UNIPROT:Q7LGC8 - FACTA Search

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Query: UNIPROT:Q7LGC8 (HSD)
3,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3 Alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3 alpha-HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co-substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3 alpha-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3 alpha-HSD. Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3 alpha-HSD seem to be members of the short-chain alcohol dehydrogenase family.
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PMID:Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. 155 29

Several mammalian livers contain monomeric 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) with A-stereospecificity in hydrogen transfer, which differs from the B-specific dimeric enzyme of human placenta in its ability to catalyze the oxidoreduction of xenobiotic trans-dihydrodiols of aromatic hydrocarbons and carbonyl compounds. Here, we report the isolation and characterization of a mouse cDNA clone encoding monomeric 17 beta-HSD of the liver. This clone had an entire coding region for a protein of 323 amino acid residues with a molecular weight of 37,055. The deduced sequence of the protein aligned with a high degree of identity with rat and rabbit 20 alpha-HSDs, rat and human 3 alpha-HSD/dihydrodiol dehydrogenases, and bovine prostaglandin F synthase, which are members of the aldoketoreductase family, but was distinct from human 17 beta-HSD and carbonyl reductase, members of the short chain dehydrogenases. The expression of the cDNA in Escherichia coli resulted in synthesis of a protein that was active toward androgens, estrogens, and xenobiotic substrates. The recombinant and mouse liver 17 beta-HSDs also exhibited low 20 alpha-HSD activity toward progestins, which is similar to bifunctional activity of human placental 17 beta-HSD. Therefore, the mouse enzyme was given the designation of estradiol 17 beta-dehydrogenase (A-specific). Northern analysis of mouse tissues revealed the existence of a single 1.7-kilobase 17 beta-HSD mRNA species in the liver, kidney, testis, and stomach. The liver mRNA content was considerably more abundant than those found in the other tissues, as 17 beta-HSD protein was mainly detected in the liver by Western analysis.
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PMID:Molecular cloning and characterization of mouse estradiol 17 beta-dehydrogenase (A-specific), a member of the aldoketoreductase family. 773 80

Screening of a mouse liver lambda gt 11 cDNA library with a rat liver 11 beta-hydroxysteroid dehydrogenase cDNA (11 beta-HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11 beta-hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11 beta-hydroxysteroid dehydrogenase structures. Open-reading-frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short-chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post-translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11 beta-dehydrogenation/11-oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P.C. (1993) Mol. Endocrinol. 7, 154-160; Agarwal, A.K., Tusie-Luna, M.T., Monder, C. & White, P.C. (1990) Mol. Endocrinol. 4, 1827-1832]. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti-(microsomal carbonyl reductase) serum and N-terminal sequence analysis of the purified protein confirms the identity of microsomal 11 beta-hydroxysteroid dehydrogenase with the previously described, microsomal-bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol. 47, 1805-1812], and points to an involvement of the 11 beta-HSD1A isoform in the reductive phase-I metabolism of xenobiotic compounds, besides its endocrinological functions. The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11 beta-HSD primary structure.
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PMID:Cloning and primary structure of murine 11 beta-hydroxysteroid dehydrogenase/microsomal carbonyl reductase. 785 87

1. The ontogenic pattern of xenobiotic carbonyl reducing activity and glucocorticoid 11 beta-oxidoreducing activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in mouse liver and kidney was examined. In addition, the expression of this enzyme was investigated by means of immunoblot analysis in the same tissues. 2. In liver, the foetus shows low or no enzyme activities. After birth both activities increase dramatically with age and remain then on a high plateau until the time of sexual maturity (4 weeks). After maturity, the enzyme activities decline to intermediate values. The developmental pattern of immunological expression of the liver enzyme corresponds well with that of the enzyme activity. 3. Considerable activities of xenobiotic carbonyl reduction and glucocorticoid 11 beta-oxidoreduction are also present after birth in all developmental stages of the kidney. However, no immunological crossreaction was found in any stages with the antibody against the liver 11 beta-HSD suggesting the presence of a structurally different isozyme form in the kidney. 4. The dramatic increase of both activities during the peri- and postnatal developmental periods suggest a potentially biological significance of the liver 11 beta-HSD isozyme in early animal life. 5. Besides being involved in 11 beta-glucocorticoid metabolism in particular the liver enzyme seems to play an additional role as xenobiotic carbonyl reductase.
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PMID:Ontogenic pattern of carbonyl reductase activity of 11 beta-hydroxysteroid dehydrogenase in mouse liver and kidney. 801 86

