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PubMed1992

Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases.

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.

Alcohol Oxidoreductases↗
PubMed1983

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells.

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Continuous-flow automated assay of steroids with nylon-tube-immobilized hydroxysteroid dehydrogenases.

Several NAD(P)+-dependent hydroxysteroid dehydrogenases, namely 3 alpha-hydroxysteroid dehydrogenase, beta-hydroxysteroid dehydrogenase, 7 alpha-hydroxysteroid dehydrogenase, and 12 alpha-hydroxysteroid dehydrogenase were separately immobilized on nylon tubes for the continuous-flow automated assay of hydroxysteroids. 3 alpha-Hydroxysteroid dehydrogenase was also immobilized on pore glass. Spectrophotometric monitoring in the visible region, where blank values were markedly reduced, was achieved through the Meldola blue catalyzed transfer of hydrogen from NAD(P)H to a tetrazolium salt. Nylon-tube-immobilized enzymes maintained 45-55% of the original activity after 1 month of intermittent use. The operational range, using the "end point" approach, was 1-25 nmol of steroid and the assay speed 10-15 samples/h. Reliable results were obtained in the determination of 3 alpha-hydroxysteroids and 3 beta, 17 beta-hydroxysteroids in urine and total bile acids in serum.

Autoanalysis↗
PubMed2005

11beta-Hydroxysteroid dehydrogenase type 2 protects the neonatal cerebellum from deleterious effects of glucocorticoids.

11beta-Hydroxysteroid dehydrogenase type 2 is a glucocorticoid metabolizing enzyme that catalyzes rapid inactivation of corticosterone and cortisol to inert 11-keto derivatives. As 11beta-hydroxysteroid dehydrogenase type 2 is highly expressed in the developing brain, but not in the adult CNS, we hypothesized that it may represent a protective barrier to the deleterious actions of corticosteroids on proliferating cells. To test this hypothesis we have investigated the development and growth of the cerebellum in neonatal C57BL/6 mice and mice lacking 11beta-hydroxysteroid dehydrogenase type 2 (-/-). 11beta-Hydroxysteroid dehydrogenase type 2-/- mice had consistently lower body weight throughout the neonatal period, coupled with a smaller brain size although this was normalized when corrected for body weight. The cerebellar size was smaller in 11beta-hydroxysteroid dehydrogenase type 2-/- mice, due to decreases in size of both the molecular and internal granule layers. When exogenous corticosterone was administered to the pups between postnatal days 4 and 13, 11beta-hydroxysteroid dehydrogenase type 2(-/-) mice were more sensitive, showing further inhibition of cerebellar growth while the wildtype mice were not affected. Upon withdrawal of exogenous steroid, there was a rebound growth spurt so that at day 21 postnatally, the cerebellar size in 11beta-hydroxysteroid dehydrogenase type 2-/- mice was similar to untreated mice of the same genotype. Furthermore, 11beta-hydroxysteroid dehydrogenase type 2-/- mice had a delay in the attainment of neurodevelopmental landmarks such as negative geotaxis and eye opening. We therefore suggest that 11beta-hydroxysteroid dehydrogenase type 2 acts as to protect the developing nervous system from the deleterious consequences of glucocorticoid overexposure.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed1999

N-sulphoconjugation of amines by human cytosolic hydroxysteroid sulphotransferase.

1. N- and O-sulphoconjugation of various substrates were studied with human liver cytosol and purified cytosolic sulphotransferase in the presence of 3-phosphoadenosine 5-phosphosulphate. 2. Human liver cytosol catalysed N-sulphoconjugation of alicyclic and aryl-amines, and O-sulphoconjugation of hydroxysteroid and phenol. Activities of amine sulphoconjugation in the cytosol correlated well with those of hydroxysteroid but not with phenol. 3. Alicyclic amine sulphotransferase in human liver cytosol was purified to homogeneity by anion exchange, affinity and hydroxyapatite chromatography. Sulphoconjugating activities of alicyclic amine co-purified with those for hydroxysteroid conjugation. Subunit molecular weight of the purified sulphotransferase was 34 kDa. Contents of the purified enzyme correlated with the sulphoconjugating activities of hydroxysteroid and alicyclic amine. From these results, we concluded that the alicyclic amine sulphotransferase purified in this study was identical to hydroxysteroid sulphotransferse in human liver cytosol. 4. The results of this study indicate that hydroxysteroid sulphotransferase in human liver cytosol catalyses N-sulphoconjugation of alicyclic and aryl-amines. Hydroxysteroid sulphotransferase in the cytosol is reported to catalyse O-sulphoconjugations of various compounds including hydroxysteroids, bile acids, cholesterol, and aliphatic and benzylic alcohols. The present and previously reported results indicate that hydroxysteroid sulphotransferase in the cytosol catalyses both N- and O-sulphoconjugations of several substrates.

