共查询到20条相似文献,搜索用时 15 毫秒
1.
Ke-Nan Qin Maria I. New K.-C. Cheng 《The Journal of steroid biochemistry and molecular biology》1993,46(6)
Rat liver 3α-hydroxysteroid dehydrogenase cDNA was previously cloned by us. In this study, 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. The sequence of 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 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. Because 3α-hydroxysteroid dehydrogenase and human chlordecone reductase belong to the aldo-keto reductase superfamily, we named these human clones HAKRa to HAKRd. Northern blot analysis showed that the liver expresses the highest levels of all four clones. Expression of all four clones was also detected in the brain, kidney, lung, and testis, whereas the placenta expressed only the messenger RNA for HAKRb. Genomic blot analysis using HAKRb as the probe detected multiple DNA fragments hybridized to the probe and a high degree of restriction fragment length polymorphism, suggesting the complexity of this supergene family. 相似文献
2.
Fraser M. Rogerson Jean-Guy LeHoux J. Ian Mason 《The Journal of steroid biochemistry and molecular biology》1995,55(5-6):481-487
The enzyme 3β-hydroxysteroid dehydrogenase/Δ5→4-isomerase (3β-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3β-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3β-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3β-HSD. Separate cDNAs encoding three isoforms of 3β-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3β-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3β-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3β-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3β-HSD isoform. These differ from the type 1 3β-HSD expressed in the adrenal cortex. 相似文献
3.
Cheng KC 《The Journal of steroid biochemistry and molecular biology》1992,43(8):1083-1088
3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase. 相似文献
4.
The 3-α-hydroxysteroid dehydrogenase and the 3-β-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyacrylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration.The pure 3-α-hydroxysteroid dehydrogenase was completely devoid of 3-β-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-β at an appreciable rate. This activity accounts for about 40 per cent of the total 17-β-estradiol dehydrogenase of the crude bacterial extract.Affinity labelling of pure 3-α-hydroxysteroid dehydrogenase was carried out using 5-β-pregnane 3,20-dione-12-α-iodoacetate and 5-α-androstane 3-one-17-β-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues. 相似文献
5.
Matsunaga T Endo S Maeda S Ishikura S Tajima K Tanaka N Nakamura KT Imamura Y Hara A 《Archives of biochemistry and biophysics》2008,477(2):339-347
Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27 kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and α-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C19/C21-steroids into 3β-hydroxysteroids. The stereospecific conversion to 3β-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor α ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3β-hydroxysteroid synthesis and xenobiotic carbonyl metabolism. 相似文献
6.
7.
L. Martini R.C. Melcangi R. Maggi 《The Journal of steroid biochemistry and molecular biology》1993,47(1-6):195-205
This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons, in the glia, and in neuroblastoma cells. The activities of the 5α-reductase (the enzyme that converts testosterone into dihydrotestosterone, DHT), and of the 3α-hydroxysteroid dehydrogenase (the enzyme that converts DHT into 5α-androstane-3α,17β-diol, 3α-diol) have been first evaluated in primary cultures of neurons, oligodendrocytes and type-1 and -2 astrocytes, obtained from the fetal or neonatal rat brain. All the cultures were used on the fifth day. The formation of DHT or 3α-diol was evaluated incubating the different cultures with labeled testosterone or DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, type-2 astrocytes and oligodendrocytes also possess considerable 5α-reductase activity, while type-1 astrocytes show a much lower enzymatic concentration. A completely different localization was observed for 3α-hydroxysteroid dehydrogenase; the formation of 3α-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3α-diol is formed in very low yields by neurons, type-2 astrocytes and oligodendrocytes. The compartmentalization of two strictly correlated enzymes (5α-reductase and 3α-hydroxysteroid dehydrogenase) in separate central nervous system (CNS) cell populations suggests the simultaneous participation of neurons and glial cells in the 5α-reductive metabolism of testosterone. Subsequently it has been shown that, similarly to what happens when testosterone is used as the substrate, the 5α-reductase which metabolizes progesterone into 5α-pregnane-3,20-dione (DHP) shows a significantly higher activity in neurons than in glial cells; however, type-1 and -2 astrocytes as well as oligodendrocytes also possess some ability to 5α-reduce progesterone. On the other hand, 3α-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5α-pregnane-3α-ol-20-one, appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3α-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoforms of the enzyme involved in androgen and progesterone metabolism is discussed.Finally, the ability of the human neuroblastoma cell line SH-SY5Y to metabolize androgens and progesterone was studied incubating the cells in the presence of labeled testosterone or progesterone to measure, respectively, the formation of DHT or DHP (5α-reductase activity). 