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is considered to confer mineralocorticoid specificity on the non-selective Type I adrenocorticoid receptor by converting active 11-hydroxyglucocorticoids to receptor-inactive 11-oxo metabolites, in mineralocorticoid target tissues like the kidney. However, 11 beta-HSD is also present in the liver, where it may regulate steroid exposure to the glucocorticoid Type II receptor. Because of the much higher activities compared to that in kidney, liver 11 beta-HSD possibly has additional functions besides the metabolism of glucocorticoids. In the present investigation we have isolated 11 beta-HSD from mouse liver microsomes and demonstrate that the homogeneously purified enzyme is also capable of catalyzing the reductive metabolism of xenobiotic carbonyl compounds such as metyrapone, p-nitroacetophenone and p-nitrobenzaldehyde. Enzyme kinetic studies revealed that, in addition to NADP+, mouse liver 11 beta-HSD also accepts NAD+ as cosubstrate for glucocorticoid 11 beta-dehydrogenation. NADH as cosubstrate for 11-oxoreduction plays only a minor role compared to that with NADPH, a fact which is also true for xenobiotic carbonyl reduction. Inhibition experiments revealed strong sensitivity of xenobiotic carbonyl reduction to glucocorticoids. The competitive nature of this inhibition suggests that both glucocorticoids and xenobiotic carbonyl substances bind to the same catalytically active site of 11 beta-HSD. High enzyme activities were also found in microsomal fractions of the ovary and adrenal gland but, although expressing considerable glucocorticoid 11-dehydrogenation activity (one third that of liver), almost no carbonyl reduction was detectable in kidney microsomes. Immunoblot analysis with polyclonal antibodies directed against the liver 11 beta-HSD did not yield an immunological crossreaction in the same tissues. In conclusion, corresponding to the cytosolic aldo-keto reductases, microsomal 11 beta-HSD of liver may be considered to play a role in the phase I biotransformation of pharmacologically relevant carbonyl substances as well as protecting organisms against toxic carbonyl compounds by converting them to less lipophilic and more soluble and conjugatable metabolites. Discrepancies in bioactivity together with the lack of response to anti-liver 11 beta-HSD antibodies strongly indicate the existence of distinct forms of 11 beta-HSD to be present in kidney, adrenal gland and ovary. The ability of xenobiotic carbonyl reduction might be another distinguishing feature among the various 11 beta-HSD isozymes.
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PMID:11 beta-hydroxysteroid dehydrogenase mediates reductive metabolism of xenobiotic carbonyl compounds. 820 97

In human liver, we previously identified one isoform of dihydrodiol dehydrogenase activity that expresses high affinity bile acid binding (HBAB) with minimal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity for bile acids. This protein may assist in the rapid intracellular transport of bile acids from the sinusoidal to the canalicular pole of the cell. We now report the cDNA cloning and bacterial expression of this novel, multifunctional protein. A 1252-base pair HBAB cDNA was cloned from a HepG2 lambda GT11 library using a rat hepatic bile acid binder cDNA probe. Bacterial expressed recombinant HBAB oxidized racemic trans dihydrodiol benzene (0.455 mumol NADPH/mg/min) with minimal 3 alpha-HSD activity for bile acids (< 0.003 mumol NADPH/mg/min). Lithocholic acid and chenodeoxycholic acid dissociation constants as determined by displacement of the fluorescent probe, bis-1-anilino-8 sulfonate, were higher than those previously reported for the native protein (1 microM versus 10 nM). Significant amino acid sequence homology was found with the human chlordecone reductase, bovine prostaglandin F synthetase, and rat hepatic-3 alpha-HSD suggesting, that HBAB is also a member of the recently identified, monomeric oxidoreductase gene family. Future studies will define the physiologic significance of this novel, multifunctional protein in bile acid transport and xenobiotic metabolism.
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PMID:cDNA cloning and expression of the human hepatic bile acid-binding protein. A member of the monomeric reductase gene family. 848 99

