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PubMed1993

Ten years experience with masculinizing genitoplasty in male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency.

An extensive survey in a highly inbred Arab community documented over 60 cases of male pseudohermaphroditism due to 17 beta hydroxysteroid dehydrogenase deficiency. During a period of 10 years (1981-1991), 16 such patients underwent masculinating genitoplasty. Two children have had a one stage corrective genitoplasty while all others (2 adults, 3 adolescents and 9 children) had staged operations. Cosmetic and functional results are very encouraging in children and adolescents but very poor among adults. Based on our experience and follow-up, we advocate early sex assignment and early masculinizing genitoplasty with preoperative hormonal therapy to male pseudohermaphrodites due to 17 beta hydroxysteroid dehydrogenase deficiency.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

The activity of 3 beta-hydroxysteroid dehydrogenase and delta 4-5 isomerase in human follicular tissue.

The steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase and delta 4-5 isomerase (3 beta-HSD) that convert delta 5-hydroxysteroids to delta 4-ketosteroids were measured in human follicular tissue collected during the follicular phase of the menstrual cycle. This study was done in order to determine whether 3 beta-HSD enzyme activity fluctuated during the follicular phase of the menstrual cycle and, if so, whether these changes were reflected by the concentration of steroids in the follicular fluid. A microsomal fraction was prepared from each of 14 follicular-phase follicles, and 3 beta-HSD enzyme activity was estimated by the amount of androstenedione synthesized in the presence of excess substrate (dehydroepiandrosterone) and cofactor (nicotinamide adenine dinucleotide). Androstenedione, dehydroepiandrosterone, and progesterone were measured in the aspirated follicular fluid of each follicle. 3 beta-HSD enzyme activity was undetectable in the smallest (3 to 5 mm) follicles, increased in 5 to 6 mm follicles to 363.2 +/- 60 pmol of androstenedione per milligram per hour, and increased still further in 9 to 11 mm follicles to 5,000 +/- 200 pmol of androstenedione per milligram per hour. The increase in 3 beta-HSD enzyme activity was accompanied by an increase in the concentration of androstenedione in the follicular fluid as well as of progesterone in larger follicles. These data indicate that 3 beta-HSD activity increases during the follicular phase of the menstrual cycle. It is suggested that the primary product of the increased enzyme activity is androstenedione. Since androstenedione is the principal C19 steroid produced by the ovulatory follicle and serves as a substrate for estradiol production, this increase in 3 beta-HSD activity may be important for the associated changes in the late follicular phase that lead to ovulation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Affinity labeling identifies histidine at the active site of human placental 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase.

OBJECTIVE: Our aim was to determine if the multifunction enzyme, 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase has one or more active sites to effect dehydrogenase and isomerase activities. STUDY DESIGN: This steroid, which we have purified to homogeneity from human placental microsomes, was inactivated by the affinity labeling steroid, 2 alpha-bromo[2'-14C]acetoxyprogesterone. The amino acids that were radioalkylated in the absence and presence of the dehydrogenase substrate pregnenolone were identified. RESULTS: Pregnenolone completely abolished the inactivation of dehydrogenase. Histidine was localized in the active site of 3 beta-hydroxysteroid dehydrogenase because the radiolabel disappeared from enzyme inactivated in the presence of pregnenolone. Cysteine, a major radiolabeled product (80%) in the absence of pregnenolone, was decreased twofold in incubations that contained pregnenolone. Neither pregnenolone nor the isomerase substrate 5-androstene-3,17-dione protected isomerase from inactivation by the affinity alkylator. CONCLUSION: This observation contradicts coexisting, separate binding sites, one for each activity. Rather, a conformation shift around one binding region prompted by products of the dehydrogenase reaction may create the isomerase activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1980

Renaturation and urea-induced denaturation of 20 beta-hydroxysteroid dehydrogenase studied in solution and in the immobilized state.

Tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) from Streptomyces hydrogenans was reactivated after inactivation, dissociation and denaturation with urea. The effect of several factors such as NAD+, NADH, substrate, sulphydryl reducing agents, extraneous proteins, pH and enzyme concentration on reactivation was investigated. The coenzymes were found to be essential for obtaining a high reactivation yield (about 90%), since in their absence the reactivation was less than 10%. NADH was effective at lower concentrations than NAD+. The reactivated enzyme, after the removal of inactive aggregates, showed physical and catalytic properties coincident with those of the native enzyme. The mechanism by which NADH affects the reconstitution of 20 beta-hydroxysteroid dehydrogenase was investigated using both soluble enzyme and enzyme immobilized on Sepharose 4B. The immobilization demonstrates that isolated subunits are inactive and incapable of binding NADH and suggests that subunit association to the tetramer is essential for enzymatic activity. NADH appears to act, after subunit assembly has taken place, by stabilizing tetramers and preventing their aggregation with monomers that would give rise to inactive polymers.

20-Hydroxysteroid Dehydrogenases↗
PubMed1988

Characterization of NADP-dependent 12 beta-hydroxysteroid dehydrogenase from Clostridium paraputrificum.

Clostridium paraputrificum D 762-06 was found to contain an NADP-dependent 12 beta-hydroxysteroid dehydrogenase, already present in uninduced cells. Its specific activity could, however, be enhanced up to about 3-fold by the inclusion of bile acids with a 12-keto group or a 12 beta-hydroxy group in the growth medium. 3 alpha-Hydroxy-12-keto-5 beta-cholanoic acid was the most effective inducer. A pH optimum of 10.0 and a molecular weight of 126,000 were estimated by molecular sieve chromatography. The enzyme preparation reduced 12-keto groups in conjugated and unconjugated bile acids and oxidized a 12 beta-hydroxy function, but oxidative activity was only about 25% of the reductive one. Disubstituted bile acids showed lower Km values than the corresponding trisubstituted ones, the lowest Km values being those observed for 3,12- and 7,12-5 beta-cholanoic acids. No measurable activity against 12 alpha-hydroxyl groups could be detected. The enzyme was found to be heat-labile (95% inactivation at 50 degrees C for 10 min), but the activity was maintained for about 4 weeks when lyophilized preparations were stored at -20 degrees C. 12 beta-Hydroxysteroid dehydrogenase activity was also demonstrated in the membrane fraction after solubilization with Triton X-100, suggesting that it was originally a membrane-bound enzyme.

Bile Acids and Salts↗
PubMed1990

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase as a 3-keto bile acid reductase from human liver cytosol.

The NADPH-dependent 3 alpha-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5 beta-cholanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak 1), 232-fold (peak 2) and 345-fold (peak 3) and the recoveries of the enzymes were 0.4% (peak 1), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34, 7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32,000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid, 7 alpha-hydroxy-5 beta-cholestan-3-one and 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one to the corresponding 3 alpha-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzyme required a sulfhydryl group for activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Oxidation and reduction of bile acid precursors by rat hepatic 3 alpha-hydroxysteroid dehydrogenase and inhibition by bile acids and indomethacin.

Enzyme kinetics of purified rat hepatic 3 alpha-hydroxysteroid dehydrogenase for bile acid precursors and effects of bile acids and indomethacin on those activities were studied. This enzyme catalyzed the oxidoreduction of the C3 position of bile acid precursors. Km for 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one (1.6 microM) was markedly lower than Km for 7 alpha-hydroxy-5 beta-cholestan-3-one (28 microM) but Vmax was similar. Km for 3 alpha, 7 alpha-dihydroxy-5 beta-cholestane (12 microM) was lower than Km for 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestane (150 microM) although Vmax/Km values were similar for both compounds. Bile acids and indomethacin inhibited the reduction of 3-oxo bile acid precursors. NADPH inhibited the binding of lithocholic acid (3 alpha-hydroxy-5 beta-cholanic acid) by 3 alpha-hydroxysteroid dehydrogenase. These data suggest that intrahepatic bile acid concentrations may affect the reduction of 3-oxo-bile acid precursors and intrahepatic redox conditions may affect intracellular bile acid transfer.