Amines↗
PubMed1977

NAD-dependent 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Eubacterium lentum ATCC no. 25559.

Eubacterium lentum (ATCC No. 25559) was shown to contain 3alpha-and 12alpha-hydroxysteroid dehydrogenases both of which were NAD-dependent and active against conjugated and unconjugated bile salts. In addition, the 3alpha-hydroxysteroid dehydrogenase was active against members of the Androstan series containing a 3alpha-hydroxyl group regardless of the stereo-orientation of the 5-H-. No measurable activity against 7alpha-, 7beta-, 11beta-, or 17beta-hydroxyl groups was demonstrated. The growth of E. lentum and the production of 3alpha- and 12alpha-hydroxysteroid dehydrogenases were greatly enhanced by the addition of L-, D- or DL-arginine to the medium. Yields of hydroxysteroid dehydrogenase were optimal in the range of 0.50-0.75% arginine; however, the growth of the organisms was further enhanced at arginine concentrations greater than 0.75%. The 12alpha-hydroxysteroid dehydrogenase was heat labile and could be selectively inactivated by heating at 50 degrees C for 45 min. Both the heated enzyme preparation (containing only 3alpha-hydroxysteroid dehydrogenase) and the unheated enzyme preparation (containing 3alpha- and 12alpha-hydroxysteroid dehydrogenases) were useful in the spectrophotometric quantification of bile salts. The optimal pH values for 3alpha- and 12alpha-hydroxysteroid dehydrogenases were 11.3 and 10.2, respectively. Kinetic studies have Km estimates of 2.10(-5) M and 1.0.10(-4) M with 3alpha,7alpha-dihydroxy-5beta-cholanoyl glycine and 7alpha,12alpha-dihydroxy-5beta-cholanoate for the two respective enzymes.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c.

A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104,000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated. The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values. 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid. Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro. Here, the pH optimum for the overall reaction was 8.5-8.7. 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Distribution and ontogeny of 3 alpha-hydroxysteroid dehydrogenase in the rat brain.

3 alpha-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3 alpha-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3 alpha-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activities were found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. No sexual dimorphism was found in either the concentration levels or the activities of the brain 3 alpha-hydroxysteroid dehydrogenase. At the time of birth, the rat brain already expresses a significant level of 3 alpha-hydroxysteroid dehydrogenase; the levels of brain 3 alpha-hydroxysteroid dehydrogenase activity in rats continue to rise during the first week after their birth, and reach a plateau thereafter.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver.

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.

17-Hydroxysteroid Dehydrogenases↗
PubMed1981

Bile induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum.

Eight strains of Clostridium absonum grown in the presence of 4 . 10(-4) M deoxycholate contained both NADP-dependent 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities. In one strain studied in detail, significant amounts of NADP-dependent 7 alpha- and 7 beta-hydroxysteroid dehydrogenase and NAD-dependent 7 alpha-hydroxysteroid dehydrogenase activities were demonstrated only when cells were grown in the presence of deoxycholate or chenodeoxycholate, both optimal at 4 . 10(-4) M. When the bile salt was deleted from the medium, only a trace of 7 alpha-hydroxysteroid dehydrogenase was present and 7 beta-hydroxysteroid dehydrogenase was absent. Other bile salts including cholate, ursodeoxycholate and keto bile salts were less effective as inducers. Addition of cholate to medium already containing deoxycholate at a suboptimal concentration enhanced the induction, while addition of ursodeoxycholate suppressed the induction. Further enhancement of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase could be obtained by additions of deoxycholate (up to a total of 6 . 10(-4) M) during the growth of the organisms (in log phase). As enzyme enhancement is blocked by addition of rifampicin to the medium, the authors conclude that the enzymes are bile salt-inducible. Growth curve studies revealed an optimal enzyme yield at a harvest time of approx. 6-9 h. We have preliminarily characterized several inducible enzyme components: an NADP-dependent 7 beta-hydroxysteroid dehydrogenase as well as both NAD- and NADP-dependent 7 alpha-hydroxysteroid dehydrogenases.

