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1.
Anne-Ccile Cotillon Robert Morfin 《The Journal of steroid biochemistry and molecular biology》1999,68(5-6)
Transformation of physiologically important 3-hydroxy-steroids by the DHEA-induced 7α-hydroxylase of F. moniliforme was investigated. Whereas DHEA was almost totally 7α-hydroxylated, PREG, EPIA and ESTR were only partially converted into their 7α-hydroxylated derivatives because hydroxylation at other undetermined positions as well as reduction of ketone at C17 or C20 into hydroxyl also occurred. Cholesterol was not transformed by the enzyme. Kinetic parameters of the 7α-hydroxylation for these substrates were determined and confirmed that DHEA was the best substrate of the 7α-hydroxylase. Inhibition studies of DHEA 7α-hydroxylation by the other 3-hydroxy-steroids were also carried out and proved that DHEA, PREG, EPIA and ESTR shared the same active site of the enzyme. Induction effects of these steroids were compared, and DHEA appeared to be the best inducer of the 7α-hydroxylase of F. moniliforme. 相似文献
2.
Lafaye P Chmielewski V Nato F Mazié JC Morfin R 《Biochimica et biophysica acta》1999,1472(1-2):222-231
Human tonsils were assessed for their ability to 7alpha-hydroxylate pregnenolone (PREG), dehydroepiandrosterone (DHEA) and 3-epiandrosterone (EPIA). Both 7alpha-hydroxy-DHEA and 7alpha-hydroxy-EPIA were produced by homogenates of either whole tonsils or of lymphocyte-depleted tonsil fractions. In contrast, isolated lymphocytes were found to be unable to carry out 7alpha-hydroxylation. When co-cultures of tonsil-derived T and B lymphocytes were set up under stimulatory conditions, IgGs were released in the supernatants and could be quantitated, and immunomodulating properties of different steroids were monitored. When PREG was added to a mixture of tonsil-derived B and T lymphocytes, a decrease of non-specific and specific IgG was observed. An increase in specific anti-tetanus toxoid and anti-Bordetella pertussis antigen IgGs was obtained with either 1 microM 7alpha-hydroxy-DHEA or 1 microM 7alpha-hydroxy-EPIA. In contrast, DHEA and EPIA were unable to trigger such an effect. When cultures of isolated tonsillar B cells were used, none of the steroids tested showed significant effects on specific IgG productions. These data led to the conclusion that human tonsillar cells transform DHEA and EPIA, but not PREG, into 7alpha-hydroxylated metabolites. These metabolites could act on target tonsillar T lymphocytes which in turn act upon B lymphocytes for increasing specific IgG production. 相似文献
3.
Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density 总被引:2,自引:0,他引:2
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《The Journal of cell biology》1993,121(1):135-143
The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3- ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha- OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta- HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
4.
In a recent publication, we reported that deoxycholic acid is 7 alpha-hydroxylated to yield glycocholate or taurocholate in vivo in the hamster (1987. Kuroki et al. Hepatology. 7: 229-234). In order to explore the possibility that amidation of free deoxycholic acid precedes the 7 alpha-hydroxylation, we assayed 7 alpha-hydroxylase activities of free and conjugated deoxycholates in vitro. 7 alpha-Hydroxylase activities of glycodeoxycholate and taurodeoxycholate were 720 +/- 132 and 640 +/- 160 pmol/mg.min-1, respectively. Activity of 7 alpha-hydroxylation of free deoxycholate was very low (60 +/- 20 pmol/mg.min-1). After treatment with phenobarbital in a dose of 100 mg/kg per day for 6 days, 7 alpha-hydroxylase activities of conjugated deoxycholates were decreased significantly (40%, P less than 0.01, n = 8), whereas that of free deoxycholate was not significantly changed. In the rat, 7 alpha-hydroxylase activities of conjugated deoxycholates were induced significantly (45% increase, P less than 0.05, n = 5) by phenobarbital treatment in sharp contrast to the hamster. There were significant correlations between the 7 alpha-hydroxylase activity of taurodeoxycholate and that of glycodeoxycholate both in the hamster and in the rat (hamsters: n = 16, r = 0.98, P less than 0.01; rats: n = 10, r = 0.82, P less than 0.01). These studies suggested that deoxycholic acid is 7 alpha-hydroxylated after amidation with glycine or taurine in vivo and that the same enzyme may well catalyze the 7 alpha-hydroxylation of glycodeoxycholate and taurodeoxycholate in the hamster.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