3α-Hydroxysteroid dehydrogenase activity was studied by evaluating the conversion of labeled DHT into 3α-diol. The results demonstrate that undifferentiated neuroblastoma cells possess a significant 5α-reductase activity, as shown by the considerable conversion of testosterone into DHT; moreover, this enzymatic activity seems to be significantly stimulated following cell differentiation induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), but not after differentiation induced by retinoic acid (RA). The 5α-reductase present in SH-SY5Y cells is also able to convert progesterone into DHP. In undifferentiated cell, this conversion is about 8 times higher than that of testosterone into DHT. Under the influences of TPA and RA, the formation of DHP follows the same pattern observed for that of DHT. SH-SY5Y cells also appear to possess the enzyme 3α-hydroxysteroid dehydrogenase, since they are able to convert DHT into 3α-diol. This enzymatic activity is not altered following TPA-induced differentiation, and appears to be decreased following treatment with RA. It is suggested that the SH-SY5Y cell line may represent a useful “in vitro” model for the study of the mechanisms involved in the control of androgen and progesterone metabolism in nervous cells. 相似文献
8.
Igor Tsigelny Michael E. Baker 《The Journal of steroid biochemistry and molecular biology》1995,55(5-6):589-600
We have used the X-ray crystallographic structures of rat and human dihydropteridine reductase and Streptomyces hydrogenans 20β-hydroxysteroid dehydrogenase to model parts of the 3-dimensional structure of human 11β- and 17β-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 11β-hydroxysteroid dehydrogenase-types 1 and 2. We also examine the structure of an -helix at catalytic site of 17β-hydroxysteroid dehydrogenase-types 1, 2, 3, and 4. This -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 11β- and 17β-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 11β- and 17β-hydroxysteroid dehydrogenases-type 2. We suggest that interactions of the dimerization surface of -helix F with proteins or membranes may be important in regulating activity of hydroxysteroid dehydrogenases. 相似文献
9.
M. Dumont V. Luu-The Y. de Launoit F. Labrie 《The Journal of steroid biochemistry and molecular biology》1992,41(3-8):605-608
The insert of 1278 bp containing the entire coding region of cDNA encoding human 17β-hydroxysteroid dehydrogenase (17β-HSD) was inserted into a pHS1 vector and expressed in HeLA human cervical carcinoma cells and COS-1 monkey kidney tumor cells. Western blot analysis indicated that the expressed protein migrates at the same position as the purified enzyme and is recognized by the antibody raised against purified human placental 17β-HSD. The expressed enzyme efficiently catalyzes the interconversion of estrone and estradiol while dehydroepiandrosterone and 5-androstene-3β,17β-diol are interconverted at a lower rate. The present data suggest the existence of two 17β-HSDs. 相似文献
10.
Trevor M. Penning Yi Jin Vladi V. Heredia Mitchell Lewis 《The Journal of steroid biochemistry and molecular biology》2003,85(2-5):247
3α-Hydroxysteroid dehydrogenases (3α-HSDs) inactivate steroid hormones in the liver, regulate 5α-dihydrotestosterone (5α-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3α-HSD isoforms exist and correspond to AKR1C1–AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3α-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3α-, 17β-, and 20α-hydroxysteroid oxidase activity. Their kcat values are 50–100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3α-HSD, bile acid binding protein and peripheral 3α-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (β-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low kcat seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate “wobble” at the plastic active site. 相似文献
11.
Carlos M. Tellería Carlos O. Stocco Ricardo P. Deis 《The Journal of steroid biochemistry and molecular biology》1995,52(6):567-573
The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea. 相似文献
12.
Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family. 相似文献
13.
