Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We isolated cDNA clones of the mRNAs for cytochromes P450scc (CYP11A1) from sheep and goat adrenocortices using the RT-PCR method. We determined the complete nucleotide sequences of the coding and 3'-flanking regions of these cDNA clones. The results confirmed the amino terminal sequence of the sheep cytochrome P450scc reported previously [Miyatake et al., Biochim. Biophys. Acta 1215,176-182 (1994)]. On the basis of comparison of the deduced amino acid sequences of various animals and rainbow trout cytochromes P450scc, and other mitochondrial cytochrome P450 isozymes, we discussed the substrate-associated and adreno-ferredoxin-binding region of cytochrome P450scc.
J Steroid Biochem Mol Biol 1996 Feb
PMID:Molecular cloning and nucleotide sequences of cDNA clones of sheep and goat adrenocortical cytochromes P450scc (CYP11A1). 864 27

The functional development of the neonatal rat adrenal cortex is characterized by a triphasic response to adrenocorticotropic hormone (ACTH), with a nadir in responsiveness around neonatal day 10 (d10). In this study, the hypothesis was tested that hyporesponsiveness to ACTH partly results from deficiencies in steroidogenic enzyme content. Immunoreactive (ir) levels of mitochondrial cytochrome P450 enzymes (side chain cleavage (P450scc) and 11 beta-hydroxylase (P450c11)) did not change during neonatal development. Immunoreactive levels of microsomal 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), however, were significantly and comparably lower in both day 1 (d1) and d10 neonates compared to adult rats. Activity of 3 beta-HSD did not parallel changes in ir 3 beta-HSD content. Enzyme activity was low on d1 (approximately 39% of adult activity), but by d10 was statistically equivalent to that of microsomes from adult adrenal glands. Immunoreactive levels of microsomal cytochrome P450 21 alpha-hydroxylase (P450c21) were significantly lower in d1 glands than in adult glands (by approximately 50%), but by d10 were statistically indistinguishable from adults. On the other hand, P450c21 activity was equivalent on d1 and d10 and both were significantly lower compared to adults (approximately 62% of adult activity). ACTH injections from d3-d10 facilitated the adrenocortical steroidogenic response to ACTH on d10. This treatment increased levels of ir 3 beta-HSD, but not ir P450c21. The results suggest that rat adrenocortical 3 beta-HSD and P450c21 are developmentally and differentially regulated, and that ir levels of the proteins are not correlated with enzyme activity during the neonatal period. One possible explanation for these observations is that multiple isoforms of the two enzymes, with different antigenic and enzymatic properties, may be expressed during development at different times. In addition, the combined decreased activities of these two enzymes can almost entirely account for the decreased steroidogenic output of rat adrenocortical cells on d1, but not during the later neonatal period.
Mol Cell Endocrinol 1995 Oct 30
PMID:Ontogeny of immunoreactive and bioactive microsomal steroidogenic enzymes during adrenocortical development in rats. 867 48

Earlier studies in immature porcine granulosa cells cultured in serum-free medium showed dual actions of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In cells incubated for 24 h, TPA inhibited follicle-stimulating hormone (FSH)-stimulated cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA accumulation. In contrast, at 4 h, TPA increased P450scc mRNA concentration in the absence and presence of FSH or 8-bromo-cAMP; in addition, TPA augmented FSH-stimulated cAMP accumulation. The actions of TPA were then examined in the presence of the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). With IBMX present, TPA caused a smaller relative augmentation of cAMP accumulation during a 4-h incubation period, suggesting that TPA may both increase cAMP synthesis and inhibit its degradation. The stimulatory effect of FSH or 8-bromo-cAMP on P450scc mRNA concentration was not modified by IBMX. However, TPA no longer augmented the FSH- or 8-bromo-cAMP-stimulated P450scc mRNA accumulation when IBMX was present. In cells treated with FSH for 24 h, IBMX augmented progesterone production, but paradoxically accentuated the inhibitory effect of TPA on steroidogenesis. These results indicate that IBMX converts TPA from a stimulatory into an inhibitory agent by an action unrelated to cAMP, and points to the need for caution in interpreting experiments with this drug.
Mol Cell Endocrinol 1996 Mar 25
PMID:Paradoxical effect of 3-isobutyl-1-methylxanthine on cytochrome P450 cholesterol side-chain cleavage mRNA accumulation in porcine granulosa cells. 873 81