Chenodeoxycholic Acid↗
PubMed1995

Structures important in mammalian 11 beta- and 17 beta-hydroxysteroid dehydrogenases.

We have used the X-ray crystallographic structures of rat and human dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase to model parts of the 3-dimensional structure of human 11 beta- and 17 beta-hydroxysteroid dehydrogenases. We use this information along with previous results from studies of Drosophila alcohol dehydrogenase mutants to analyze the structures in binding sites for NAD(H) and NADP(H) in 11 beta-hydroxysteroid dehydrogenase-types 1 and 2. We also examine the structure of an alpha-helix at catalytic site of 17 beta-hydroxysteroid dehydrogenase-types 1, 2, 3, and 4. This alpha-helix contains a highly conserved tyrosine and lysine. Adjacent to the carboxyl side of this lysine is a site proposed to be important in subunit association. We find that 11 beta- and 17 beta-hydroxysteroid dehydrogenases-type 1 have the same residues at the "anchor site" and conserve other stabilizing features, despite only 20% sequence identity between their entire sequences. Similar conservation of stabilizing structures is found in the 11 beta- and 17 beta-hydroxysteroid dehydrogenases-type 2. We suggest that interactions of the dimerization surface of alpha-helix F with proteins or membranes may be important in regulating activity of hydroxysteroid dehydrogenases.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1988

High-performance liquid chromatography of hydroxysteroids detected with post-column immobilized enzyme reactors.

Assaying the low concentrations of steroid hormones in extracts of body fluids requires detectors that are both highly sensitive to the steroid and relatively insensitive to interfering compounds usually present in much higher concentrations. To explore the use of moderately specific enzymes in post-column reactors, we immobilized 3 alpha- and beta-hydroxysteroid dehydrogenase on controlled pore glass beads, 37 microns in diameter, and constructed 4.6-mm diameter reactor columns, 3-cm long, packed with one of the two kinds of these beads. Hydroxysteroids eluted from the analytical column were mixed with the coenzyme, nicotinamide adenine dinucleotide (NAD), before passing through the reactor. The effluent from the reactor was passed through the 70-microliters flow cell of a fluorometer in which the fluorescence of the NADH produced in the enzyme-catalyzed oxidation of the hydroxysteroid was monitored. At the conventional high-performance liquid chromatography flow-rates used, oxidation of the steroids was almost complete. The yield depended on both the residence time of steroids in the reactor column and the concentration of organic modifier in the reaction mixture. Maximal yield was obtained with buffer having a low organic solvent concentration and passing through the reactor slowly. In assays of mixtures of epimeric hydroxysteroids, the 3 alpha-hydroxysteroids were detected with the 3 alpha-hydroxysteroid dehydrogenase reactor; the beta-hydroxysteroids were not, confirming the specificity of the enzymatic detection. With the fluorometer used, picomole quantities of steroids could easily be distinguished from noise.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Distribution of 3 alpha-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans.

3 alpha-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3 alpha-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3 alpha-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activity was found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. We used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. One of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5'-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3 alpha-hydroxysteroid dehydrogenase and human chlordecone reductase. We are currently investigating the functional relationship between the enzymes encoded by these human cDNAs and 3 alpha-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

Molecular cloning of a novel type of rat cytoplasmic 17beta-hydroxysteroid dehydrogenase distinct from the type 5 isozyme.

Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD+ as the preferred coenzyme at pH 7.4, and showed low activity towards 20alpha- and 3alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3alpha-hydroxysteroid dehydrogenase.

17-Hydroxysteroid Dehydrogenases↗
PubMed1987

Occurrence of hydroxysteroid oxidoreductases in liver of turtles.

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.

17-Hydroxysteroid Dehydrogenases↗
PubMed1976

Behavior of 3alpha- and 7alpha-hydroxysteroid dehydrogenases on chenodeoxycholate substituted Sepharose.

