their 3 beta-hydroxy metabolites. The aim of the present study was to further characterize the enzymatic properties of rat types I, III and IV, especially their role in the formation and degradation of DHT after transient expression in intact human HeLa cervical carcinoma, JEG-3 choriocarcinoma or SW-13 adrenal cortex adenocarcinoma cells in culture. The expressed type III 3-KSR in intact HeLa cells catalyzed the reduction of DHT into 3 beta-diol, whereas expression of type I 3 beta-HSD in these cell lines had no significant effect on the basal conversion of DHT into 3 beta-diol, but it did increase the formation of DHT from 3 beta-diol. A-dione is the predominant product obtained when DHT and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) are used as substrates in intact JEG-3 and SW-13 cells transfected with rat type I 3 beta-HSD. Furthermore, this predominant 17 beta-HSD activity was also observed in SW-13 cells transfected with the novel rat type IV 3 beta-HSD. The predominance of this 'secondary' 17 beta-HSD activity is also reflected in HeLa cells transfected with type I 3 beta-HSD by the deduced predominant pathway 3 beta-diol-->DHT-->5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol)-->androsterone (ADT), in which formation of 3 alpha-HSD activity of HeLa cells, whereas the other reactions are catalyzed by the type I 3 beta-HSD isoenzyme. This observation thus demonstrates that rat type I 3 beta-HSD may also catalyze the conversion of 3 alpha-diol into ADT through its intrinsic 17 beta-HSD activity. The predominant metabolic pathways observed in the present study could be attributed to preponderant bioavailability of NAD+ and NADPH in the intact transfected cells used.
Mol Cell Endocrinol 1994 Jul
PMID:Formation and degradation of dihydrotestosterone by recombinant members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase family. 795 95

Structures of the Fab' fragment of the anti-progesterone antibody DB3 in complex with five cross-reactive steroids (aetiocholanolone, 5 beta-androstane-3,17-dione, 5 alpha-pregnane-20-one-3 beta-ol-hemisuccinate, progesterone-11 alpha-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 A. These different steroids compete with progesterone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional structures. Comparison of the unliganded DB3 Fab' and these five steroid-Fab' complexes reveals that all the steroid ligands bind to an "open" conformation of the Fab' as defined by the orientation of the indole side-chain of TrpH100, whereas in the unliganded or "closed" form the binding site is occluded by TrpH100. Small but significant conformational changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various cross-reactive ligands are accommodated in the binding site in two distinct orientations. We term these binding modes syn and anti, as they are defined by the orientation of the steroid beta face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainly by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 alpha position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specificity, such as linker position and the paucity of functional groups on the immunogenic hapten.
J Mol Biol 1994 Sep 02
PMID:Structural analysis of antibody specificity. Detailed comparison of five Fab'-steroid complexes. 807 92