3-Hydroxysteroid Dehydrogenases↗
PubMed1976

Determination of urinary dehydroepiandrosterone using 3beta-hydroxysteroid dehydrogenase.

A method for the determination of urinary dehydroepiandrosterone (DHA) using 3beta-hydroxysteroid dehydrogenase is described. The method includes extraction with ethylacetate, solvolysis, and color development using 3beta-hydroxysteroid dehydrogenase. A study of comparison with gas-liquid chromatography method and recovery test was made, and results were sufficient for routine tests.

Chromatography, Gas↗
PubMed1988

Evidence of 3 beta-hydroxysteroid dehydrogenase activity in several murine embryonal carcinoma cell lines.

This report describes, for the first time to our knowledge, a possible steroidogenic activity in established murine embryonal carcinoma cell lines (PCC3, PCC4, F9), revealed by a 3 beta-hydroxysteroid dehydrogenase activity (revelation of NADH2 by staining, and RIA assessment of delta 4-androstenedione). The remarkable analogy between such totipotent cells and embryonal cells may suggest that this activity could be present before histologic organization of the embryonal testis. Nonmalignant embryonal cells such as fibroblasts (3/A/1/D-3) or myoblasts (T984) were also found to possess a 3 beta-hydroxysteroid dehydrogenase activity, thus suggesting that this enzyme is not specific to hormone-secreting cells, but the sign of a more general phenomenon.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Prokaryotic 20 beta-hydroxysteroid dehydrogenase is an enzyme of the 'short-chain, non-metalloenzyme' alcohol dehydrogenase type.

The primary structure of 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was determined after FPLC purification of a commercial preparation. Peptides obtained from different proteolytic cleavages were purified by reverse phase HPLC. The 255-residue structure deduced was found to be distantly homologous to those of Drosophila alcohol dehydrogenase and several other dehydrogenases, establishing that prokaryotic 20 beta-hydroxysteroid dehydrogenase as a member of the 'short-chain alcohol dehydrogenase family'. With the enzymes characterized, the identity is greatest (31-34%) towards 4 other prokaryotic dehydrogenases, but the family also includes mammalian steroid and prostaglandin dehydrogenases. These enzymes are low in Cys and have a strictly conserved Tyr residue that appears to be important.

20-Hydroxysteroid Dehydrogenases↗
PubMed1992

Characterization of bovine liver cytosolic 3 alpha-hydroxysteroid dehydrogenase and its aldo-keto reductase activity.

1. 3 alpha-Hydroxysteroid dehydrogenase was purified to homogeneity from bovine cytosolic fraction, which was monomeric and its molecular weight was estimated to be about 35 kDa. 2. The enzyme had ability to catalyze NADP(H)-dependent oxidoreduction of position 3 alpha-hydroxy and keto group of steroids and also could catalyze the reduction of some ketones and quinones. 3. In addition, benzenedihydrodiol was one of the substrates of dehydrogenase activity with NADP+. 4. Indomethacin, synthetic steroids and SH-reagents were potent inhibitors for this enzyme. 5. Inactivation of the enzyme by GSSG-treatment was restored to its original activity by the addition of DTT. 6. The presence of coenzyme, 0.33 mM NADP+, completely protected from the DTNB-inactivation. 7. Bovine liver cytosolic enzyme immunologically crossreacted with rat liver 3 alpha-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Properties of 5-ene-3 beta-hydroxysteroid dehydrogenase in granulosa tissue from the domestic fowl (Gallus domesticus).

