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PubMed1978

A direct effect of medroxyprogesterone acetate on 17 beta-hydroxysteroid dehydrogenase in adult rat testis.

Sixty-day-old rats were divided into four groups and treated for 30 days with either medroxyprogesterone acetate (Provera), gonadotropins (bovine LH and ovine FSH), Provera plus gonadotropins, or saline. The progestin treatment resulted in a lowering of plasma levels of testosterone, androstenedione, and LH, as well as in a reduction of epididymal sperm counts and accessory sex organ weights. The progestin-treated groups showed markedly lower levels of testicular 17 beta-hydroxysteroid dehydrogenase activity (35% of controls) and delta 5,3 beta-hydroxysteroid dehydrogenase activity (70% of controls). Rats treated with only gonadotropins exhibited reduced 17 beta-hydroxysteroid dehydrogenase but increased delta 5,3 beta-hydroxysteroid dehydrogenase activities. It was concluded from these results that progestins may affect testicular steroidogenesis and spermatogenesis not only by reducing LH secretion but also by a direct effect on the testis, as LH suppression could not account for the inhibition of 17 beta-hydroxysteroid dehydrogenase activity. Long term progestin treatment did not alter the steroidogenic response of the testis to acute administration of LH, although the testosterone to androstenedione ratio in plasma was decreased.

17-Hydroxysteroid Dehydrogenases↗
PubMed2002

5 alpha-reductase and 11 beta-hydroxysteroid dehydrogenase activity in prepubertal Hispanic girls with premature adrenarche.

Girls with idiopathic premature adrenarche, characterized by the early appearance of pubic hair and adrenal hyperandrogenism, may be at an increased risk for polycystic ovarian syndrome and its associated complications. Alterations of peripheral metabolism of adrenal steroids, specifically increased 5 alpha-reductase and 11 beta-hydroxysteroid dehydrogenase activities, have been documented in patients with polycystic ovarian syndrome and proposed as an underlying mechanism for the adrenal hyperandrogenism in this syndrome. We sought to investigate whether alterations in 5 alpha-reductase and 11 beta-hydroxysteroid dehydrogenase activities are present in girls with premature adrenarche, suggesting a possible role in the pathogenesis of the hyperandrogenism of this condition. We studied C19 and C21 urinary steroid metabolites, 5 alpha/5 beta and 11 oxo/11 hydroxy metabolite pairs as well as the ratios of the total 5 alpha/total 5 beta and total 11 oxo/total 11 hydroxy metabolites in 24-h urine samples from 17 prepubertal Hispanic girls with premature adrenarche and seven controls. We found no differences in the 5 alpha-reductase or 11 beta-hydroxysteroid dehydrogenase activities in the prepubertal girls with premature adrenarche, compared with the controls. When age and body mass index Z-score were controlled for in the statistical analysis, the results did not change. Total cortisol metabolites were not different in the girls with premature adrenarche, compared with the controls. In conclusion, we did not demonstrate a difference in the peripheral steroid metabolism, specifically 5 alpha-reductase and 11 beta-hydroxysteroid dehydrogenase activities, in prepubertal Hispanic girls with premature adrenarche, compared with controls. Therefore, in this group of young girls, alterations in 5 alpha-reductase or 11 beta-hydroxysteroid dehydrogenase activities do not appear to contribute to their early pubic hair development.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1983

Androgen 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in ventral prostate epithelial and stromal cells from immature and mature rats.

