Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3beta-hydroxysteroid dehydrogenase/steroid delta5-->4-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3beta-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3beta-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3beta-HSD activity. The 3beta-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3beta-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme.
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PMID:Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase. 974 38

Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms. Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 A. The resolution limit for these crystals is 3.9 A. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212. The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 A, and the observed resolution limit is 2.2 A. Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 A, beta = 91.9 degrees. This last crystal form has a diffraction limit of 2.7 A resolution.
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PMID:Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae. 976 13

3Beta-hydroxysteroid dehydrogenase/steroid delta5-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding the human wild-type I (placental) enzyme and the human type I mutant- Y253F. The wild-type and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells. Ultraviolet (UV) spectral analyses showed that the wild-type enzyme induced changes in the UV spectrum of the competitive isomerase inhibitor, 19-nortestosterone, and the Y253F mutant did not. The wild-type isomerase required activation by coenzyme to produce the spectral shift. Activation of isomerase by NADH produced a greater change in the 19-nortestosterone spectrum than activation by NAD+. These observations provide direct evidence that Tyr253 functions as the general acid (proton donor) in the isomerase reaction mechanism. Furthermore, the coenzyme-activation profiles support our proposed two-step enzyme mechanism in which NADH produced by the 3beta-HSD activity induces the enzyme to assume the isomerase conformation.
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PMID:Functional activity of 3beta-hydroxysteroid dehydrogenase/isomerase. 988 36

NAD+-dependent 15-hydroxy-PG dehydrogenase (PGDH) is the major enzyme involved in the initial inactivation of PGs, and its activity is reduced by glucocorticoids, cortisol (F), and dexamethasone (DEX). In turn, glucocorticoid regulation of PGDH activity in placenta and chorion could be regulated indirectly by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity. In the placenta, 11beta-HSD2 is the dominant isoform, acting as a dehydrogenase [F to cortisone (E)]; and in chorion, 11beta-HSD1 predominates as a reductase (E to F). The present study was designed to determine whether glucocorticoid regulation of PGDH activity in placenta and chorion could be regulated indirectly by 11beta-HSD activity. We obtained Percoll-purified human placental and chorion trophoblast cells from uncomplicated term pregnancies, cultured them for 72 h, then treated the cells with cortisol (100 nmol/L), cortisone (1 micromol/L), or DEX (100 nmol/L), in the presence or absence of carbenoxolone (CBX, 800 nmol/L), an 11beta-HSD inhibitor, for 24 h. Activity of PGDH was assessed by incubation (4 h) with PGF2alpha (282 nmol/L) and measurement of conversion to 13,14-dihydro-15-keto PGF2alpha. CBX alone had no effect on PGDH activity in either placenta or chorion trophoblast cells. In chorion, E significantly inhibited PGDH activity, and this effect was reversed by addition of CBX. F and DEX significantly inhibited PGDH, and this effect was unaltered by coadministration of CBX. In contrast, in placenta, there was no effect of E, or of E with CBX, on PGDH activity. However, F and DEX inhibited PGDH, and the effect of F (but not DEX) was greater in the presence of CBX. In conclusion, we suggest that effects of E and F on PGDH are modified by the tissue-specific expression of 11beta-HSD isoforms.
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PMID:Local modulation by 11beta-hydroxysteroid dehydrogenase of glucocorticoid effects on the activity of 15-hydroxyprostaglandin dehydrogenase in human chorion and placental trophoblast cells. 1002 89

The kidney (11-HSD2 or 11-HSDK) isozyme of 11beta-hydroxysteroid dehydrogenase confers specificity for aldosterone on mineralocorticoid receptors in target tissues. In rodent kidney, this isozyme is expressed mainly in cortical collecting ducts and is undetectable in proximal tubules. Using mouse M-1 and rabbit RCD cortical collecting duct cells, we analyzed the 5'-flanking region of the human HSD11K gene encoding this enzyme in an attempt to identify transcriptional regulatory elements responsible for gene expression in the kidney. M-1 and RCD cells had high levels of NAD+ dependent 11-HSD activity with corticosterone as the substrate. Luciferase reporter constructs containing 1785 or 327 nucleotides (nt) upstream of the initiator ATG codon were expressed at similar levels in each cell line, but deletion to 167 nt almost completely abolished expression in both cell types. This region is GC-rich and contain Sp1 binding sites. Electrophoretic mobility shift assays of the region containing the putative Sp1 sites showed several DNA-protein complexes in both the cell types. Mutations of the Sp1 sites decreased transcriptional activity in M-1 cells; however, these mutations had a marginal effect in the RCD cells. These results suggest that elements controlling renal cell type expression are located in the proximal 327 nucleotides of the 5' flanking region of HSD11K.
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PMID:Expression of HSD11K (NAD+ dependent 11beta-hydroxysteroid dehydrogenase) promoter constructs in renal cell lines. 1092 54

