Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha), aromatase cytochrome P-450 (P-450AROM) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)(+)-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of approximately 1.9 kb was detected at low levels, while 3 beta-HSD mRNA of 1.7 kb was in relatively high abundance throughout the oestrous cycle. While P-450(17) alpha mRNA of 1.9 kb and P-450AROM of 2.7, 2.2 and 1.7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14-21 of gestation). Levels of P-450(17) alpha mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3 beta-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450(17) alpha mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Endocrinol 1991 Apr
PMID:Expression of mRNA species encoding steroidogenic enzymes in the rat ovary. 171 Apr 61

Site-directed mutagenesis of the arginine-rich region within the putative active site of the rat testicular P45017 alpha (17 alpha-hydroxylase cytochrome P-450, the product of P450XVII gene) was performed to identify specific amino acids that contribute to either the hydroxylase or lyase activities catalyzed by this enzyme. The conversion of Arg346 to alanine differentially abolished lyase activity without affecting hydroxylase activity, resulting in an accumulation of the 17 alpha-hydroxylated intermediate, and partial lyase activity was recovered by conversion of this mutant to Lys346. Similar results were obtained with the conversion of Arg357 to alanine, although this mutant also diminished hydroxylase activity, and full lyase and hydroxylase activities were recovered with a lysine in this position. Major reductions in hydroxylase activity were apparent with the conversion of Arg363 to Ala, and this inhibition was reversed by a lysine at position 363. In contrast, differential effects were not observed with the mutants Arg361 Ala, Arg361 Lys, or Tyr334Phe. Both mutations at the Arg361 position resulted in a total loss of hydroxylase and lyase activities, and mutation at the Tyr334 position had no effect on either activity. The identification of specific amino acids that are essential for either the hydroxylase or lyase reaction indicates that the steroid substrate-protein interaction changes during the course of the two consecutive reactions and reveals the potential for separation of the two activities by chemical and biological modulators within the active site region of the P45017 alpha.
Mol Endocrinol 1991 Oct
PMID:Dissociation of hydroxylase and lyase activities by site-directed mutagenesis of the rat P45017 alpha. 172 39

We used adult rat hepatocytes in primary culture (HPC) as a model system to study the hepatic phase II metabolism of the anticoagulant warfarin. Hepatocytes were isolated by a collagenase perfusion technique and maintained for 24 hr in Waymouth's medium containing 0.1 mM (R)-warfarin. When HPC medium was analyzed by reverse phase high performance liquid chromatography with diode-array detection, 4'-, 6-, and 7-hydroxywarfarin were identified. Several putative conjugates were observed eluting between 13 and 18 min. Treatment of hepatocyte medium with beta-glucuronidase and sulfatase resulted in the loss of five putative conjugates and concomitant increases in 4'-, 6-, and 7-hydroxywarfarin and warfarin, suggesting that these metabolites and warfarin were conjugated. Use of the beta-glucuronidase inhibitor saccharic acid 1,4-lactone enabled the determination of the relative extents of conjugation of each metabolite by glucuronic acid and sulfate. Glucuronidation was the predominant pathway for 4'-hydroxywarfarin, whereas 6-hydroxywarfarin and warfarin occurred mainly as sulfate conjugates. In contrast, 7-hydroxywarfarin was converted to both glucuronide and sulfate conjugates. Exposure of HPC to phenobarbital resulted in a decrease in cytochrome P-450-mediated production of hydroxylated warfarin metabolites; however, an increase in the production of 8-hydroxywarfarin was observed when HPC were exposed to beta-naphthoflavone. Unique conjugation patterns were found when hydroxylated warfarins were substituted for warfarin in HPC medium. Both 7- and 8-hydroxywarfarin were converted to one sulfate and two glucuronide conjugates, whereas 4'-hydroxywarfarin was converted to a single glucuronide conjugate. A spectral library of these conjugates was used to identify the major conjugates of warfarin formed by rat HPC.
Mol Pharmacol 1992 Jan
PMID:Phase II metabolism of warfarin in primary culture of adult rat hepatocytes. 173 19

Incubation of 11-deoxycortisol with a cytochrome P-450(11 beta)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydroxycortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to 1H-NMR analysis. The spectrum was essentially identical to that of 18-hydroxycortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occurring steroids. The one- and two-dimension 1H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occurring steroids. The 1H-NMR spectrum showed the presence of signals of 19-CH3 and 18-CH2 protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-deoxycortisol. From these results, we conclude that bovine P-450(11 beta) can catalyze the hydroxylation of 11-deoxycortisol at 11 beta-, 18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.
J Steroid Biochem Mol Biol 1991 Dec
PMID:Bovine adrenal cytochrome P-450(11 beta)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR. 175 90