Using the reverse transcription polymerase chain reaction, mRNAs encoding steroidogenic P450s as well as NADPH-cytochrome P450 reductase (P450 reductase), adrenodoxin and the transcription factor steroidogenic factor 1 (SF-1) were all detected in rodent brain, but their distribution between brain regions varied. Adrenodoxin and P450 reductase were detected in all regions, suggesting the presence of both mitochondrial and microsomal P450s throughout the brain. Messenger RNAs encoding P450scc (CYP11A1) and P45017 alpha (CYP17) were also detected in all brain regions, this being the first report of CYP17 in the brain. P450c21 (CYP21) was detected only in the brain stem. P45011 beta (CYP11B1) and P450aldo (CYP11B2) are expressed in rat brain, but not in mouse brain; CYP11B1 primarily in the cerebrum, whereas CYP11B2 was detected in all brain regions. In both species, highest levels of aromatase P450 (CYP19) mRNA were detected in the cerebrum. SF-1 expression was restricted to the cerebrum minus cortex. Thus, although SF-1 is required for high level expression of the steroidogenic enzymes in adrenals and gonads, other factors may influence the expression of these genes in the brain. If the mRNAs detected by RT-PCR are indeed translated into functional enzymes, these studies suggest that different brain regions have different capacities for local steroid hormone production and metabolism. This raises the technical challenge of locating the specific sites of synthesis as well as the function of such locally produced ligands.
Brain Res Mol Brain Res 1995 Dec 01
PMID:Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain. 875 Aug 63

Langmuir-Blodgett films of cytochrome P450scc were prepared on the solid supports and their spectral properties were investigated. Being immobilized, hemoprotein changes its spin state from initially high to low spin. This transition is reversible since after the solubilization of hemoprotein, the spin state equilibrium is shifted towards high-spin state. Anaerobic reduction of film incorporated cytochrome P450scc by electron transfer chain (NADPH-->adrenodoxin reductase-->adrenodoxin) revealed the low rate of the reaction that coincides well with the content of the hemoprotein low-spin form. We suggest that particularly regular orientation of solid cytochrome P450scc are of crucial importance for this phenomenon.
Biochem Mol Biol Int 1996 May
PMID:Cytochrome P450scc spin state transitions in the thin solid films. 879 43

Testicular tumorigenesis was observed in transgenic mice expressing the 6-kb mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (SV40 Tag) fusion gene. The tumors were confined to Leydig cells using immunohistochemistry with anti-Tag antibody, specific binding of biotinylated hCG and histochemistry for 3 beta-hydroxysteroid dehydrogenase. Leydig cell hyperplasia and presence of Tag protein in the testicular interstitial tissue were already evident at 5 and 6.5 days of age, respectively. An immortalized cell line, BLT-1, was established from one testicular tumor. These cells expressed the LH receptor and P450scc mRNAs, and displayed LH-responsive cAMP and progesterone production, and low testosterone production. The cells also specifically bound 125I-labeled recombinant human LH with high affinity (36000 binding sites/cell), and the binding was regulated by 8Br-cAMP and hCG. This gonadal tumor model is valuable for further studies on endocrine functions of Leydig cells and their tumorigenesis in vivo and in vitro.
Mol Cell Endocrinol 1996 May 31
PMID:The mouse inhibin alpha-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice. 880 33

