UNIPROT:Q8NEX9 - FACTA Search

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

A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5 alpha cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter-1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein)-1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to approximately 70% homogeneity [nominally 15 nmol P450 (mg protein)-1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.
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PMID:Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. 834 45

Previous studies reported that, in the absence of drug exposure, multidrug resistance, including resistance to Adriamycin (ADR), could develop in primary rat hepatocyte cultures (B. Carr, Proc. Am. Assoc. Cancer Res., 29:1158, 1988). However, the hepatocytes in that report were cultured on plastic without the benefit of an extracellular matrix (ECM). Because the ECM regulates hepatic gene expression, we have critically evaluated in primary cultures of rat hepatocytes how the ECM affects hepatic ADR resistance, the level of the drug efflux transporter associated with MDR, P-glycoprotein (pgp), and transport of a prototypical pgp substrate, vincristine. Hepatocytes cultured on type I collagen (Vitrogen) had greater resistance to ADR toxicity accompanied by parallel increases in the level of pgp mRNA, decreased drug accumulation, and enhanced drug efflux when compared with the hepatocytes maintained on the basement membrane matrix Matrigel. The development of ADR resistance coincided with the time course of increased pgp mRNA but was not coincident with the time course of expression of either the placental isozyme of glutathione S-transferase or P-450 reductase, proteins associated with MDR in some resistance models. Southern blot analysis revealed neither gross changes in pgp gene structure or gene copy number to account for the increase in pgp RNA levels for hepatocytes cultured on Vitrogen. ECM also regulated xenobiotic-inducible expression of hepatic pgp, since chemotherapeutic agents, including vincristine and colchicine, induced pgp mRNA exclusively in hepatocytes cultured on Vitrogen. The critical matrix proteins in Matrigel responsible for regulation of pgp were determined by the selective addition of its components to the culture environment. The presentation of the individual matrix elements as a rigid substratum to the hepatocyte did not decrease pgp mRNA. In contrast, the presentation to the same hepatocytes of either laminin or type IV collagen in a nonrigid state (solubly in the medium) selectively decreased hepatocellular pgp mRNA. We conclude that primary rat hepatocytes develop ADR resistance with time in culture due to increased expression of pgp and that ECM proteins represent endogenous physiological modulators of both basal and chemotherapeutically inducible expression of hepatic P-glycoprotein.
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PMID:Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes. 842 4

The enzyme family carbonyl reductase, which catalyses the reduction of xenobiotic as well as endogenous ketones and aldehydes, has not been very well studied in terms of its biological functions and structural aspects. The aim of the present study was to check for the occurrence of inter-individual variability of carbonyl reductase activity in human liver. In vitro metabolism of p-nitroacetophenone (PNAP, a prototype substrate) indicated the presence of a high- and low-affinity enzyme site. The reductase activity of 17 kidney donor livers was screened at two concentrations (0.05 and 0.5 mM PNAP, below and above Km). The rates of reductase activity at 0.05 mM suggested a normal distribution. In contrast, at 0.5 mM the rates indicated a non-normal distribution, i.e. bi- or tri-modality. As an index of variability of enzyme affinity, ratios of velocities at 0.5 to 0.05 mM PNAP were calculated in order to check their frequency distributions. Three out of 17 kidney donor livers showed an atypical ratio. In these three cases, the high ratio was due to the low activity of the high affinity form of carbonyl reductase. Autopsy livers are a more readily available tissue source and about half the activity of the kidney donor livers was found in 43 autopsy livers indicating that they are a useful source of human tissue for studies of carbonyl reductase.
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PMID:Carbonyl (phenone) reductase in human liver: inter-individual variability. 851 35