Xingwang Li Guoxin Hu Xiaoheng Li Yi-Yan Wang Yuan-Yuan Hu Hongyu Zhou Syed A. Latif David J. Morris Yanhui Chu Zhiqiang Zheng Ren-Shan Ge 《PloS one》2015,10(11)
Background
11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) interconverts active 11β-hydroxyl glucocorticoids and inactive 11keto forms. However, its directionality is determined by availability of NADP+/NADPH. In liver cells, 11β-HSD1 behaves as a primary reductase, while in Leydig cells it acts as a primary oxidase. However, the exact mechanism is not clear. The direction of 11β-HSD1 has been proposed to be regulated by hexose-6-phosphate dehydrogenase (H6PDH), which catalyzes glucose-6-phosphate (G6P) to generate NADPH that drives 11β-HSD1 towards reduction.Methodology
To examine the coupling between 11β-HSD1 and H6PDH, we added G6P to rat and human liver and testis or Leydig cell microsomes, and 11β-HSD1 activity was measured by radiometry.Results and Conclusions
G6P stimulated 11β-HSD1 reductase activity in rat (3 fold) or human liver (1.5 fold), but not at all in testis. S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11β-HSD1 reductase activity. We compared the extent to which 11β-HSD1 in rat Leydig and liver cells might be coupled to H6PDH. In order to clarify the location of H6PDH within the testis, we used the Leydig cell toxicant ethane dimethanesulfonate (EDS) to selectively deplete Leydig cells. The depletion of Leydig cells eliminated Hsd11b1 (encoding 11β-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter). H6pd mRNA level and H6PDH activity were barely detectable in purified rat Leydig cells. In conclusion, the availability of H6PDH determines the different direction of 11β-HSD1 in liver and Leydig cells. 相似文献14.
Hiroshi Itoh Toshiaki Katada Michio Ui Hiroshi Kawasaki Koichi Suzuki Yoshito Kaziro 《FEBS letters》1988,230(1-2)
We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2α gene and rat Gi2α cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1α and Goα cDNAs, respectively. 相似文献
15.
Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H. With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound βαβα-structures of the CHCRs suggests the presence of a typical NADP+-binding motif and is similar to the corresponding region of 3α,20β-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase. The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%). However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE2 and PGF2α from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster. 相似文献
16.
Anita H. Payne Trent R. Clarke Paul A. Bain 《The Journal of steroid biochemistry and molecular biology》1995,53(1-6):111-118
The classical form of the enzyme 5-ene-3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3β-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3βHSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3βHSD I and III function as 3β-hydroxysteroid dehydrogenases and 5-en→4-en isomerases using NAD+ as a cofactor. The enzymatic function of 3βHSD II has not been completely characterized. Mouse 3βHSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3βHSD I is expressed in the gonads and adrenal glands of the adult mouse. 3βHSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1. 相似文献
17.
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11β-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90–95% pure and exhibited dehydrogenase and reductase activities with KM values in agreement with those reported in the literature. 相似文献
18.
Giacomo Carrea Roberto Bovara Renato Longhi Sergio Riva 《Enzyme and microbial technology》1985,7(12):597-600
12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP+ was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD+ oxidoreductase, EC 1.1.1.176), and the regeneration of NADP+ was obtained through the glutamate dehydrogenase (l-glutamate:NADP+ oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained 70% of the initial activity after 2 months of continuous use. 相似文献
19.
Dirk Classen-Houben Daniela Schuster Thierry Da Cunha Alex Odermatt Gerhard Wolber Ulrich Jordis Bernhard Kueenburg 《The Journal of steroid biochemistry and molecular biology》2009,113(3-5):248-252
Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA. 相似文献
20.
Thierry Normand Bettina Husen Frauke Leenders Hlne Pelczar Jean-Luc Baert Agns Begue Anne-Claire Flourens Jerzy Adamski Yvan de Launoit 《The Journal of steroid biochemistry and molecular biology》1995,55(5-6):541-548
17β-hydroxysteroid dehydrogenases (17β-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17β-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17β-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal β-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17β-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17β-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17β-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17β-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17β-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels. 相似文献