Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 in Spodoptera frugiperda (Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05-0.20 nmol/mg cell protein; 0.05-0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000-1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10-20 pmol/mg cell protein. The K(m) and K(cat) values for bufuralol metabolism were estimated as 4.7 microM and 12.23 min-1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the "late" basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme.
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PMID:Functional high level expression of cytochrome P450 CYP2D6 using baculoviral expression systems. 863 23

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.
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PMID:Involvement of cytochrome P-450 in the 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii. 921 26

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatographic step on an adenosine 2',5'-diphosphate-agarose affinity column. Analysis on SDS/PAGE indicated single bands with apparent molecular masses of 53, 82, and 80 kDa for purified equine testicular cytochrome P-450 aromatase (eAROM), equine testicular reductase and rat liver reductase respectively. eAROM shows a time- and concentration-dependent activity that was stable for at least 2 months when stored at -78 degrees C. It is a highly hydrophobic protein composed from 505 residues and direct sequencing of its N-terminal part showed good homology when compared with human aromatase. When deglycosylated by N-glycosidase-F the apparent molecular mass of eAROM was decreased from 53 to 51 kDa as revealed by electrophoresis, its activity, however, was not impaired. eAROM exhibits much higher affinity for androgens than for 19-norandrogens, Km values were approximately 3, 16 and 170 nM for androstenedione (A), testosterone (T) and 19-nortestosterone (19-NT) respectively. However, it aromatizes 19-norandrostenedione (19-NA) slightly more efficiently than A, the estrone (E1) formed was 4.27 vs 3.54 pmol min-1 micrograms-1 respectively (P < 0.01). After incubation of eAROM with radiolabelled A and separation of steroids on HPLC, E1, 19-hydroxyandrostenedione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-hydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was increased in the presence of EDTA (up to 50 mM) and decreased in the presence of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glycoprotein, however, deglycosylation by N-glycosidase-F does not appear to impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the intermediary compounds of aromatization 19-OHA and 19-oxoA appear to be synthesized by the same active site that synthesizes E1 as the final product, (d) the inhibition of eAROM by increasing concentrations of Mg2+ and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity.
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PMID:Purification and characterization of equine testicular cytochrome P-450 aromatase: comparison with the human enzyme. 941 12

Acute toxicity of a neem pesticide (Vepacide-Tech) was studied in male Wistar rats by oral (single) intubation for 7 days. Vepacide was found to be moderately toxic to rat based on LD50 value. Subacute toxicity of Vepacide-Tech was also studied in male rats by oral (multiple) intubation of low (80 mg Kg-1 day-1), medium (160 mg Kg-1 day-1) and high dose (320 mg Kg-1 day-1) for 90 days. High dose caused a significant decrease in Cytochrome P-450 (Cyt. P-450) concentration at 45 and 90 days and the medium dose caused same effect at 90th day in liver and lung. Kidney showed similar effect at 90 days by the three doses. Cytochrome b5 (Cyt. b5) concentration was significantly decreased in liver, lung and kidney at 45 and 90 days at medium and high doses. Brain Cyt.b5 concentration was decreased on 90th day at high dose. Cytochrome P-450 reductase (Cyt.P-450 reductase) concentration was decreased significantly in liver and brain at 45 and 90 days, respectively at medium and high doses. The withdrawal study (28 days) has shown significant recovery. These results demonstrate that low levels exposure of Vepacide may have significant effect on the xenobiotic detoxification mechanism of different tissues of rat.
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PMID:The effect of subacute administration of a neem pesticide on rat metabolic enzymes. 967 51

The effect of cholesterol and protein diet on mixed-function oxidase activity and desaturation of fatty acids were studid as well as phenobarbital inducibility of those parameters. Investigations were carried out on Wistar adult male rats. The animals were on the cholesterol (0.25%) and protein (32%) diet for 45 days. Cytochrome P-450 and cytochrome b5 contents, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, aniline hydroxylase and 4-aminopyrine N-demethylase activities were measured in a hepatic microsomal fraction. The cholesterol diet exerted a negative effect on all parameters except NADH-cytohrom b5 reductase. The protein diet did not change the activity or levels of enzymes under study. The only exception applied to induction of NADH-cytochrome b5 reductase and 4-aminopyrine N-demethylase activities. Simultaneous treatment with phenobarbital had a heterogenous effect depending on the type of diet and parameters examined.
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PMID:[Effect of the cholesterol and protein diet on cytochrome P-450 dependent monooxygenase activity]. 973 43

NADPH-Cytochrome P-450 Reductase is the major part of MFO (mixed function oxidase) system of liver. A simple and economic method of preparation of reductase was introduced in this paper and it was certified that xenobiotics benzene, bichloroethylene, and asbestos can be oxidized by reductase and reconstituted cytochrome P-450 2B1. But the metabolites of the 3 compounds can not cause the changes of plasmid PBR322 DNA.
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PMID:[Preparation of NADPH-cytochrome P-450 reductase and the role in metabolism of xenobiotics]. 1032 90