The hypogonadal (hpg) mouse, which lacks circulating gonadotrophins during development, has been used (a) to determine whether initial expression of steroidogenic enzyme activity is dependent upon gonadotrophins and (b) to examine the responsiveness of these enzymes to luteinizing hormone (LH) stimulation. Activities of 17 alpha-hydroxylase, 17-ketosteroid reductase and 5 alpha-reductase were very low but detectable in the hpg testis while cholesterol side-chain cleavage (CSCC) activity was undetectable. In contrast, 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity was high (11% of normal testis). Treatment with LH increased CSCC and 17 alpha-hydroxylase activity more than 11-fold within 24 h. 5 alpha-Reductase activity was increased 3-fold after 3 days treatment while 17-ketosteroid reductase and 3 beta HSD activities did not respond until after 10 days of treatment. The overall increases in 5 alpha-reductase (4-fold) and 3 beta HSD (6-fold) activities were low while changes in 17-ketosteroid reductase (20-fold) and, particularly, CSCC (greater than 130-fold) and 17 alpha-hydroxylase (153-fold) were more marked. Results show (1) that expression of 3 beta HSD activity may be independent of gonadotrophins, (2) that activity of 17 alpha-hydroxylase, 17-ketosteroid reductase and 5 alpha-reductase is expressed, though at low levels, in the absence of gonadotrophins and (3) that prior exposure to gonadotrophins is not required for a rapid response to LH stimulation, particularly with respect to the cytochrome P-450 enzymes.
J Steroid Biochem Mol Biol 1991 Dec
PMID:Steroidogenic enzyme activity in the hypogonadal (hpg) mouse testis and effect of treatment with luteinizing hormone. 175 91

The constitutive expression of 25-hydroxyvitamin D3-24-hydroxylase (25-(OH)D3-24-hydroxylase) activity has been studied in an adherent variant (Ad-HL60) of the human promyelomonocytic leukaemia cell line HL60. The Ad-HL60 cells have a more differentiated phenotype than the non-adherent cells from which they were derived, and synthesized 1.88 +/- 0.07 (+/- S.E.M.) pmol 24,25-(OH)2D3/h per 10(6) cells following culture in RPMI-1640 medium containing less than 0.02 nM 1 alpha,25-(OH)2D3. They also synthesized 1.66 +/- 0.05 pmol 24,25-(OH)2D3/h per 10(6) cells following culture in 1 alpha,25(OH)2D3-free medium supplemented with 1 g bovine serum albumin/l instead of 10% serum. In contrast, non-adherent HL60 cells required exposure to 10-100 nM 1 alpha,25-(OH)2D3 to induce equivalent 24,25-(OH)2D3 synthesis. The 25-(OH)D3-24-hydroxylase expressed by Ad-HL60 cells had an apparent Michaelis constant of 1 microM and maximal rate of 20 pmol/h per 10(6) cells with substrate concentrations from 0.012 to 1.2 microM/incubation (5-500 ng/ml). Furthermore, 24,25-(OH)2D3 synthesis was inhibited in a dose-dependent manner by ketoconazole (0.01-10 microM), suggesting that the enzyme is cytochrome P-450 dependent. Ad-HL60 cells expressed approximately 3500 specific receptors for 1 alpha,25-(OH)2D3/cell with a dissociation constant of 40 pM. Following exposure to 0.1-100 nM 1 alpha,25-(OH)2D3, Ad-HL60 cell proliferation was significantly inhibited compared with controls grown in medium containing less than 0.02 nM 1 alpha,25-(OH)2D3 for 96 h. Expression of 25-(OH)D3-24-hydroxylase was also inhibited in a dose- and time-dependent manner; however, expression of nonspecific esterase was not induced. Both of these findings are contrary to those previously demonstrated for non-adherent HL60 cells, whereas the dose-dependent inhibition of cell proliferation by 1 alpha,25-(OH)2D3 occurs in both adherent and non-adherent phenotypes. These observations on Ad-HL60 cells represent the first description of a cell type in which 1 alpha,25-(OH)2D3 appears to inhibit 25-(OH)D3-24-hydroxylase activity. The Ad-HL60 cells also constitutively metabolized 1 alpha,25-(OH)2D3 in a manner consistent with formation of 1 alpha,25-(OH)2D3 without previous exposure to 1 alpha,25-(OH)2D3. In contrast, many other cell types, including non-adherent HL60 cells, require exposure to 1 alpha,25-(OH)2D3 to induce metabolism of 1 alpha,25-(OH)2D3 to 1 alpha,24,25-(OH)3D3, a reaction that represents the initial step for catabolism of 1 alpha,25-(OH)2D3 to calcitroic acid.
J Mol Endocrinol 1991 Dec
PMID:Studies on an adherent variant of the human promyelomonocytic HL60 leukaemia cell line that constitutively expresses 25-hydroxyvitamin D3-24-hydroxylase activity which is inhibited by 1 alpha,25-dihydroxyvitamin D3. 177 40

