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)

Previous work has shown an increase in rat liver enzyme activities after chronic exposure to gasoline vapor. In the present study, male Sprague-Dawley rats were pretreated with unleaded gasoline at 1 and 5 ml/kg, i.p., and selected hepatic microsomal monooxygenase activities were determined at 18, 48, and 72 h. At 18 h, moderate increases were observed in P450 content (1.3-fold), cytochrome c-reductase activity (1.25-fold), and in N-nitrosodimethylamine demethylation rate (1.25- to 1.6-fold). Pentoxyresorufin dealkylase activity (an activity displayed primarily by P450IIB1) was significantly elevated at 18 and 48 h (30- to 60-fold), and ethoxyresorufin dealkylase activity (an activity displayed by P450 IA1) was elevated (2- to 4-fold). Immunoblot analysis revealed no change in P450IIE1 at these time points, but an elevation in P450IIB1 in agreement with the pentoxyresorufin dealkylase activity measurements.
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PMID:Alteration of rat liver microsomal monooxygenase activities by gasoline treatment. 209 37

Cytochrome P450-dependent arachidonic acid metabolism in human kidney cortex from several postmortem subjects has been characterized. Using HPLC and GC/MS, four cytochrome P450-arachidonic acid metabolites were tentatively but not unequivocally identified as epoxyeicosatrienoic acid (EET), dihydroxyeicosatrienoic acid (DHT) and 19- and 20-hydroxyeicosatetraenoic acids, suggesting the involvement of two major cytochrome P450 enzymes, epoxygenase and omega/omega-1 hydroxylases. This pattern of metabolism was similar to that found in rabbit and rat kidneys. The formation of these metabolites was dependent on the presence of NADPH and inhibited by IgG of NADPH-cytochrome P450 (c) reductase. Immunologic studies of renal cytochrome P450 epoxygenase demonstrated that antibodies prepared against human-purified hepatic cytochrome P450 epoxygenase recognized renal enzyme protein and inhibited the enzyme activity by 92%. In contrast, control immunoglobulin did not inhibit renal cytochrome P450 epoxygenase. Antibody inhibition of renal cytochrome P450 epoxygenase demonstrated a degree of conservation of both enzyme proteins between liver and kidney. Antibodies against lauric acid omega/omega-1 hydroxylases (P450 omega) inhibited the formation of omega/omega-1 hydroxylase products, 19- and 20-HETEs. Identical qualitative patterns of arachidonic acid metabolites were observed in all cortical microsomes studied. Interindividual variations were observed in the cytochrome P450-dependent arachidonic acid metabolism, and the activities ranged from 0.031 to 5.027 nmol arachidonic acid converted/mg protein/30 min. which is about a 150-fold difference. However, when the specific activities for total cytochrome P450-dependent arachidonic acid metabolism were calculated, two separate groups could be distinguished, high and low metabolizers of arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytochrome P450-dependent arachidonic acid metabolism in human kidney. 210 7

Cytochrome P450IIE1 (P450IIE1) is responsible for the metabolic activation of N-nitrosodimethylamine (NDMA), a potent environmental carcinogen. This P450 enzyme displays a high-affinity NDMA demethylase (NDMAd) activity and is known to be induced by fasting and acetone administration. In the present work, the effects of pituitary hormones on the regulation of P450IIE1 in the liver were investigated and compared in rats and mice. There was no difference in liver microsomal NDMAd activity (nmol/min/mg protein) in rats in the intact (0.38 +/- 0.12), sham-operated (0.44 +/- 0.06), and hypophysectomized (0.52 +/- 0.04) groups. However, hypophysectomy caused a 2-fold increase in hepatic P450IIE1 protein levels as determined by immunoblot analysis. The P450IIE1 mRNA level in hypophysectomized rat was also significantly increased. The levels of blood ketone bodies (acetone, acetoacetate, and beta-hydroxybutyrate) were not different in the intact, sham-operated, and hypophysectomized groups, suggesting that ketone bodies are not involved in the induction of P450IIE1 protein and its mRNA by hypophysectomy. The discrepancy between the NDMAd activity and the increased P450IIE1 protein in rat liver by hypophysectomy can be partially explained by the lower hepatic NADPH-P450 reductase activity (50% that of the control) in the hypophysectomized rats. Upon the induction of liver NDMAd activity by fasting and acetone, hypophysectomy attenuated the effect of acetone but abolished the effect of fasting completely. Nevertheless, fasting still caused a 3-fold increase in the liver P450IIE1 mRNA level. An involvement of pituitary hormones in the regulation of liver microsomal P450IIE1 in mouse, however, was not observed. There was no difference in constitutive NDMAd activity between genetically growth hormone-deficient (lit/lit) mice and their phenotypically normal heterozygotes (lit/+). Fasting for 48 h caused 1.5- to 2-fold induction and acetone caused 2- to 3-fold induction, in both groups. The above changes in enzyme activity were due to the changes of P450IIE1 levels as verified by the immunoblot analysis. In male BALB/c mice, neither the hepatic NDMAd activity nor the P450IIE1 protein level was altered by hypophysectomy. The effects of acetone on the liver NDMAd activity were also similar in hypophysectomized and sham-operated mice. The results suggest that pituitary hormones are important in the regulation of the expression and activity of hepatic P450IIE1 in rats but not in the mouse strains investigated.
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PMID:Roles of pituitary hormones in the regulation of hepatic cytochrome P450IIE1 in rats and mice. 211 67