After the incubation of minced mammary tissues from non-lactating/non-pregnant (NL/NP), nonlactating/pregnant (NL/P), fully lactating (FL) and late-lactating (LL) cows with [14C]-labelled pregnenolone or progesterone and dehydroepiandrosterone (DHEA), the following metabolites were identified at all stages: 20alpha-dihydropregnenolone, progesterone (from pregnenolone), 5alpha-pregnanedione, 5alpha-pregnan-3beta-ol-20-one, 20alpha- and 20beta-dihydroprogesterone (from progesterone), 5-androstene-3beta,17beta-diol, 5alpha-androstanedione, 5alpha-androstan-3beta-ol-17-one, androstenedione, testosterone and DHEA acyl ester (from DHEA). These products indicate the occurrence of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase, 17beta-hydroxysteroid oxidoreductase (17beta-HOR), 20alpha- and 20beta-hydroxysteroid dehydrogenases, steroid 5alpha-reductase and acyl transferase activities. Incubation of mammary tissue homogenates with [1,2,6,7-(3)H]androstenedione and testosterone confirmed the presence of a 17beta-HOR acting prevalently in a reductive way but failed to show evidence of any aromatase activity beyond background level. When total RNA from mammary tissues of NL/NP and LL cows was reverse-transcribed and amplified by polymerase chain reaction (PCR) with three sets of primers specific for bovine P450scc, P450c17 and P450arom cDNAs, no fragment of the expected size could be detected on gel. Southern analysis with corresponding digoxigenin-labelled ovarian probes, however, gave a positive signal for P450arom cDNA in five out of eight samples of LL mammary tissue. These data indicate that the bovine mammary gland has very limited steroidogenic capabilities that are essentially compatible with the terminal activation of circulating steroids from steroidogenic endocrines. It is uncertain, however, whether this conclusion applies to anestrous or ovariectomized lactating cows as well.
J Steroid Biochem Mol Biol 1996 Nov
PMID:Occurrence of steroidogenic enzymes in the bovine mammary gland at different functional stages. 901 Mar 26

Cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene expression is regulated by gonadotropins via cAMP in the ovary and by ACTH via cAMP in adrenal cortical cells. Previously, we have characterized a response element located at -118 to -101 bp in the 5'-flanking region of the bovine P450scc gene required for cAMP-stimulated transcription in both mouse adrenocortical Y1 cells and bovine ovarian cells in primary culture. It was shown that this region contains a binding site for the transcription factor Sp1. Deletion of this sequence abolished cAMP-stimulated transcription in both Y1 cells and bovine ovarian luteal cells. Another sequence element located at -57 to -32 bp upstream from the transcription initiation site, which is highly conserved in CYP11A of other species, contains the motif TAGCCTTG, similar to the consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. In the present study, gel shift analysis using nuclear extracts of either Y1 cells or bovine luteal cells demonstrated that the sequence between -57 and -32 bp bound SF-1. A mutation of the SF-1-binding site that abolished binding of the nuclear protein to DNA reduced markedly the basal transcription of the reporter gene as well as the responsiveness to cAMP, when the mutated fragments containing the region from -186 to +12 bp were cloned into a luciferase construct and transfected into mouse adrenal Y1 cells and bovine luteal cells. The role of SF-1 in P450scc transcription was further confirmed by transactivation of the -186/+12Luc construct employing an SF-1 expression vector after transfection into nonsteroidogenic COS-1 cells. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites, and from a construct containing both Sp1 and SF-1 elements upstream of the CYP11A TATA box, indicated that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y1 cells. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 can associate in vivo. These results establish that basal and cAMP-stimulated activity of the bovine P450scc promoter in both Y1 cells and bovine luteal cells requires the combined action of at least two transcription factors, Sp1 and SF-1.
Mol Endocrinol 1997 Feb
PMID:Steroidogenic factor 1 (SF-1) and SP1 are required for regulation of bovine CYP11A gene expression in bovine luteal cells and adrenal Y1 cells. 901 60