Microsomal 5-ene-3 beta-hydroxysteroid dehydrogenase activity from fowl preovulatory granulosa tissue was solubilised by treatment with 2.0 M KCl. The efficiency of the solubilisation procedure was assessed by measuring the amount of activity remaining in the supernatant after centrifugation at 105,000 g for 8 h. The microsomal activity was resolved into two components: a fraction solubilised by the salt treatment and a fraction remaining membrane bound. The results indicate that the enzyme exists in two different environments within the membrane. The time course for the thermal inactivation of the microsomal enzyme supported this conclusion. The properties of the solubilised enzyme were compared with the untreated microsomal preparation, with respect to steroidal inhibition. Progesterone, 4-androstene-3,17-dione and oestradiol-17 beta all inhibited the enzyme and no difference was observed between the two preparations. The results are discussed in relation to the involvement of 5-ene-3 beta-hydroxysteroid dehydrogenase in the control of progesterone production in fowl granulosa tissue.

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Solubilization of 17 beta-hydroxysteroid dehydrogenase from rat liver microsomes.

The conditions for the solubilization of 17 beta-hydroxysteroid dehydrogenase from a rat liver microsomal preparation with the non-ionic detergent Triton X-100 were studied. The recoveries of 17 beta-hydroxysteroid dehydrogenase activity and of proteins in the solubilized form were determined as a function of detergent concentration, of pH and temperature, of incubation time and of saline concentration. The soluble fraction obtained under the optimal conditions contained 80% of the proteins and 75% of the enzymatic activity of initial microsomes. The presence of Triton X-100 in the solubilized proteins was not essential for enzyme activity.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

Kinetic mechanism of porcine testicular 17 beta-hydroxysteroid dehydrogenase.

A study on product inhibition of 17 beta-hydroxysteroid dehydrogenase from porcine testes was carried out by measuring the initial velocities of NADPH formation using testosterone as the substrate steroid. Type of inhibition by NADPH against NADP+ was competitive in both saturated and unsaturated concentrations of testosterone. In the saturated concentration of NADP+, activity of the enzyme was not inhibited by NADPH against testosterone. In the unsaturated concentrations of NADP+, however, NADPH brought mixed type inhibition against testosterone. The similar modes of inhibition by the product steroid, androstenedione were observed in the saturated and unsaturated concentrations of NADP+ and testosterone. The fluorescence of NADPH was increased in the presence of the enzyme, and fluorometric titration indicated that 1 mol of NADPH was bound to 1 mol of the 17 beta-hydroxysteroid dehydrogenase. Addition of testosterone to enzyme-NADPH complex reduced the intensity of fluorescence of NADPH, suggesting formation of testosterone-enzyme-NADPH complex as a ternary dead end complex. From the analyses of product inhibition and spectral changes of NADPH, the kinetic mechanism of the enzyme was revealed as rapid equilibrium random system with two dead end complexes which consisted of the two reduced reactants bound to the enzyme and the two oxidized ones bound to it.

17-Hydroxysteroid Dehydrogenases↗
PubMed1984

Helix pomatia induced conversion of some 3 beta-hydroxysteroids.

After incubating a dehydroepiandrosterone solution with an enzymatic preparation from Helix pomatia, the steroid recovery was markedly decreased. The transforming activity resulted specific for some 3 beta-hydroxysteroids and it appeared to lower after storing the preparation at 4 degrees C for some days. The presence of 4-androsten-3,17-dione within the incubation products suggests that the enzymatic preparation contains at least 3 beta-hydroxysteroid: NAD oxydoreductase and 3-oxosteroid-5-4-ene isomerase. The use of large amounts of freshly dissolved Helix pomatia juice solution may lead to unreliable results in steroid analyses.

Animals↗
PubMed1986

Sex steroids and 5-en-3 beta-hydroxysteroids in specific regions of the human brain and cranial nerves.