Kim SB Chalbot S Pompon D Jo DH Morfin R 《The Journal of steroid biochemistry and molecular biology》2004,92(5):383-389
The cytochrome P4507B1 (P4507B1) in the human hippocampus is responsible for the production of 7alpha-hydroxylated derivatives of dehydroepiandrosterone (DHEA) and other 3beta-hydroxylated neurosteroids. Minor quantities of the 7beta-hydroxylated derivatives are also produced. Neuroprotective action of these 7-hydroxysteroids was reported. Recombinant human P4507B1 was prepared from yeast coexpressing the human hippocampal P450 cDNA and the human P450 reductase genes. Microsomal P4507B1 activity was tested in the presence of NADPH and (14)C-labeled steroid substrates to deduce kinetic parameters and to study inhibitor responses. The K(M) values obtained for DHEA, pregnenolone, epiandrosterone, 5alpha-androstane-3beta,17beta-diol and estrone were 1.90 +/- 0.06, 1.45 +/- 0.03, 1.05 +/- 0.12, 0.8 +/- 0.04 and 1.20 +/- 0.26 microM, respectively. Production of limited amounts of 7beta-hydroxylated derivatives was also observed, but only with DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone. K(M) values determined for 7beta-hydroxylation were identical to those for 7alpha-hydroxylation. The DHEA 7alpha-hydroxylation was inhibited by estrone and estradiol (mixed type inhibition) and by the [25-35] beta-amyloid peptide (non-competitive inhibition). These results indicate that in human, the 7-hydroxylation catalysed by P4507B1 preferentially takes place on DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone with major and minor formation of 7alpha- and 7beta-hydroxylated derivatives, respectively. Both estrogens and a beta-amyloid component inhibit the P4507B1-mediated production of the 7-hydroxysteroid metabolites. 相似文献
6.
M Axelson J Shoda J Sj?vall A Toll K Wikvall 《The Journal of biological chemistry》1992,267(3):1701-1704
The metabolism of cholesterol in isolated intact pig liver mitochondria has been investigated. Six major cholesterol metabolites were identified by gas-liquid chromatography-mass spectrometry, the metabolic end product being 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. Incubations with the synthesized intermediates suggested that the major pathway from cholesterol to this acid proceeds via the sequence of 26-hydroxylation, 7 alpha-hydroxylation, further oxidation of the side chain and oxidation/isomerization in the A-ring. The observed reactions prove that in addition to a sterol 26-hydroxylase, pig liver mitochondria contain significant amounts of a 7 alpha-hydroxylase active on side chain oxygenated 3 beta-hydroxy-delta 5-C27 steroids, an oxidoreductase active in the side chain of 26-hydroxylated steroids and a 3 beta-hydroxy-delta 5 steroid oxidoreductase active on 7 alpha-hydroxylated C27 steroids. Since 7 alpha-hydroxy-3-oxo-4-cholestenoic acid is believed to be an important precursor of chenodeoxycholic acid, this study shows that the first reactions in the biosynthesis of bile acids can be exclusively mitochondrial and thereby bypass microsomal cholesterol 7 alpha-hydroxylase as the rate-limiting enzyme. 相似文献
7.
Dehydroepiandrosterone (DHEA) has been shown to decrease the dexamethasone (DEX)-induced apoptosis of thymocytes and to be one of the native 3beta-hydroxysteroids extensively 7alpha-hydroxylated in thymus. This led us to question whether DHEA or 7alpha-hydroxy-DHEA is responsible for the decrease in DEX-induced apoptosis of thymocytes and whether this property is shared with other native 3beta-hydroxysteroids and their 7alpha-hydroxylated metabolites. Treatment of mice with DHEA or 7alpha-hydroxy-DHEA prior to DEX led to a smaller decrease in thymus weight than with DEX alone and to a disappearance of the DEX-induced changes in thymocyte phenotypes. Thymocyte apoptosis induced by DEX treatment was significantly lowered in DHEA- and 7alpha-hydroxy-DHEA-treated thymi, even after 18 h culture with additional 10-6 mol/L DEX. Extensive apoptosis of thymocytes cultured with 10-7 mol/L DEX was brought back to control levels when 10-5 mol/L 7alpha-hydroxy-DHEA or 10-5 mol/L 7alpha-hydroxy-epiandrosterone was added. After use of DHEA and epiandrosterone or pregnenolone, less significant and no significant changes were obtained, respectively. These findings imply that the 7alpha-hydroxylation of 3beta-hydroxysteroids may be a prerequisite for an exquisite regulation of the thymocyte-positive selection driven by the glucocorticoids produced in thymic epithelial cells. 相似文献
8.