Recognition and analysis of distinct mechanisms by which primaquine and other hemolytic drugs activate the hexose monophosphate shunt (HMS) have suggested a hitherto unsuspected pharmacogenetic interaction between daunorubicin metabolism and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Because this deficiency is very common, and because anthracyclines are indispensable antitumor antibiotics that are biotransformed mainly by carbonyl reductase, we have compared the reductase-mediated conversion of daunorubicin to daunorubicinol and the conversion of doxorubicin to doxorubicinol in G6PD-deficient and nondeficient erythrocytes. We found that even without G6PD deficiency, the HMS dehydrogenases selectively limited daunorubicin metabolism, as contrasted with that of doxorubicin. The milder GdA- variety of G6PD deficiency restricted the biotransformation of daunorubicin at therapeutic levels, in hemolysates and intact erythrocytes, within 15 minutes, for at least 24 hours. The bioconversion defect was even more severe in Gd Mediterranean G6PD deficiency. Primaquine aldehyde competed with daunorubicin as a substrate for carbonyl reductase. These studies show that HMS dehydrogenase activity controls carbonyl reductase-dependent biotransformation. New issues arise concerning possible effects of G6PD deficiency on the oncolytic and toxic properties of anthracyclines that are effective substrates for carbonyl reductase and also on non-xenobiotic reactions catalyzed by this enzyme.
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PMID:Glucose-6-phosphate dehydrogenase deficiency severely restricts the biotransformation of daunorubicin in human erythrocytes. 864 64

The influence of the quinone-reducing enzyme, DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase], on the genotoxicity of quinones was examined in two cell lines, namely a human hepatoma cell line, HepG2 and a brown bullhead fibroblast cell line, BB. The quinone-reductive characteristics of these two cell lines were examined using an acetylated cytochrome c reduction assay for enzymatic reductase activity. Subsequently, the influence of DT diaphorase on the genotoxicity of two model quinones, menadione (MND) and 9,10-phenanthrenequinone (PQ) was examined in an alkaline unwinding assay for DNA single-strand breaks. Results revealed that DT diaphorase was the predominant quinone reductase in cytosols of both cell lines, and that levels of specific DT diaphorase activity were generally equivalent in the two species. Despite these similarities, results revealed marked qualitative differences between the two species in terms of the influence of DT diaphorase on quinone-mediated genotoxicity. When pretreated with the DT diaphorase inhibitor, dicoumarol, HepG2 cells exhibited a marked exacerbation of genotoxicity in the presence of either MND or PQ, indicating protective influence of the enzyme. In contrast, quinone genotoxicity in BB cells was not affected by DT diaphorase inhibition, indicating the lack of a protective effect of DT diaphorase. This study illustrates the manner in which functionally analogous enzymes may have markedly distinct influences on xenobiotic toxicity in different cellular systems.
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PMID:Influence of DT diaphorase on quinone-mediated genotoxicity in human and fish cell lines. 865 9

Proteins implement most biological functions at the molecular level. As one might expect based on this fact, it appears that the altered functional states associated with toxic effects involve changes in the abundance or structure of proteins. Although numerous specific assays exist to measure changes in the abundance of individual proteins, practical limitations have prevented widespread use of multiple protein assays for the global characterization of toxicity. Recent developments in protein analytical technology are rapidly changing this picture. Two-dimensional gel electrophoresis, a technique capable of resolving and quantitating hundreds of proteins simultaneously, is becoming an automated, high-throughput tool. In parallel, techniques have been developed that allow the resulting deluge of protein measurements to be organized into a prototype Molecular Effects Database describing xenobiotic effects in rodent liver. This database can detect, classify, and characterize a broad range of liver toxicity mechanisms. It currently contains approximately 10 million protein measurements, including data on the liver effects of 43 compounds, with a further 50 compounds to be added in 1995. Observed effects range from very broad (sex steroids alter levels of 45% of all liver proteins) to very specific (e.g., hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors). Companion 2-dimensional databases describing rodent brain and kidney have been initiated, as have linkages to the genomic sequence databases. Assimilation of this approach into research and regulatory toxicology poses an interesting challenge--one that is likely to lead to a radically more sophisticated understanding of toxicity and its biological basis.
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PMID:Simultaneous measurement of hundreds of liver proteins: application in assessment of liver function. 883 83

Since the brain is not a homogeneous organ, but one dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monoxygenase system in brain, as well as the regulation of its expression, is important in elucidating its function in that organ. In order to study these issues, female rats-both ovariectomized and not-were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and P450 reductase determined using polymerase chain reaction. Results of this investigation indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR was utilized to demonstrate the effects of xenobiotics (phenobarbital, beta-naphthoflavone, imipramine) and sex hormones (testosterone, estrogen) on the levels of these messages in the female rat brain. Significant induction of message for P4502D forms was noted with testosterone in the absence of estrogen. The level of mRNA for reductase was not significantly influenced by any of the treatments, however. These results raise the issue of a sexual dimorphism in the rat regarding P4502D expression in brain.
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PMID:Anatomical distribution of NADPH-cytochrome P450 reductase and cytochrome P4502D forms in rat brain: effects of xenobiotics and sex steroids. 890 23