We studied the contents of aldosterone and cortisol (F) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with primary aldosteronism. The aldosterone content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while F content in adenomas was similar to the level in normal adrenal glands. The aldosterone-producing adenomas showed a markedly higher level of P-450c11 mRNA, a slightly but not significantly increased level of P-450c21 mRNA and a significantly decreased level of P-450c17 mRNA, compared with those in normal adrenal glands. The expression of P-450scc mRNA in adenomas was similar to the level in normal adrenal glands. These results suggested that the renin-independent overproduction of aldosterone in adrenocortical adenomas from the patients with primary aldosteronism results from increasing expression of the mRNA for P-450c11 and decreasing expression of the mRNA for P-450c17.
Mol Cell Endocrinol 1991 Apr
PMID:Expression of cytochrome P-450 mRNAs in steroidogenesis of adrenocortical adenomas from patients with primary aldosteronism. 182 Sep 78

We studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21 alpha-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with Cushing's syndrome. The F content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while the DHEA level was similar to that in normal adrenal glands. The adrenal adenomas showed a markedly higher level of P-450c17 mRNA, and a slightly but not significantly increased level of P-450c21 mRNA, compared with normal adrenal glands. The expression of P-450scc and P-450c11 mRNA in the adenomas was similar to that in normal adrenal glands. These results suggest that the overproduction of cortisol in adrenocortical adenomas associated with Cushing's syndrome results from an increased expression of P-450c17 and P-450c21 mRNA.
Mol Cell Endocrinol 1991 Sep
PMID:Markedly increased expression of cytochrome P-450 17 alpha-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome. 183 46

In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3-beta-hydroxysteroids. Herein, we used primary cultures of ovine adrenocortical (OAC) cells to study the effects of ACTH and transforming growth factor beta (TGF-beta) on 3 beta-HSD activity, protein and mRNA levels. TGF-beta has been previously reported to be a potent inhibitor of steroid formation in OAC cells. By using an antibody against human placental 3 beta-HSD, we showed that ACTH-treatment had a dose- and time-dependent stimulatory effect on 3 beta-HSD protein amount. This effect was maximal using 10(-9) M ACTH after a 48 h treatment. When included in the treatment medium, TFG-beta inhibited this stimulation by ACTH in a dose- and time-dependent manner. We also used a human 3 beta-HSD cDNA probe to demonstrate that the effect of both ACTH and TFG-beta were exerted at the mRNA level with maximal effects observed using 10(-9) M for ACTH and 1 ng/ml for TGF-beta. Bu2cAMP mimicked the effects of ACTH, and TGF-beta had an inhibitory effect on this stimulation. It appears from these data that TGF-beta is a negative regulator of 3 beta-HSD expression in OAC cells. The inhibitory effect of TGF-beta on 3 beta-HSD was contrasted to the TGF-beta effect on 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha). While the levels of both enzymes decreased, that of 3 beta-HSD was less sensitive than that of P-45017 alpha which decreased following TGF-beta treatment to non-detectable levels. The different sensitivities of steroidogenic enzymes to factors which regulate growth and differentiation such as TGF-beta may play a role in determining the nature of steroids released from adrenocortical cells.
Mol Cell Endocrinol 1991 Mar
PMID:Corticotropin regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase in ovine adrenocortical cells: inhibition by transforming growth factor beta. 185 Nov 15

Purification and immunoinhibition studies have suggested that the hydroxylations of (S)-mephenytoin and tolbutamide are catalyzed by rather similar forms of human liver cytochrome P-450 (P-450). However, the two activities are not well correlated in vivo; sulfaphenzaole is a selective inhibitor of tolbutamide hydroxylation, and expression of P-450 2C10 cDNA in yeast yields a protein that hydroxylates tolbutamide but not (S)-mephenytoin. The P-450 2C8, 2C9, and 2C10 cDNAs have all been isolated, and their sequences are known to be closely related (greater than 80%). Highly sensitive radiochromatographic assays were set up, and tolbutamide and (S)-mephenytoin hydroxylation activities were monitored during chromatography of human liver microsomal fractions. The two activities could be separated by chromatography, and proteins were purified to near-homogeneity that catalyzed either tolbutamide hydroxylation (P-450TB) or (S)-mephenytoin 4'-hydroxylation (P-450MP) but not both. Approximately 16 and 45% of the primary sequences of P-450TB and P-450MP, respectively, were determined by analysis of the tryptic peptides. The sequences of the P-450TB peptides matched those predicted by the P-450 2C9 and 2C10 cDNAs exactly; the P-450MP peptides showed two mismatches (of 219 residues) with the P-450 2C10 sequence. Proteins with the P-450 2C10 and P-450 2C9 sequences were expressed in Saccharomyces cerevisiae grown under different nutritional conditions, and both were found to be proficient in the hydroxylation of tolbutamide but not (S)-mephenytoin. We conclude, on the basis of this and previous work, that 1) P-450s 2C8, 2C9, and 2C10 all catalyze the hydroxylation of tolbutamide and 2) the protein involved in polymorphic (S)-mephenytoin 4'-hydroxylation is closely related to but distinct from P-450 2C8, 2C9, and 2C10.
Mol Pharmacol 1991 Jul
PMID:Separation of human liver microsomal tolbutamide hydroxylase and (S)-mephenytoin 4'-hydroxylase cytochrome P-450 enzymes. 185 42


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