Rose K Allan A Gauldie S Stapleton G Dobbie L Dott K Martin C Wang L Hedlund E Seckl JR Gustafsson JA Lathe R 《The Journal of biological chemistry》2001,276(26):23937-23944
9.
The oxidation of dehydroepiandrosterone (DHEA), 4-androstene-3, 17-dione, and estrone with Streptomyces roseochromogenes NRRL B-1233 was studied. The oxidation products were isolated and identified as as 16alpha-hydroxy-DHEA, 16alpha-hydroxy-4-androstene-3,17-dione and 16alpha-hydroxyestrone. The yields of these three products were 85%, 41% and 18%, respectively. This indicates the substrate stereospecificity of 16alpha-hydroxylase of the organism. An interrelationship between cell growth and the formation of 16alpha-hydroxylated steroid was observed in any case. For formation of 16alpha-hydroxy-DHEA, 16alpha-hydroxylase showed good activity at DHEA concentration of 3.47 x 10(-4)M. In the case of DHEA, 16alpha-hydroxy-4-androstene-3,17-dione and 5-androstene-3beta, 16alpha, 17beta-triol were obtained after the yield of 16alpha-hydroxy-DHEA reached the maximum yield for about 30 hr. The oxidation pathway of DHEA is discussed. 相似文献
10.
Torimoto N Ishii I Hata M Kobayashi Y Nakamura H Ariyoshi N Kitada M 《Biochimica et biophysica acta》2007,1774(2):223-232
CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than alpha-hydroxytriazolam at concentrations of more than 60 muM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the alpha-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both alpha- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited alpha-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and alpha-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe-O-C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4. 相似文献
11.
Elizabeth Sujkovic† Radmila Mileusnic Jonathan P. Fry† 《Journal of neurochemistry》2009,109(2):348-359
Metabolism of the neuroactive steroids pregnenolone (PREG), progesterone (PROG), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) was investigated in day-old chick brain following direct injection of the 3 H-labelled compounds into the intermediate medial mesopallium and sampling at times known to be crucial for memory formation in this brain region. 3 H-label from these steroids was cleared rapidly from the brain, decreasing to barely detectable levels within 5 h. Following extraction and fractionation, the 3 H-labelled brain steroids were identified by TLC, coupled with acetylation and/or separation in different solvent systems. PREG and PROG were converted within 10 min mostly to 20β-dihydropregnenolone (20β-DHPREG) and 5β-dihydroprogesterone, respectively. There was no detectable metabolism of DHEA. Label from DHEAS persisted for longer (half-time 18.9 min) than the free steroid but with no detectable metabolism other than a small amount (4%) of desulphation to DHEA. Further investigation of chick brain steroid metabolism by incubation of subcellular fractions (1–3 h, 37°C) with PREG, PROG or DHEA plus NADPH led to the formation of the following compounds: 20β-DHPREG from PREG (particularly in cytosol); 5β-dihydroprogesterone and 3α,5β-tetrahydroprogesterone from PROG and no detectable metabolism of DHEA. Following incubation of the same brain fractions and labelled steroids with NAD+ , there was no detectable metabolism of PREG or PROG but some conversion of DHEA to androstenedione, especially in the nuclear fraction. The results suggest direct actions of DHEA(S) on the early stages of memory formation in the chick and introduce the possibility that PREG may act indirectly via 20β-DHPREG. 相似文献
12.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(3):306-312
Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-GA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities. 相似文献
13.