In the peripheral nervous system, progesterone (PROG) has a stimulatory effect on myelination. It could be derived from local synthesis, as Schwann cells in culture express the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and convert pregnenolone (PREG) to PROG. Although 3beta-HSD mRNA can be detected by RT-PCR in peripheral nerves, the activity of the enzyme has so far not been demonstrated and characterized in nerve tissue. In this study, we show that homogenates prepared from rat sciatic nerves contain a functional 3beta-HSD enzyme and we have analysed its kinetic properties and its regulation by steroids. The activity of 3beta-HSD in homogenates was evaluated using 3H-labelled PREG as a substrate and NAD+ as a cofactor, the levels of steroids formed were calculated either by extrapolating the relationship between tritiated peaks obtained by TLC to the initial amount of PREG, or by gas chromatography/mass spectrometry determination. A rapid increase in PROG formation was found between 0 and 50 min of incubation and no further significant changes were observed between 1 and 4 h. The calculated Km value (1.06 +/- 0.19 microm) was close to the values described for the 3beta-HSD type-I and type-IV isoforms. Trilostane, a competitive inhibitor of the 3beta-HSD caused a potent inhibition of the rate of conversion of PREG to PROG (IC50 = 4.06 +/- 2.58 microm). When the effects of different steroids were tested, both oestradiol and PROG significantly inhibited the conversion of PREG to PROG.
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PMID:Characterization and regulation of the 3beta-hydroxysteroid dehydrogenase isomerase enzyme in the rat sciatic nerve. 1248 8

The actions of natural and synthetic glucocorticoids are in part determined by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). We examined whether carbenoxolone, a potent inhibitor of 11beta-HSD, would potentiate the inhibitory action of dexamethasone on interleukin-8 release from BEAS-2B cells, and whether prolonged treatment with dexamethasone at therapeutic doses would upregulate 11beta-HSD2 in the cells. We found that carbenoxolone increased the potency of dexamethasone almost 10-fold. Reverse transcription-polymerase chain reaction and Western blot revealed that BEAS-2B cells expressed 11beta-HSD2, but not 11beta-HSD1. An enzyme activity assay of the cell homogenate demonstrated only NAD+-dependent dehydrogenase activity. The Km value for cortisol in intact BEAS-2B cells was estimated to be 42 nM. When the cells were incubated with dexamethasone for up to 72 hours at increasing concentrations (10(-9) to 10(-5) M), there were considerable increases in mRNA and protein levels of 11beta-HSD2. Prolonged treatment with dexamethasone also increased the enzyme activity of 11beta-HSD in the cells in a dose- and time-dependent manner, with complete inhibition by RU38486. These results suggest that bronchial epithelial cells possess an autoregulatory system for glucocorticoids in the control of their own bioactive levels by inducing the expression of 11beta-HSD2, and that 11beta-HSD2 in the bronchial epithelium may play a role in the local regulation of inhaled glucocorticoid actions.
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PMID:Dexamethasone upregulates 11beta-hydroxysteroid dehydrogenase type 2 in BEAS-2B cells. 1257 78

Dithiocarbamates (DTCs), important therapeutic and industrial chemicals released in high quantities into the environment, exhibit complex chemical and biological activities. Here, we demonstrate an effect of DTCs on glucocorticoid action due to inhibition of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 2, converting cortisol to cortisone in the kidney, but not 11 beta-HSD1, catalyzing the reverse reaction in liver and adipose tissue. Thus, DTCs may locally increase active glucocorticoid concentrations. Preincubation with the DTC thiram abolished 11 beta-HSD2 activity, suggesting irreversible enzyme inhibition. The sulfhydryl protecting reagent dithiothreitol blocked thiram-induced inhibition and NAD+ partially protected 11 beta-HSD2 activity, indicating that DTCs act at the cofactor-binding site. A 3D-model of 11 beta-HSD2 identified Cys90 in the NAD(+)-binding site as a likely target of DTCs, which was supported by a 99% reduced activity of mutant Cys90 to serine. The interference of DTCs with glucocorticoid-mediated responses suggests a cautious approach in the use of DTCs in therapeutic applications and in exposure to sources of DTCs such as cosmetics and agricultural products by pregnant women and others.
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PMID:Inhibition of 11 beta-hydroxysteroid dehydrogenase type 2 by dithiocarbamates. 1290 62

3Beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is essential for the synthesis of all classes of steroid hormones, converting various delta5-3beta-hydroxysteroids into hormonally active delta4-3-ketosteroids in NAD+ -dependent reactions. Certain 3beta-HSD isoforms have been reported to exhibit additional dehydrogenase character (e.g., 17-hydroxysteroid dehydrogenase/reductase). We have investigated whether mouse type I (adrenal/gonadal) and type VI 3beta-HSDs (uterine/embryonic) display significant 17beta-HSD-like activity. Nonsteroidogenic HEK 293T cells were transiently transfected with pCMV-based expression vectors containing mouse type I and type VI 3beta-HSDs. Transfected cells expressing either mouse type I or type VI 3beta-HSD converted testosterone to androstenedione, albeit at rates one-tenth of those of pregnenolone to progesterone in similarly transfected 293T cells. Our findings demonstrate that the mouse 3beta-HSD I and VI isoforms can inactivate testosterone within an intact cell milieu. These findings are important not only in establishment of structure-function relationships, but also whenever murine systems are used for developmental/reproductive paradigms associated with human disorders.
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PMID:Promiscuous 3beta-hydroxysteroid dehydrogenases: testosterone 17beta-hydroxysteroid dehydrogenase activities of mouse type I and VI 3beta-hydroxysteroid dehydrogenases. 1566 15

Glucocorticoids are involved in the regulation of spermatogenesis in the boar testis by initiating apoptosis in early stages of germ cell development. Because cortisol activity is modulated by the 11beta-hydroxysteroid dehydrogenase system (11beta-HSD), the present study determined both 11beta-activating (reductive) and inactivating (oxidative) enzyme activities in testicular tissue preparations of control boars (n = 5), GnRH-immunized boars (n = 5), and immunized, estradiol-infused boars (n = 6) by radioenzyme assay based on the conversion of tritiated cortisol to cortisone in the presence of NAD+ (inactivation) or tritiated cortisone to cortisol in the presence of NADPH (activation). The presence of both isoforms, 11beta-HSD 1 and 11beta-HSD 2, was confirmed by RTPCR in testicular tissue of control boars. Additionally, cortisol, testosterone, estradiol, and LH were determined in blood plasma sampled twice before killing. Immunization led to a drop of LH from 4.3 +/- 0.3 pmol/L to 1.0 +/- 0.3 pmol/L (testosterone: 11.63 +/- 0.83 nmol/L vs. 0.28 +/- 0.07 nmol/L; 17beta-estradiol: 512.61 +/- 47.60 pmol/L vs. 77.05 +/- 14.00 pmol/L). Low 11beta-HSD reductive activity was found in boars (1 pmol steroid x min (-1) x mg (-1)). It decreased to trace amounts in immunized boars (0.07 pmol steroid x min (-1) x mg (-1)). Oxidative activity was found in boars with 10.19 +/- 2.28 pmol steroid x min (-1) x mg (-1) protein. Immunization led to a sharp decrease (0.08 +/- 0.03 pmol x min (-1) x mg (-1)). Infusion of 17beta-estradiol significantly elevated peripheral estradiol concentrations to 752.07 +/- 24.19 pmol/L which still is a physiological concentration in this species. The infusion led to a minimal reductive activity (0.04 pmol steroid x min (-1) x mg (-1)), but led to a 6-fold rise of the 11beta-HSD oxidative activity to 0.47 +/- 0.14 pmol x min (-1) x mg (-1) compared to immunized boars. It is concluded that the 11beta-HSD system is involved in the regulation of cortisol activity in the testis and thus in the regulation of spermatogenesis.
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PMID:Characterization of 11beta-hydroxysteroid dehydrogenase activity in testicular tissue of control and GnRH-immunized boars as a possible regulator of spermatogenesis. 1592 11


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