To study androgen-mediated differentiation in the rat ventral prostate, we separated the two principal cell types (epithelial and stromal) derived from prostates of immature and mature rats on two continuous Percoll gradients. Cells were immediately placed in culture medium. Testosterone metabolism by the two prostatic cell types was evaluated using [3H]testosterone and quantifying the formation of 5 alpha-[3H]dihydrotestosterone (5 alpha-DHT) and 5 alpha-[3H]androstane-(3 alpha or 3 beta), 17 beta-diols. In epithelial cells from both immature and mature rat prostates the major testosterone metabolites were 5 alpha-DHT and 5 alpha-androstane-3 alpha, 17 beta-diol. Stromal cells metabolized less testosterone than did the epithelial cells. Differences in the relative levels of the various metabolites were observed for the two age groups. To examine in more detail the changes in testosterone metabolism observed in vitro both types of cells and unfractionated cells from immature and mature rat prostates were assayed for testosterone 5 alpha-reductase (using testosterone as substrate) and 3 alpha-hydroxysteroid dehydrogenase (using 5 alpha-DHT as substrate) activities (expressed as pmol substrate reduced/min per 10(6) cells). In immature rats both 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities were localized in the epithelial cell fraction (17 and 52 respectively); stromal cells showed lower 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activity (4 and 4). Relative to epithelial cells from immature rats epithelial cells from mature rats showed a decrease in 5 alpha-reductase (7) and an increase in 3 alpha-hydroxysteroid dehydrogenase (160) activity while stromal 5 alpha-reductase showed little change (3) and 3 alpha-hydroxysteroid dehydrogenase increased to 22.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

11Beta-hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules.

Mineralocorticoid hormones regulate salt transport along the distal nephron by binding to intracellular receptors and activating gene transcription. Previous experiments showed that systemic aldosterone infusions stimulate thiazide-sensitive Na and Cl transport by distal convoluted tubule (DCT) cells; this effect could have been direct or secondary to systemic hormonal effects. Aldosterone target tissues express both mineralocorticoid receptors and the metabolic enzyme 11beta-hydroxysteroid dehydrogenase type 2. Mineralocorticoid receptors have been localized to the DCT in some experiments, but not in others. Expression of 11beta-hydroxysteroid dehydrogenase type 2 by DCT cells has not been investigated. The present experiments were designed to test the hypothesis that rat DCT cells are targets of aldosterone action. Patterns of mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase, thiazide-sensitive Na-Cl cotransporter, and Na/Ca exchanger expression along the distal tubule were examined. A polyclonal antibody was generated to localize the thiazide-sensitive Na-Cl cotransporter. Thiazide-sensitive Na-Cl cotransporter and 11beta-hydroxysteroid dehydrogenase expression were examined using both in situ hybridization and immunocytochemistry; Na/Ca exchanger and mineralocorticoid receptor expression were examined by immunocytochemistry. The results indicate that 11beta-hydroxysteroid dehydrogenase is expressed by DCT cells, as well as connecting tubule cells and principal cells of the collecting duct; expression levels are low near the junction with the thick ascending limb and rise near the transition to the connecting tubule. Mineralocorticoid receptors are expressed by DCT cells, as well as along the thick ascending limb, connecting tubule, and collecting duct. The results indicate that components of the mineralocorticoid receptor system are expressed by DCT cells, suggesting that these cells are targets of aldosterone action.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1989

3(20)alpha-hydroxysteroid dehydrogenase activity of monkey liver indanol dehydrogenase.

Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.

20-Hydroxysteroid Dehydrogenases↗
PubMed1987

Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: hydroxysteroid dehydrogenase activity.

The activity of hydroxysteroid dehydrogenases was histochemically quantified in corpora lutea (CL) from prepuberal gilts induced to ovulate and mature gilts. Prepuberal (P) gilts, 120 to 130 d of age were induced to ovulate with 1,500 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG). Three P gilts and three mature (M) gilts each were ovariectomized on d 10, 14, 18, 22 and 26 (d 0 = day of hCG for P gilts and onset of estrus for M gilts). Gilts ovariectomized on d 14, 18, 22 and 26 were hysterectomized on d 6 to ensure luteal maintenance. At the time of ovariectomy, CL were frozen in liquid nitrogen and then stored at -80 C until analysis. Cryostat sections (12 microns) were histochemically analyzed for delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHSD), 17 alpha-hydroxysteroid dehydrogenase (17 alpha OHSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha OHSD). The intensity of staining (greater enzyme activity resulted in darker staining) was quantified using a Zeiss SF microscope integrated with a Zonax photometer, which measured the percentage of light transmitted through a given area (22,500 microns 2) of the tissue section. Data were subjected to analysis of variance using the general linear models procedure of Statistical Analysis System (SAS). The 3 beta OHSD activity did not change over days, but the mean activity (throughout all days) in the P gilts (32.6 +/- 1.8) tended (P less than .08) to be elevated above that of M gilts (27.9 +/- 1.7).(ABSTRACT TRUNCATED AT 250 WORDS)