Cytochrome P-450 CYP4F3A catalyzes the inactivation of leukotriene B(4) by omega-hydroxylation, an activity of which is specifically expressed in human neutrophils. Here, we examined expression of the LTB(4) omega-hydroxylating activity during the differentiation of HL60 cells, an acute promyelocytic leukemia cell line, in the presence of various inducers. Among the inducers used, all-trans-retinoic acid (ATRA) most strongly induces the LTB(4) omega-hydroxylating activity in a dose-dependent manner. The time course of the induction of the omega-hydroxylating activity correlates well with that of the superoxide-generating activity, indicative of cell differentiation. ATRA-treated cell microsomes convert LTB(4) to its 20-hydroxyl derivative under aerobic conditions in the present of NADPH. The reaction is inhibited by carbon monoxide, an inhibitor of cytochrome P-450, and by antibodies raised against NADPH-P-450 reductase. CYP4F3A appears to be responsible for the LTB(4) omega-hydroxylase activity, based on the following observations: expression of the mRNA for CYP4F3A is observed together with the induction of LTB(4) omega-hydroxylating activity in ATRA-treated HL60 cells; and the apparent K(m) values for the omega-hydroxylation of LTB(4) and lipoxin B(4) by ATRA-treated cell microsomes are essentially the same as those of CYP4F3A in human neutrophil microsomes.
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PMID:Induction of cytochrome CYP4F3A in all-trans-retinoic acid-treated HL60 cells. 1471 52

Cytochrome P-450 and cytochrome b(5) at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b(5) reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b(5) and DPNH-cytochrome b(5) reductase activity were largely separated from one another and from an overlapping mixture of TPNH-cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination.The possible participation of these components in the mixed function oxidation of ent-kaur-16-ene and a number of its oxidized derivatives catalyzed by these microsomes is discussed in relation to the model which has been developed to explain the function of analogous components in mixed function oxidase reactions in mammalian liver microsomes.
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PMID:Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Electron Transfer Components. 1665 1

Biological production of p-hydroxycinnamic acid (pHCA) from glucose can be achieved via deamination of the aromatic amino acids l-tyrosine or l-phenylalanine. Deamination of l-phenylalanine produces trans-cinnamic acid (CA) which is further hydroxylated in the para position to produce pHCA. However, when tyrosine is used as the substrate, trans-pHCA is produced in one step. This reaction is accomplished by phenylalanine ammonia-lyase (PAL)/tyrosine ammonia-lyase (TAL). Various bacteria and eukaryotic microorganisms were screened for their ability to produce a PAL/TAL enzyme with high TAL activity. Cell-free extracts of the yeast Rhodotorula glutinis possessed the highest level of TAL activity (0.0143U/mg protein) and the lowest PAL/TAL ratio (1.68) amongst species examined. The gene for this enzyme was cloned and expressed in Escherichia coli and the kinetics of the purified PAL/TAL determined. The recombinant PAL/TAL possessed characteristics similar to those of the wild-type enzyme. Functional expression of R. glutinis PAL/TAL enzyme in Saccharomyces cerevisiae cells containing the plant C4H P-450 and P-450 reductase enzymes from Helianthus tuberosus allowed conversion of glucose to pHCA. Addition of l-phenylalanine to these cultures increased pHCA production confirming its production via the PAL route. When R. glutinis PAL/TAL was synthesized in an E. colil-phenylalanine producing strain (ATCC 31882) and grown on glucose, pHCA was formed in the absence of the Cytochrome P-450 and the P-450 reductase enzymes underlining its production via the TAL route without CA intermediacy.
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PMID:Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi. 1720 42

The nitrofuran, furazolidone, is metabolized by rat liver microsomes under both aerobic and anaerobic conditions, the rates for microsomes from 3-methylcholanthrene-induced male rats being 3.52 and 4.26 nmol/mg protein/min, respectively. The two major metabolites formed are the well-known 3-(4-cyano-2-oxobutylideneamino)-2-oxazolidone, and 2,3-dihydro-3-cyanomethyl-2-hydroxy-5-nitro-1alpha,2-di-(2-oxo-oxazolidin-3-yl)iminomethylfuro[2,3-b]furan (accounting for 16.6 and 16.4% of total extractable radioactivity, respectively). Cytochrome P-450 is not involved in the conversion of furazolidone, which was converted by rat liver microsomes to products identical to those obtained upon incubation with purified NADPH-cytochrome P-450 reductase, which is a microsomal reductase. During metabolism, a considerable amount of material (2-3% of total metabolites) became covalently bound to microsomal protein. This covalent binding could be inhibited by addition of glutathione, which also resulted in an almost complete shift from non-polar to water-soluble metabolites. No interaction of furazolidone with added calf thymus DNA was detected.
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PMID:Quantitative studies of the metabolism of furazolidone by rat liver microsomes. 2064 73


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