A new form of the NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs), present in the gram-negative bacterium Comamonas testosteroni ATCC 11996, was isolated from a testosterone-induced bacterial extract and characterized. The enzyme (HSD 28) has a monomeric molecular mass of 28 kDa. It belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDR) as established by N-terminal sequence analysis. Along with the 3 alpha-hydroxysteroid dehydrogenase and 3-oxo-reductase activities towards a variety of cis or trans fused A/B ring steroids, it also reduces several xenobiotic carbonyl compounds, including a metyrapone-based class of insecticides, to the respective alcohol metabolites. No dihydrodiol dehydrogenase activity towards trans- or cis-benzene-dihydrodiols could be detected, thus distinguishing it from the indomethacine-sensitive, mammalian liver type 3 alpha-HSDs. Subcellular fractionation revealed that the enzyme is localized in the cytoplasm of the bacterial cell. Proteins similar to the 3 alpha-HSD were detected and characterized from Comamonas testosteroni strain ATCC 17454 and from a commercially available steroid-induced extract of a patent Pseudomonas strain. The N-terminal amino acid sequence of the 3 alpha-HSD from the latter strain (HSD 29) is highly similar (94% identity over 15 residues) to a previously determined primary structure of a Pseudomonas species 3 alpha-HSD. However, no similarities could be detected between HSD 28 and a recently determined 3 alpha-HSD sequence from the ATCC 11996 Comamonas strain. The specific crossreaction of antibodies directed against mammalian liver type I 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD I) with the isolated 3 alpha-HSDs suggests the existence of a functionally and structurally related subgroup within the SDR superfamily. The broad substrate specificities of the characterized 3 alpha-HSD enzymes lead to the conclusion that they might participate in the intestinal bioactivation or inactivation of hormones, bile acids and xenobiotics since Comamonas testosteroni and related species are found in the intestinal tract of vertebrates including man.
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PMID:Characterization of a 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase from the gram-negative bacterium Comamonas testosteroni. 894 61

Detoxification of aldehydes and ketones generally proceeds via reduction to their corresponding alcohols, which are then conjugated and eliminated. We focused our interest on 11beta-hydroxysteroid-dehydrogenase type 1 (11beta-HSD 1), a pluripotent enzyme which physiologically performs the interconversion of active and inactive glucocorticoid hormones, and which also participates in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized. 11beta-HSD 1 is a glycosylated membrane protein which is very difficult to purify in an active state. In addition, expression levels in humans differ in a wide range. In order to facilitate biochemical and molecular studies on the significance of human 11beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli. Recombinant 11beta-HSD 1 from E. coli was purified to homogeneity and used to generate a polyclonal antibody. The enzyme had no enzymatic activity, possibly due to the lack of glycosylation and/or incorrect folding in E. coli. In contrast, 11beta-HSD 1 overexpressed in P. pastoris was enzymatically active towards its physiological glucocorticoid substrates as well as towards xenobiotic carbonyl compounds. In western blot experiments the antibody crossreacted with both recombinant 11beta-HSD 1 forms and with the native enzyme from mouse and human liver. In conclusion, recombinant 11beta-HSD 1 from P. pastoris serves as a valuable tool for future studies on carbonyl compound detoxification.
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PMID:Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli. 1078 78

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.
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PMID:Human 11beta-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form. 1102 92

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification. Since 11beta-HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon expression experiments in Escherichia coli, 11beta-HSD 1 turns out to be hardly soluble without detergents. This study describes attempts to increase the solubility of 11beta-HSD 1 via mutagenesis experiments by generating several truncated forms expressed in E. coli and the yeast Pichia pastoris. Furthermore, we investigated if the codon for methionine 31 in human 11beta-HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment. Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme. In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed in E. coli nor with that expressed in P. pastoris. Hydrophobicity plots proved the hydrophobic nature of 11beta-HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure.
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PMID:Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment? 1130 91

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