Microbiological Aspects in the Hydroxylation of Estrogens by Fusarium moniliforme 总被引:3,自引:1,他引:2
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A strain of Fusarium moniliforme (IH4), isolated from soil, showed outstanding enzymatic abilities to hydroxylate a number of estrogens. Estrone and estradiol were transformed into the 15alpha-hydroxy derivatives, and estradiol 3-methyl ether was transformed into the corresponding 6beta-hydroxy derivative. Delta(6)-Estrone was not hydroxylated. The accumulation of 15alpha-hydroxyestrone was influenced by the nutritional conditions of the fungus. Maximal yield was obtained when the organism grew in Czapek solution supplemented with yeast extract, although good conversion was also found in a peptone-corn molasses medium. Substitution of NO(3)-N in Czapek medium with NH(4)-N, lactalbumin hydrolysate, Casitone, or Casamino Acids resulted in limited hydroxylation of estrone. A remarkable strain specificity was demonstrated in this conversion. Of 13 strains of F. moniliforme and Gibberella fujikuroi under investigation, only 2 strains (IH4 and ATCC 9851) accumulated substantial amounts of the 15alpha-hydroxylated product. However, marked quantitative variations were observed which are attributable to a different ability of the organisms to degrade the steroid nucleus. Biochemical instabilities were also found through the appearance of spontaneous variants lacking steroid-hydroxylating activity. Replacement culture studies revealed that 15alpha-hydroxylation of estrone was dependent on the supply of external phosphate; exogenous nitrogen or energy sources were not required. Most of the enzymatic activity was confined to the mycelia. Microconidia showed a very limited hydroxylating activity, even in the presence of supplements or energy sources. 相似文献
14.
Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM. Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase. In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3. However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3. These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E. coli cells. 相似文献
15.
Philippe Liere Antoine Pianos Bernard Eychenne Annie Cambourg Karl Bodin William Griffiths Michael Schumacher Etienne-Emile Baulieu Jan Sj?vall 《Journal of lipid research》2009,50(12):2430-2444
Pregnenolone (PREG) and dehydroepiandrosterone (DHEA), and their respective sulfated forms PREGS and DHEAS, were among the first steroids to be identified in rodent brain. However, unreliable steroid isolation and solvolysis procedures resulted in errors, particularly in the case of brain steroid sulfates analyzed by radioimmunology or GC-MS of liberated free steroids. By using a solid-phase extraction recycling/elution procedure, allowing the strict separation of sulfated, free, and fatty acid esters of PREG and DHEA, PREGS and DHEAS, unlike free PREG, were not detected in rat and mouse brain and plasma. Conversely, considerable amounts of PREG and DHEA were released from unknown precursor(s) present in the lipoidal fraction, distinct from fatty acid ester conjugates. Chromatographic and mass spectrometric studies of the nature of the precursor(s) showed that autoxidation of brain cholesterol (CHOL) was responsible for the release of PREG and DHEA from the lipoidal fraction. When inappropriate protocols were used, CHOL was also the precursor of PREG and DHEA obtained from the fraction assumed to contain sulfated steroids. In contrast, free PREG was definitely confirmed as an endogenous steroid in rat brain. Our study shows that an early removal of CHOL from brain extracts coupled to well-validated extraction and fractionation procedures are prerequisites for reliable measurements of free and conjugated PREG and DHEA by GC-MS or other indirect methods. 相似文献
16.
Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes 总被引:2,自引:0,他引:2
Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes. 相似文献
17.
Lathe R 《Steroids》2002,67(12):967-977
B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway. 相似文献
18.
Norlin M Andersson U Björkhem I Wikvall K 《The Journal of biological chemistry》2000,275(44):34046-34053
A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed. 相似文献
19.
P B Hylemon E J Studer W M Pandak D M Heuman Z R Vlahcevic J Y Chiang 《Analytical biochemistry》1989,182(2):212-216
The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase. 相似文献
20.
Tritium-labeled 24(R)-hydroxyvitamin D3 and 24(S)-hydroxyvitamin D3 were chemically synthesized and the 1 alpha-hydroxylation of these compounds by chick kidney homogenates was studied. A marked stereospecific preference with regard to the orientation of the hydroxyl functionality on carbon-24 was noted: while the 24(R)-epimer could be 1 alpha-hydroxylated in readily detectable amounts, the 24(S)-epimer was not hydroxylated. Thus, 1.2 micrograms of 1 alpha,24(R)-dihydroxyvitamin D3 was isolated and its structure confirmed by mass spectrometry. The relative rate of 1 alpha-hydroxylation of 125 nM substrate tritiated 24(R)-hydroxyvitamin D3 and 25-hydroxyvitamin D3 (the presumed natural substrate for the renal 1 alpha-hydroxylase) was 1:6.7. 相似文献