A new form of the NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs), present in the gram-negative bacterium Comamonas testosteroni ATCC 11996, was isolated from a testosterone-induced bacterial extract and characterized. The enzyme (HSD 28) has a monomeric molecular mass of 28 kDa. It belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDR) as established by N-terminal sequence analysis. Along with the 3 alpha-hydroxysteroid dehydrogenase and 3-oxo-reductase activities towards a variety of cis or trans fused A/B ring steroids, it also reduces several xenobiotic carbonyl compounds, including a metyrapone-based class of insecticides, to the respective alcohol metabolites. No dihydrodiol dehydrogenase activity towards trans- or cis-benzene-dihydrodiols could be detected, thus distinguishing it from the indomethacine-sensitive, mammalian liver type 3 alpha-HSDs. Subcellular fractionation revealed that the enzyme is localized in the cytoplasm of the bacterial cell. Proteins similar to the 3 alpha-HSD were detected and characterized from Comamonas testosteroni strain ATCC 17454 and from a commercially available steroid-induced extract of a patent Pseudomonas strain. The N-terminal amino acid sequence of the 3 alpha-HSD from the latter strain (HSD 29) is highly similar (94% identity over 15 residues) to a previously determined primary structure of a Pseudomonas species 3 alpha-HSD. However, no similarities could be detected between HSD 28 and a recently determined 3 alpha-HSD sequence from the ATCC 11996 Comamonas strain. The specific crossreaction of antibodies directed against mammalian liver type I 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD I) with the isolated 3 alpha-HSDs suggests the existence of a functionally and structurally related subgroup within the SDR superfamily. The broad substrate specificities of the characterized 3 alpha-HSD enzymes lead to the conclusion that they might participate in the intestinal bioactivation or inactivation of hormones, bile acids and xenobiotics since Comamonas testosteroni and related species are found in the intestinal tract of vertebrates including man.
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PMID:Characterization of a 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase from the gram-negative bacterium Comamonas testosteroni. 894 61

NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention. We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NQO1 activity. Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme. Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity. Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line. Expression and purification of recombinant wild-type and mutant protein from E. coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein. PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells. Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded. The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation. These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.
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PMID:Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase). 900 Jun

The small intestine is the major portal of entry of ingested xenobiotics. Previous studies from this and other laboratories indicated that at least 6 of the 33 xenobiotic metabolizing forms of P450 currently identified are expressed in rat small intestinal epithelial cells. In the present study, a previously unidentified rat P450, designated CYP2J4, was identified in rat small intestine using PCR. The full-length CYP2J4 cDNA contains an open reading frame for a protein of 501 residues and is 72.5 and 75.8% identical to rabbit CYP2J1 and human CYP2J2, respectively, in deduced amino acid sequences. The coding region of CYP2J4 cDNA has been cloned into a baculoviral expression vector (pVL1392) and expressed in cultured Spodoptera frugiperta (SF9) cells. The heterologously expressed CYP2J4 protein displayed a typical p450 CO-difference spectrum, with maximum absorbance at 449 nm. When purified to near electrophoretic homogeneity, it was active toward arachidonic acid in a reconstituted system with NADPH-P450 reductase and phospholipid, producing both hydroxyeicosatetraenoic and epoxyeicosatrienoic acids. RNA blot analysis with CYP2J4 cDNA as a probe detected two mRNA species, about 2.0 and 2.4 kb, respectively, in RNA preparations from liver, intestine, olfactory mucosa, kidney, heart, and lung. The 2.0-kb mRNA species was abundant in liver, small intestine, and olfactory mucosa, whereas the 2.4-kb mRNA species was predominant only in the olfactory mucosa. Immunoblot analysis of microsomal fractions from different rat tissues with a polyclonal anti-peptide antibody to CYP2J4 detected a protein with the same electrophoretic mobility as purified CYP2J4 most abundantly in small intestine and to a lesser extent in liver and other immunoreactive proteins with slightly higher electrophoretic mobility than purified CYP2J4 in a number of tissues, including small intestine, liver, kidney, lung, and olfactory mucosa. The predominant distribution of CYP2J4, which has activity toward arachidonic acid, is provocative, but its physiological function is as yet unknown.
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PMID:CDNA cloning, heterologous expression, and characterization of rat intestinal CYP2J4. 914 31

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