Sex steroids and 5-en-3 beta-hydroxysteroids were determined by radioimmunoassay in specific regions of the human brain, in the anterior and posterior pituitary, in one sensory organ, the retina and in the cranial nerves. Progesterone, androstenedione, testosterone and estrone were found in all areas of the brain and in all the cranial nerves but not in all cases. There was no sex difference except in the case of androstenedione where values were higher in women in some brain areas. Estrone values were always higher than those of estradiol in both men and women. No 5 alpha-dihydrotestosterone was detected in any of the samples studied. The values for pregnenolone, dehydroepiandrosterone and their sulfates were much higher than those of the sex steroids in all areas of the brain and in all the cranial nerves. Values for pregnenolone were greater than those of its sulfate while those of dehydroepiandrosterone were in general equal to or higher than those of its sulfate. The values for pregnenolone were greater than those of dehydroepiandrosterone. There were no obvious regional differences in the concentrations of the 5-en-3 beta-hydroxysteroids either in specific areas of the brain or in the cranial nerves. But there was a definite trend for the free dehydroepiandrosterone values to be higher in women. The possible significance of these observations is discussed.

Androstenedione↗
PubMed1986

The affinity alkylators, 11 alpha-bromoacetoxyprogesterone and estrone 3-bromoacetate, modify a common histidyl residue in the active site of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase.

Purified human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (native enzyme) was completely inactivated by the affinity alkylator, estrone 3-bromoacetate, in the presence of cofactor (NADPH). The inactivated enzyme was reactivated to 100% activity by base-catalyzed hydrolysis of the steroidal ester-enzyme conjugate and then repurified by dialysis. Control enzyme in mixtures which contained estrone in place of alkylator was treated the same as the reactivated enzyme. 11 alpha-Bromo[2'-14C]acetoxyprogesterone, an active site-directed affinity alkylator of the enzyme, produced 5.0-fold less radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine plus 1.4-fold more 1,3-bis(carboxymethyl)-histidine in the reactivated enzyme than in the control enzyme. The lesser amount of S-(carboxymethyl)cysteine and greater amount of 1,3-bis(carboxymethyl)histidine resulted from nonspecific interactions between the reactivated enzyme and the progestin radioalkylator. The nonradiolabeled 3-(carboxymethyl)histidine originally produced by estrone 3-bromoacetate in the enzyme active site hindered radioalkylation of this amino acid by 11 alpha-bromo[2'-14C]acetoxyprogesterone to yield 5-fold less radiolabeled 3-(carboxymethyl)histidine in the reactivated enzyme relative to control enzyme. Thus, the estrogen and progestin affinity alkylators modified a common histidyl residue in the active site. These studies are direct evidence that the estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities reside at a common locus on a single protein.

Alkylation↗
PubMed1986

ACTH and thyroid hormone regulation of 3 beta-hydroxysteroid dehydrogenase activity in human fetal adrenocortical cells.

The regulation of 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase (3 beta-HSD) and hydroxysteroid sulfotransferase (HST) activities by ACTH and thyroid hormones was investigated in cell cultures from the fetal zone or definitive zone of the human fetal adrenal cortex, using a serum-free, defined medium. ACTH alone maximally stimulated the 3 beta-HSD activity several-fold, whereas triiodothyronine (T3) alone had no effect on this enzyme activity in cell cultures from each zone. However, treatment of cultures with maximal concentrations of 10 nM ACTH plus 1 nM T3 significantly increased the 3 beta-HSD activity an additional 59-115% over that for ACTH alone, without alteration of the ACTH ED50 (0.3 nM). The T3 ED50 was 31 pM for this interaction with ACTH. Thyroxine at a reduced sensitivity had the same interaction with ACTH. T3 similarly increased the stimulation of 3 beta-HSD activity by the steroidogenic agents, cholera toxin and a cAMP analog. The HST activity was not affected by T3 alone but was stimulated by ACTH alone. This stimulation was an order of magnitude less than that for the 3 beta-HSD activity in the same cultures. ACTH plus T3 did not have the synergistic effect on HST activity as observed for the 3 beta-HSD activity. These studies show an interaction between ACTH and thyroid hormone for the stimulation of 3 beta-HSD activity in cell cultures of the human fetal adrenal cortex.

3-Hydroxysteroid Dehydrogenases↗