17-Hydroxysteroid Dehydrogenases↗
PubMed1990

A possible mechanism for increased breast cell proliferation by progestins through increased reductive 17 beta-hydroxysteroid dehydrogenase activity.

We have investigated whether progestins may be able to regulate breast cell proliferation by altering the fraction of oestradiol relative to oestrone, using the human breast cancer cell line MCF-7. The ability of the two oestrogens, oestradiol and oestrone, to stimulate breast tumour cell proliferation was investigated. Oestradiol in concentration was of 10-fold greater proliferative potency than oestrone. The progestin MPA increased both reductive and oxidative 17 beta-hydroxysteroid oxidoreductase activity when the tissue culture media pH indicator phenol red was included in the media. When phenol red was excluded from the tissue culture media, MPA increased predominantly the reductive 17 beta-hydroxysteroid oxidoreductase activity, and to a far greater extent than in the presence of phenol red. Other progestins such as levonorgestrel, norethisterone and norethisterone acetate also increased predominantly reductive 17 beta-hydroxysteroid oxidoreductase activity in the absence of phenol red. The action of MPA on reductive 17 beta-hydroxysteroid oxidoreductase activity was increased by treatment with oestradiol to a small but significant extent. We propose that the progestational increase of reductive 17 beta-hydroxysteroid oxidoreductase activity is a possible mechanism by which progestins may increase breast cell proliferation in vivo.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalysed by a human placental 17beta-hydroxysteroid dehydrogenase.

The human placental 17beta-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17beta-estradiol and other estrogens. Since 19 alpha-estradiol is a competitive inhibitor of 9,10-phenanthrenequinone by the 17beta-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17beta-hydroxysteroid dehydrogenase, the equilibrium between 17beta-estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17beta-hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans-dihydrodiols tested.

17-Hydroxysteroid Dehydrogenases↗
PubMed1975

NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.

Bacteroides↗
PubMed1994

Major hydroxysteroid sulfotransferase STa in rat liver cytosol may consist of two microheterogeneous subunits.

The possible existence of two microheterogeneous subunits, designated ST-40P and ST-41P, of hydroxysteroid sulfotransferases in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNAs, both isolated from two rat liver cDNA libraries. These subunits consisted of an equal number of amino acid residues with only one amino acid substitution. ST-40P and ST-41P expressed as homodimers from the ST-40 and ST-41 cDNAs in Escherichia coli had enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and the carcinogen 5-hydroxymethylchrysene (5-HCR), with formation of the reactive metabolite 5-HCR sulfate, at rates very similar to those by STa, the major hydroxysteroid sulfotransferase in rat liver cytosol. This strongly suggested that they are essential components of STa. The present study carried out by using the recombinant enzymes provides the first direct evidence for the identity of sulfotransferases catalysing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing hydroxysteroid sulfotransferases from bile acid sulfotransferases should be improved.

Amino Acid Sequence↗
PubMed1992

Expansion of the mammalian 3 beta-hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP-galactose-4-epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus.