The concentrations of five 16-androstene steroids were determined, by a GC-MS method, in freshly-produced apocrine sweat (adrenaline-induced), in 8 men and 2 women. The ranges of concentrations (nmol/microliter) in apocrine sweat were: 5 alpha-androst-16-en-3-one (5 alpha-A), 0.1-2.0 and 4,16-androstadien-3-one (androstadienone), 0-1.9, 5,16-Androstadien-3 beta-ol (androstadienol) was also found in 5 of the subjects (range 0.05-1.05). 5 alpha-Androst-16-en-3 alpha- or 3 beta-ols [3 alpha (beta)-androstenols] were only found in small amounts (< 0.1 nmol/microliters) in a few subjects. In the second study, prior to apocrine sweat collection (adrenaline injection), the axillary skin of 6 of the male subjects was washed with diethyl ether on an adjacent site of the axillary vault. The concentrations of 16-androstenes were compared in the ethereal extracts and apocrine sweat. The former contained detectable levels (pmol/cm2) of androstadienone (17.9 +/- 2.4), 3 alpha-androstenol (6.9 +/- 3.7), 3 beta-androstenol (1.8 +/- 1.0) and androstadienol (1.9 +/- 0.5) (means +/- SEM) in all 6 subjects. All but 1 subject also had 5 alpha-androstenone, the mean value for the others being 2.5 +/- 0.6. The axillary skin levels of 3 alpha- and 3 beta-androstenols, androstadienol and, in 3 subjects, androstadienone exceeded those in the apocrine sweat obtained from the same subjects, whereas levels of 5 alpha-androstenone in the skin extracts were all lower than in apocrine sweat samples, when related to the corresponding areas of skin sampled. The metabolism of 16-androstenes was studied in vitro in the presence of two aerobic coryneform bacteria, previously shown to metabolize testosterone as well as being capable of producing odour from extracts of axillary sweat in an odour-generation test. Although both coryneforms caused complex metabolic reactions and were capable of oxidation or reduction at C-3 and C-4, the overall direction favoured reduction. For example, large quantities of the more odorous 5 alpha-androstenone and 3 alpha-androstenol were formed from androstadienol and androstadienone. In contrast, strains of corynebacteria, unable to produce odour and incapable of metabolizing testosterone, were also unable to metabolize 16-androstenes.(ABSTRACT TRUNCATED AT 400 WORDS)
J Steroid Biochem Mol Biol 1994 Mar
PMID:Comparison of 16-androstene steroid concentrations in sterile apocrine sweat and axillary secretions: interconversions of 16-androstenes by the axillary microflora--a mechanism for axillary odour production in man? 814 19

The synthesis of 5,16-androstadien-3 beta-ol from pregnenolone occurs via a cytochrome P450-dependent reaction (andien-beta synthase) that is analogous to the C17-hydroxylase/lyase reaction. It is not known whether the andien-beta synthase activity in adult porcine testis involves cytochrome P450c17 or is unique to porcine testis. Andien-beta synthase activity in testis microsomes was inhibited by high pH and concentration of salt, while C17-hydroxylase/lyase activity was stimulated under these conditions. Cytochrome P450c17 purified from adult porcine testis and adrenal glands and bovine adrenal glands had only C17-hydroxylase/lyase activity in the absence of cytochrome b5. However, when cytochrome b5 isolated from porcine testis was added, andien-beta synthase activity was detected in all three preparations of cytochrome P450c17, with the highest activity found in the porcine preparations. The andien-beta synthase activity was further increased from 2.5 to 6 times when NADH cytochrome b5 reductase was added along with cytochrome b5. Levels of mRNA for cytochrome b5 relative to cytochrome P450c17 mRNA were five times higher in porcine testis than in porcine adrenal. It appears that the andien-beta synthase activity is catalysed by cytochrome P450c17, which is not unique to the porcine testis and is dependent upon adequate levels of cytochrome b5.
J Steroid Biochem Mol Biol 1993 Nov
PMID:Cytochrome P450c17 from porcine and bovine adrenal catalyses the formation of 5,16-androstadien-3 beta-ol from pregnenolone in the presence of cytochrome b5. 824 Sep 78

The enzymatic synthesis of [3H]5-pregnen-3 beta-ol-20-one sulfate using [3H]5-pregnen-3 beta-ol-20-one, 3'-phosphoadenosine-5'-phosphosulfate and hydroxysteroid sulfotransferase 1 purified from rat liver is reported. The described procedure allowed the obtainment of high specific activity [3H]5-pregnen-3 beta-ol-20-one sulfate in yields ranging from 78 to 86% with respect to [3H]5-pregnen-3 beta-ol-20-one. Two-dimensional thin-layer chromatography was used to purify [3H]5-pregnen-3 beta-ol-20-one sulfate which upon solvolysis resulted in the formation of [3H]5-pregnen-3 beta-ol-20-one. The identity both of the synthesized compound and the solvolysed one was confirmed by reversed-phase high pressure liquid chromatography, and 2-dimensional thin-layer chromatography.
J Steroid Biochem Mol Biol 1993 Nov
PMID:Enzyme-catalyzed synthesis of radiolabeled 5-pregnen-3 beta-ol-20-one sulfate. 824 Sep 86