Two methods for the purification of cytochromes-P450 from microsomes of human liver are described. Method A: Cyt-P450 were solubilized from microsomes using a non ionic detergent, the Lubrol. The Cyt-P450 were purified by affinity, hydrophobicity followed by ion-exchange chromatography on DEAE-5PW column (HPLC) with an overall yield of 18% and a specific activity of 10 nmole/mg of protein. The recovery of NADPH Cyt-P450 reductase by method A (affinity) is about 60% with a specific activity of 16.2 U.I./mg of protein. Method B: Cyt-P450 were solubilized from microsomes using a zwitterionic detergent, the CHAPS. Cyt-P450 were filtered and separated by chromatofocusing on Mono-P column (HPLC). By this method it was possible to increase strongly the specific activity keeping a yield of 50% of Cyt-P450. Also it was possible to apply this method to small samples of human liver like biopsies (0.5 to 2.5 g).
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PMID:[Purification of cytochrome P-450 and NADPH cytochrome p-450 reductase from human liver]. 212 60

The NADPH-cytochrome P450 reductase gene isolated from the yeast Saccharomyces cerevisiae [Yabusaki et al., J. Biochem. 103, 1004-1010 (1988)] was expressed on a multi-copy plasmid in the yeast. The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3 kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300 bp. The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase. When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level. These recombinant yeast strains showed enhanced cytochrome c reductase activity with increased cellular reductase levels. A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450(17 alpha) resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species. These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450(17 alpha)-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450(17 alpha) in the yeast microsomes.
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PMID:Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae. 212 33

The p-nitrophenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits was inhibited by 3-amino-1,2,4-triazole in a time- and NADPH-dependent manner. The loss of p-nitrophenol hydroxylation, an activity catalyzed predominately by P450IIE1, displayed a number of characteristics consistent with suicide inhibition of the enzyme. These include irreversibility, saturability, similarity of the effect of pH on the rate constant for inactivation and catalysis by the isozyme, protection by substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. At pH 6.8, the KI for 3-amino-1,2,4-triazole for inactivation was 57 mM and the maximal rate of inactivation was 0.43 min-1. The inactivation of hepatic microsomes resulted in a loss of spectrally detectable P450 which was correlated with the concentration of P450IIE1 in various microsomal preparations. Purified P450IIE1 was rapidly autoinactivated (kinact of about 0.1 min-1) in the presence of NADPH and P450 reductase. However, the autoinactivation was completely prevented by the addition of catalase. In the presence of catalase, purified P450IIE1 was inactivated in a time- and concentration-dependent manner by 3-amino-1,2,4-triazole (KI was 10 mM and the maximal rate of inactivation was 0.44 min-1). The inactivation resulted in the loss of spectrally detectable P450 but did not cause the formation of P420 or a loss of heme as determined by the reduced pyridine hemochrome. The spectrum of the inactivated enzyme exhibited a decreased extinction in the Soret region with a broad maximum at 378 nm and a shoulder around 428 nm. Inactivated P450IIE1 did not show a characteristic low-spin spectrum in the presence of 4-methylpyrazole. When 3-amino-1,2,4-[5-14C]triazole was used in the inactivation reaction, there was no significant incorporation of radioactivity into the protein or heme; these results suggest that the inactivation may be due to covalent binding of the heme to the protein or the modification of residues near the heme, which prevent substrate interaction. The effective inhibition of P450IIE1 by 3-amino-1,2,4-triazole suggests that the compound may be useful for the identification of P450IIE1-dependent microsomal catalysis.
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PMID:Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino-1,2,4-triazole. 213 88

One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here D1, was purified to a specific content of 12.1 +/- 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 +/- 1.6 and 2.3 +/- 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancreas microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the D1 or D2 forms of spiny lobster P450 were measured by mixing D1 or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and D1 was an efficient catalyst of progesterone 16 alpha-hydroxylation, with a turnover number of 43 per minute. Other good substrates for D1 and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by D1 or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species.
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PMID:Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver. 217 37