Mammalian 3 beta-hydroxysteroid dehydrogenase and plant dihydroflavonol reductases are descended from a common ancestor. Here we present evidence that Nocardia cholesterol dehydrogenase, E. coli UDP-galactose-4 epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus are homologous to 3 beta-hydroxysteroid dehydrogenase and dihydroflavonol reductase. Analysis of a multiple alignment of these sequences indicates that viral ORFs are most closely related to the mammalian 3 beta-hydroxysteroid dehydrogenases. The ancestral protein of this superfamily is likely to be one that metabolized sugar nucleotides. The sequence similarity between 3 beta-hydroxysteroid dehydrogenase and the viral ORFs is sufficient to suggest that these ORFs have an activity that is similar to 3 beta-hydroxysteroid dehydrogenase or cholesterol dehydrogenase, although the putative substrates are not yet known.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies.

In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.

17-Hydroxysteroid Dehydrogenases↗
PubMed2005

Effect of streptozotocin-induced diabetes on the gene expression and biological activity of 3beta-hydroxysteroid dehydrogenase in the rat spinal cord.

Abnormal secretion of steroids by the adrenals and gonads is one of the disturbances occurring in diabetics but the impact of diabetes on steroid formation in the nervous system has never been studied. However, it is well known that numerous actions of peripheral steroids on the nervous system require their conversion into neuroactive metabolites within the neural tissue. As this in situ steroid synthesis/metabolism is crucial for the control of several neurobiological functions, we investigated the effects of streptozotocin-induced diabetes on the gene expression and activity of 3beta-hydroxysteroid dehydrogenase in the spinal cord, a pivotal structure involved in sensorimotor and neurovegetative mechanisms. 3beta-Hydroxysteroid dehydrogenase is a key enzyme which participates to the biosynthesis of all classes of steroids by converting delta5-3beta-hydroxysteroids such as pregnenolone and dehydroepiandrosterone into delta4-3-ketosteroids as progesterone and androstenedione, respectively. Reverse transcription coupled with quantitative real-time polymerase chain reaction revealed that 3beta-hydroxysteroid dehydrogenase gene was over-expressed in the spinal cord of streptozotocin-treated rats compared with controls. Pulse-chase experiments combined with high performance liquid chromatography and continuous flow detection of newly-synthesized steroids showed an increase of 3beta-hydroxysteroid dehydrogenase activity responsible for a hyper-production of progesterone in the spinal cord of diabetic rats. This up-regulation of progesterone biosynthesis was concomitant with a decrease of its transformation into tetrahydroprogesterone, a process which facilitated progesterone accumulation in the spinal cord of streptozotocin-treated rats. Since progesterone is a potent neuroprotective steroid, increase of its production appeared as an endogenous molecular and biochemical mechanism triggered by spinal nerve cells to cope with degenerative effects of streptozotocin-induced diabetes. Our results constitute the first direct evidence showing an impact of diabetes on steroid biosynthetic and metabolic pathways in the nervous system. The data open new perspectives for the modulation of deleterious effects of diabetes by neuroprotective steroids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Aromatase and 17beta-hydroxysteroid dehydrogenase inhibition by flavonoids.

A method for estimating in the same assay both aromatase and 17beta-hydroxysteroid dehydrogenase activities in human placental microsomes using radiolabelled [1,2,6,7-3H]4-androstene-3,17-dione was proposed. In this assay, estrone (E1) and estradiol (E2) produced were separated by HPLC and estimated using a radioactive flow detector. Using this method, the inhibitory effect of various flavonoids, including flavone, flavanone and isoflavone, on the human placental aromatase and 17beta-hydroxysteroid dehydrogenase was studied. Flavonoids were shown to be potent inhibitors of both aromatase and 17beta-hydroxysteroid dehydrogenase activities. We found that 7-hydroxyflavone and apigenin are the most effective aromatase and 17beta-hydroxysteroid dehydrogenase inhibitors, respectively. Experiments showed that a hydroxyl group in position 7 was essential for anti-17beta-hydroxysteroid dehydrogenase activity. However, flavonoids with 7-methoxy or 8-hydroxyl groups on the A ring showed only anti-aromatase activity. Structure-activity relationships were discussed.

17-Hydroxysteroid Dehydrogenases↗
PubMed1998

Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase.