The microsomal fraction from the testes of immature pigs can convert pregnenolone to 17-hydroxypregnenolone and also to 5,16-androstadien-3 beta-ol (andien-beta). The available evidence supports the hypothesis that both these reactions are catalysed by one enzyme, cytochrome-P450(17 alpha). In the absence of cytochrome b5, 17-hydroxypregnenolone will be the major product but that if cytochrome b5 is present in sufficient quantity, andien-beta becomes a major product. The point of divergence between the conversion of pregnenolone to either 17-hydroxypregnenolone or andien-beta was investigated using enzyme kinetic analysis to determine whether 16 alpha-hydroxypregnenolone, 20 beta-hydroxypregnenolone or 16-dehydropregnenolone could be specific intermediates to one reaction or the other. Product inhibition by 17-hydroxypregnenolone and andien-beta was competitive for both 17-hydroxylase and "andien-beta synthetase" supporting the current view of a common active site for both reactions. 16 alpha-Hydroxypregnenolone was a very poor competitive inhibitor of 17-hydroxylase and andien-beta synthetase with Ki(app) values many fold greater than the Km(app) for pregnenolone or the Ki(app) for reaction product, rendering it unlikely that 16 alpha hydroxylation is a key intermediary step in either pathway. 20 beta-Hydroxypregnenolone was a more potent inhibitor of andien-beta synthetase than of 17-hydroxylase and for the latter enzyme activity, the Ki(app) was lower than that for 17-hydroxypregnenolone itself. However, for andien-beta synthetase, 20 beta-hydroxypregnenolone may be an early intermediate as the Ki(app) was consistent with the affinity for the active site being intermediate between the Km(app) for pregnenolone and the Ki(app) for andien-beta. 16-Dehydropregnenolone was equipotent at inhibiting 17-hydroxylase and andien-beta synthetase activities suggesting that 16-dehydropregnenes may be involved in the stages immediately prior to C21 side-chain cleavage.
J Steroid Biochem Mol Biol 1993 Jul
PMID:17-Hydroxylase and andien-beta synthetase activities in immature pig testis microsomal fraction: kinetic studies of the pregnenolone binding site and possible intermediates of the reactions. 833 93

Pregnanolone [5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha)] and allopregnanolone [5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha)] are neuroactive steroids that are reduced metabolites of progesterone. Both 5 beta 3 alpha and 5 alpha 3 alpha are potent positive modulators of the gamma-aminobutyric acid response that enhance the binding of [3H]flunitrazepam ([3H] FNZ) to the gamma-aminobutyric acid type A receptor. Chronic (48 hr) exposure of brain neurons in culture to 5 beta 3 alpha or 5 alpha 3 alpha abolishes potentiation of [3H]FNZ binding by these steroids. This uncoupling, or loss of allosteric interactions between steroid and benzodiazepine recognition sites, is dose dependent, stereospecific, and reversible. The number and affinity of [3H]FNZ binding sites are unaffected. In contrast, the steroids 5 beta-pregnan-3 beta-ol-20-one, beta-estradiol, testosterone, progesterone, deoxycorticosterone, and dexamethasone, which show little capacity to potentiate [3H]FNZ binding, are also much less effective in inducing uncoupling of steroid and benzodiazepine recognition sites. These results suggest a mechanism whereby neurons could become refractory to long term modulation by neuroactive steroids. The results are discussed in terms of their possible relevance to premenstrual anxiety and enhanced frequency of seizures in certain women.
Mol Pharmacol 1993 Jul
PMID:Gamma-aminobutyric acidA receptor regulation: chronic treatment with pregnanolone uncouples allosteric interactions between steroid and benzodiazepine recognition sites. 839 20

The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such "secondary" activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Structure-function relationships and molecular genetics of the 3 beta-hydroxysteroid dehydrogenase gene family. 854 74

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in "non-target" tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17 beta-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17 beta-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of beta-actin. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17 beta-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Expression and regulation of aromatase and 17 beta-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells. 854 82


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