HL60 cells exposed to increasing paraquat concentrations were screened for clones without increased superoxide dismutase activities in an effort to examine cytotoxic events occurring after superoxide production. The resulting resistance to paraquat was not associated with alterations in paraquat uptake, catalase, or NADPH-P450 reductase activity, but to alterations in glutathione-dependent enzyme activities. While increases in glutathione-dependent enzymes upon exposure to paraquat have been reported, the increases were considered a secondary response to increases in superoxide dismutase activities. Our results demonstrate that glutathione-dependent enzymes alone provide protection against paraquat toxicity, and their increase upon exposure to paraquat can be independent of the response of superoxide dismutases. This supports a previous finding that cells resistant to Adriamycin, based on elevated glutathione peroxidase and transferase activities are also cross-resistant to paraquat. Unlike this previous report, the increase in glutathione peroxidase was not a persistent genetic event, as activities returned to normal upon removal of paraquat. An isolated increase in glutathione peroxidase without accompanying increases in superoxide dismutases was a rare event, as nearly all clones examined after exposure to paraquat had increased superoxide dismutase.
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PMID:Glutathione-dependent enzymes alone can produce paraquat resistance. 217

Seven P450/reductase fused enzymes were produced in Saccharomyces cerevisiae by expressing fused cDNAs consisting of bovine cytochrome P450c17 (P450c17) and yeast NADPH-cytochrome P450 reductase (reductase). These fused enzymes differed in the length and amino acid sequence of the hinge region between the P450 and reductase moieties. Expression of the fused constructs under the control of the yeast alcohol dehydrogenase I promoter and terminator of expression vector pAAH5 in S. cerevisiae AH22 cells resulted in the production of about 2-8 X 10(4) molecules per cell of the seven corresponding fused enzymes. Six of the fused enzymes incorporated a protoheme, as confirmed by reduced CO-difference spectra. Recombinant yeast strains producing each of the fused hemoproteins showed P450c17-dependent 17 alpha-hydroxylase activity toward progesterone. The most active fused enzyme, delta N23FE, which lacked the amino-terminal 23 amino acids of the reductase, showed about 10 times higher 17 alpha-hydroxylase activity than bovine P450c17, although the fused enzyme (delta N23FE)' with an amino acid sequence in the hinge region different from delta N23FE was less active than delta N23FE. The fused enzyme delta N0FE, consisting of P450c17 and whole reductase, showed about 1.8 times higher activity than bovine P450c17. No activity was found with delta N84FE lacking the amino-terminal 84 amino acids of the reductase moiety. P450c17-dependent C17,(20)-lyase activity toward 17 alpha-hydroxyprogesterone was detected to lesser extents in the recombinant yeast. Fused bovine P450c17/yeast reductase enzymes show enhanced 17 alpha-hydroxylase activity, and the length and amino acid sequence in the hinge region between the P450c17 and yeast reductase moieties can be important for efficient intramolecular electron transfer in the fused enzymes.
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PMID:Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes. 218 Apr 29

The regulation of heme oxygenase activity in the developing neonate is essential to the control of bilirubin production as well as intracellular heme and hemoprotein metabolism. The coordinated activity of the microsomal enzymes, heme oxygenase and NADPH-cytochrome c (P450) reductase, and the cytosolic enzyme biliverdin reductase is responsible for the degradation of heme. The complete reaction sequence requires oxygen and NADPH, and produces bilirubin and carbon monoxide in equimolar amounts. Although heme oxygenase expresses a rather broad range of substrate affinities, the oxidative degradation of heme is exclusively alpha-specific. Heme oxygenase is found in several tissues, with significant activity levels in the liver, spleen, and erythropoeitic tissue. Heme oxygenase activity is inducible by heme and other metalloporphyrins, hormones, starvation, stress, toxins, and xenobiotics. Heme oxygenase induction is generally considered to be the result of an increased protein synthesis and gene transcription. This hypothesis is supported by recent studies of the heme oxygenase gene that identified inducer element binding sites responsive to metal administration, heat shock, and nutrient availability. In the developing fetus and neonate, hepatic heme oxygenase activity and mRNA levels are elevated above that of the adult. This suggests that the elevated heme catabolism observed in neonates may be associated with an increased transcription of the heme oxygenase gene. The apparent induction of hepatic heme oxygenase during the neonatal period is probably the result of tissue-specific and time-dependent transcriptional regulating factors including potentially hormones and heme. Several metalloporphyrins, such as the tin and zinc porphyrin complexes, inhibit heme oxygenase activity and thus have therapeutic potential for the treatment of neonatal jaundice. Recent studies suggest that the meso- and bis-glycol derivatives of these metalloporphyrins may be more potent inhibitors of heme oxygenase activity in vitro and in vivo than the protoporphyrin structures. As structural analogues of heme, however, these compounds may also have other less desirable effects on the regulation of heme and hemoprotein metabolism, particularly in the developing neonate.
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PMID:Developmental biology of heme oxygenase. 219 31


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