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3alpha/beta,20beta-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20beta-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4-1% and the mutant with arginine-198 has a catalytic efficiency of 4-23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3alpha/beta,20beta-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.

20-Hydroxysteroid Dehydrogenases↗
PubMed2000

Mutations in the type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) gene can cause premature pubarche in girls.

OBJECTIVE: Most previous studies have failed to demonstrate any mutations in the type II 3beta hydroxysteroid dehydrogenase (HSD3B2) gene in patients satisfying the hormonal criteria of nonclassic 3beta-hydroxysteroid dehydrogenase deficiency, suggesting that a mutant 3beta-hydroxysteroid dehydrogenase protein is not the cause of this disorder. We screened the HSD3B2 gene for mutations in girls with premature pubarche and a hormonal diagnosis of 3beta-hydroxysteroid dehydrogenase deficiency. DESIGN: From 30 girls with premature pubarche, we selected 9 whose ACTH-stimulated 17-hydroxypregnenolone levels were elevated (> or =6 SD) and screened the HSD3B2 gene for mutations. MEASUREMENTS: All patients were submitted to a standard ACTH stimulation test. Serum steroids were measured and compared to the mean level of pubertal stage matched control subjects. The four exons and exon-intron boundaries of the HSD3B2 gene were amplified by polymerase chain reaction and screened for mutations by denaturing gradient gel electrophoresis. The fragments with abnormal migration on denaturing gradient gel electrophoresis were directly sequenced. RESULTS: A homozygous T259M mutation was identified in one girl and a new compound heterozygous G129R/P222H mutation was identified in two sisters. The highest ACTH-stimulated 17-hydroxypregnenolone levels, 147, 339 and 351 nmol/l, were found in those patients with mutations in the HSD3B2 gene. In the patients without mutations, ACTH-stimulated 17-hydroxypregnenolone ranged from 48 to 111 nmol/l. ACTH-stimulated dehydroepiandrosterone levels had an overlap among the girls with and without mutations and the normal controls. CONCLUSIONS: Premature pubarche can be caused by mutations in the type II 3beta hydroxysteroid dehydrogenase gene.

17-alpha-Hydroxypregnenolone↗
PubMed1988

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.

17-Hydroxysteroid Dehydrogenases↗
PubMed1987

Oxidation of primary bile acids by a 7 alpha-hydroxysteroid dehydrogenase elaborating Clostridium bifermentans soil isolate.

A gram-positive, rod-shaped anaerobe (strain F-6) was isolated from soil. This organism was identified by cellular morphology as well as fermentative and biochemical data as Clostridium bifermentans. Strain F-6 formed 7-ketolithocholic acid from chenodeoxycholic acid and 7-ketodeoxycholic acid from cholic acid in whole cell cultures, but did not transform deoxycholic acid, ursodeoxycholic acid, or ursocholic acid. This reaction is reversible. The structures of 7-ketolithocholic acid and 7-ketodeoxycholic acid were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. When incubated with the strain F-6 glycine and taurine conjugates of the primary bile acids were partially hydrolyzed and transformed to 7-keto products. Optimal yields of 7-ketolithocholic acid and 7-ketodeoxycholic acid were obtained after 78 h of incubation. Culture pH changed with time and was characterized by an initial drop (1.1 pH units) and a gradual increase back to the starting pH (7.3). Corroborating these observations, an inducible, NADP-dependent, 7 alpha-hydroxysteroid dehydrogenase was demonstrated in cell extracts of strain F-6. A trace of NAD-dependent 7 alpha-hydroxysteroid dehydrogenase was also found. A substantial increase in the specific activity of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was observed when either 7-ketolithocholic acid, chenodeoxycholic acid, or deoxycholic acid was included in the growth medium. Optimal induction of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was achieved with 0.3-0.4 mM 7-ketolithocholic acid. Production of the enzyme(s) was optimal at 6-8 h of growth and the 7 alpha-hydroxysteroid dehydrogenases had a pH optimum of approximately 11.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile Acids and Salts↗