The prothoracic glands in vitro convert 25-hydroxycholesterol (25C) to 25-hydroxy-7-dehydrocholesterol (7d25C) and to ecdysteroids at a greater rate than cholesterol (C) is converted to ecdysteroids via 7-dehydrocholesterol (7dC). Mediated via a cytochrome P450 most probably located in the endoplasmic reticulum (ER), both intact and extensively homogenized prothoracic glands, as well as crude subcellular fractions, were able to 7,8-dehydrogenate 25C to 7d25C eight-fold more efficiently than they could convert C to 7dC. However, less than a two-fold difference was observed in the subsequent monooxygenase mediated conversion of these two intermediates formed in situ into ecdysteroids, mainly ecdysone (E) and 2-deoxyecdysone (2dE) and/or their 3-dehydroderivatives. When 7dC, and particularly 7d25C, were made directly available to these tissue preparations, their conversion to ecdysteroids greatly exceeded that of the in situ conversion of either C or 25C, via 7dC or 7d25C, respectively. Indeed, there was an eight-fold increase in the VMAX for 25C dehydrogenation by homogenized glands relative to the dehydrogenation of C. Most important, however, was the 1000-fold increase in the VMAX observed for the direct production of E from emulsified 7d25C by gland homogenates relative to E production from 25C via 7d25C synthesized in situ. Thus, it is apparent that even after the rapid and efficient conversion of 25C to 7d25C within the ER, the subsequent rate of conversion of this intermediate to E is greatly retarded relative to that observed following the direct incubation of emulsified 7d25C with gland homogenates. These differential kinetics of direct and indirect 7d25C incorporation into E are interpreted as evidence for the existence of a barrier to the efficient translocation of the delta 5,7-sterol intermediates from the ER to another site where the subsequent, uncharacterized initial conversions leading to ecdysteroids take place. On the basis of studies on mammalian adrenal cortical steroidogenesis, this site is postulated to be the inner membrane/matrix of the mitochondria. The present data support the hypothesis that the translocation of both 7dC and 7d25C, first from the site of their probable synthesis within the ER membranes, next through the cytosol to the outer mitochondrial membrane, and then across the intramitochondrial aqueous space to the inner membrane/matrix compartment, may be analogous to the translocation in the adrenal cortex of ER-derived C, first to the plasma membrane and/or to the outer mitochondrial membrane and then to the inner mitochondrial membrane/matrix for P450scc-mediated conversion into pregnenolone.
Insect Biochem Mol Biol
PMID:Metabolism in vitro of cholesterol and 25-hydroxycholesterol by the larval prothoracic glands of Manduca sexta. 901 37

Supraphysiological doses of LHRH-Analogue blocked the C21 to C19 steroid conversion in the mature Wistar rats testis. It was associated with inhibition of the NAD-dependent secondary alcohol-dehydrogenase (A-D II) histochemical reaction in the Leydig cells. Under this condition the treated group exhibited lower testis, seminal vesicle and prostate weights, intratesticular (IT) and plasmatic (PL) increased progesterone (P4) and decreased testosterone (T) concentrations. We also observed a decrease in the IT androstenedione (delta 4) concentration without pregnenolone (P5) change. All these data confirm a chemical castration pointing to a blockade at the level of the P450C21scc (17 alpha-hydroxylase/17-20 desmolase) enzyme complex. After hCG administration there is no difference in sexual gland weights, while steroid's biosynthesis are stimulated and all IT and PL steroid concentrations increase. A-D II showed a lower optical density in the LHRH-A treated groups and no differences in the hCG rats. The hydroxylase or lyase activity of the P450C21scc may change under certain hormonal conditions as occurs in adrenarche, probably due to conformational changes in the active site of the enzyme system since it is encoded by only one gene. We suppose that the secondary alcohol itself and not the coenzyme reacts with the enzyme active site inhibited by the LHRH-A, since the NAD dependent 3 beta, hydroxysteroid-dehydrogenase (3 beta HOST-D) is affected in the opposite sense. This study shows A-D II reaction as a marker of the mediated P450C21scc enzyme complex activity in the rat testis Leydig cells.
Cell Mol Biol (Noisy-le-grand) 1997 May
PMID:Secondary alcohol dehydrogenase as a marker of the conversion between progestagens and androgens in the rat testis. 919 84


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