Chenodeoxycholate (3alpha-, 7alpha-dihydroxy-5beta-cholanoate) was linked to Sepharose 4B by an ethylenediamine bridge. When 3alpha-hydroxysteroid dehydrogenase and 7alpha-hydroxysteroid dehydrogenase preparations were applied to a column of covalently linked chenodeoxycholate, both enzymes were retarded at pH 6.7; the 7alpha-OH oriented enzyme more than the 3alpha-OH enzyme. Approximately forty-fold purification of 7alpha-hydroxysteroid dehydrogenase was achieved in one step. Although no significant purification of 3alpha-hydroxysteroid dehydrogenase occurred, the background value in the fluorometric enzymatic estimation of bile acids by eluted 3alpha-hydroxysteroid dehydrogenase was markedly reduced. Molecular weight estimation by Sephadex G-200 gave the values of 47,000 for 3alpha-hydroxysteroid dehydrogenase and 105,000 for 7alpha-hydroxysteroid dehydrogenase.

Chenodeoxycholic Acid↗
PubMed2001

Troglitazone is a competitive inhibitor of 3beta-hydroxysteroid dehydrogenase enzyme in the ovary.

OBJECTIVE: Troglitazone is a potent inhibitor of progesterone release from porcine granulosa cells. This is associated with a marked increase in pregnenolone secretion, implicating inhibition of the 3beta-hydroxysteroid dehydrogenase enzyme. This study determined whether troglitazone is a direct inhibitor of 3beta-hydroxysteroid dehydrogenase activity. STUDY DESIGN: Homogenates of porcine granulosa cells underwent classic enzyme kinetic analysis through Lineweaver-Burke and Dixon plotting. Human ovarian homogenates were also assayed for the effects of troglitazone on 3beta-hydroxysteroid dehydrogenase enzyme activity. Enzyme kinetics data were analyzed by the HyperKinetics software program. Analysis of variance was used to determine statistical significance for human ovarian homogenate experiments. RESULTS: In porcine granulosa cells Lineweaver-Burke analysis found that troglitazone inhibition of 3beta-hydroxysteroid dehydrogenase enzyme activity was competitive in nature, with 5 microg/mL troglitazone increasing the apparent Michaelis constant from 1.3 to 4.3 micromol/L (no change in maximum velocity). Dixon plot analysis demonstrated that the inhibition constant for troglitazone of 3beta-hydroxysteroid dehydrogenase is approximately 6.5 microg/mL, which is in the same order of magnitude as its therapeutic concentration in blood. Troglitazone also significantly decreased the activity of 3beta-hydroxysteroid dehydrogenase in homogenates of human ovarian tissue. CONCLUSION: We conclude that troglitazone can inhibit steroidogenesis in the ovary by direct competitive inhibition of 3beta-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Pseudomonas 3 beta-hydroxysteroid dehydrogenase. Primary structure and relationships to other steroid dehydrogenases.

The 3 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni commercially available was purified by an FPLC step and submitted to sequence determination by peptide analysis. The structure obtained reveals a 253-residue polypeptide chain, with an N-terminal, free alpha-amino group, and a low cysteine content. Comparisons with other hydroxysteroid dehydrogenases recently characterized reveal distant similarities with prokaryotic and, to some extent, also eukaryotic forms of separate specificities. Residue identities with a Streptomyces 20 beta-hydroxysteroid dehydrogenase are 35% and distributed over the entire molecule, whereas residue identities with the mammalian 17 beta-hydroxysteroid dehydrogenase only constitute 20%, and are essentially limited to the N-terminal and central parts, Nevertheless, all these enzymes exhibit a conserved tyrosine residue (position 151 in the present enzyme) noted as possibly having a functional role in some members of this protein family. Combined, the results establish the prokaryotic 3 beta-hydroxysteroid dehydrogenase as belonging to the family of short-chain alcohol dehydrogenases, reveal that the hydroxysteroid dehydrogenases are no more closely related than dehydrogenases with other enzyme activities within the family (e.g. glucose, ribitol, hydroxyprostaglandin dehydrogenases), show several of the mammalian hydroxysteroid dehydrogenases to have subunits of longer size with different patterns of similarity than those of the prokaryotic family members characterized, and define important segments of the coenzyme-binding region for this enzyme group.

3-Hydroxysteroid Dehydrogenases↗