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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major increase in P450 reductase protein or activity was detectable over a 3-day period. Together, these studies establish that thyroid hormone regulates P450 reductase expression by pretranslational mechanisms. They also suggest that other regulatory mechanisms, which may involve changes in P450 reductase protein stability and/or changes in the translational efficiency of its mRNA, are likely to occur.
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PMID:Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms. 173 85

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."
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PMID:The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases. 174 31

Chain shortening via beta-oxidation from the omega-end has been recognized as the major pathway for the degradation of cysteinyl leukotrienes as well as leukotriene B4 (LTB4). The metabolic compartmentation of this pathway was studied using peroxisomes purified from normal and clofibrate-treated rat liver. beta-Oxidation products of omega-carboxy-LTB4, including omega-carboxy-dinor-LTB4 identified by gas chromatography-mass spectrometry, were formed by the isolated peroxisomes. The reaction was dependent on CoA, ATP, and NAD and was stimulated by FAD. NADPH was necessary for the further metabolism of omega-carboxy-dinor-LTB4. Together with microsomes a degradation of omega-carboxy-LTB4 also proceeded in isolated mitochondria in the presence of CoA, ATP, and carnitine. beta-Oxidation of the cysteinyl leukotriene omega-carboxy-N-acetyl-leukotriene E4 was observed only with isolated peroxisomes in combination with lipid-depleted microsomes. Direct photoaffinity labeling using omega-carboxy-[3H] LTB4 and omega-carboxy-N-[3H]acetyl-LTE4 served to identify peroxisomal leukotriene-binding proteins. The bifunctional protein (EC 4.2.1.17 and 1.1.1.35) and 3-ketoacyl-CoA thiolase (EC 2.3.1.16) of the peroxisomal beta-oxidation system were the predominantly labeled polypeptides as revealed by precipitation with monospecific antibodies. In vivo studies with N-acetyl-[3H2]LTE4, N-acetyl-[3H8]LTE4, and N-[14C]acetyl-LTE4 after treatment with the peroxisome proliferator clofibrate indicated formation and biliary excretion of large amounts of metabolites more polar than omega-carboxy-tetranor-N-acetyl-LTE3 including omega-carboxy-tetranor-delta 13-N-acetyl-LTE4 and omega-carboxy-hexanor-N-acetyl-LTE3. Increased formation of beta-oxidized catabolites of N-acetyl-LTE4 and LTB4 was also observed in hepatocytes isolated after clofibrate treatment. Our results indicate that peroxisomes play a major role in the beta-oxidation of leukotrienes from the omega-end. Whereas omega-carboxy-LTB4 was beta-oxidized both in isolated peroxisomes and mitochondria, the cysteinyl leukotriene omega-carboxy-N-acetyl-LTE4 was exclusively degraded in peroxisomes.
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PMID:Peroxisomal degradation of leukotrienes by beta-oxidation from the omega-end. 176 71

Trimethylaminuria (TMAuria), the excessive urinary excretion of the odorous trimethylamine (TMA), accompanies elimination of TMA in sweat and corresponding "fish-odor" syndrome. TMA was oxidized in vitro in rat liver microsomes from male Sprague-Dawley rats to TMA N-oxide and N-demethylated to dimethylamine (DMA). Both reactions were inhibited to 1-3% of normal activity by preincubation of microsomes without NADPH-generating system at 37 degrees C for 10 minutes indicating the FAD-containing monooxygenase-catalyzed reactions. On the other hand, the reactions were not inhibited by gas phase containing up to 80% carbon monoxide/20% oxygen mixture. The results are compatible with the hypothesis that in rat liver microsomes the N-oxygenation and N-demethylation of TMA are catalyzed only or predominantly by FAD-containing monooxygenases, and the cytochrome P-450 monooxygenases play a negligible, if any, role.
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PMID:The FAD-containing monooxygenase-catalyzed N-oxidation and demethylation of trimethylamine in rat liver microsomes. 182 76

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
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PMID:NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. 184 86

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.
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PMID:Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia. 189 14

Hepatic flavin-containing monooxygenases catalyze NADPH-dependent oxygenation of a wide variety of drugs that possess a nucleophilic heteroatom. Two forms of these enzymes (form 1 and 2) have been isolated from rabbit liver microsomes and partially characterized (Ozols, J., 1989, Biochem. Biophys. Res. Commun. 163, 49-55). The complete amino acid sequence of form 2 is presented here. Sequence determination was achieved by pulsed liquid-phase and solid-phase sequencing of 40 peptides generated by chemical and enzymatic cleavages, including CNBr cleavage of tryptophanyl residues. Form 2 monooxygenase contains 533 amino acid residues and has a molecular weight of 60,089. The COOH terminus of this enzyme is very hydrophobic and presumably functions to anchor the protein to the membrane. Form 2 is readily degraded, since a form lacking residues 1 to 278 and a form without the COOH-terminal segment were also isolated from solubilized membrane preparations. The amino acid sequence of form 2 is 52% identical to that of form 1 and shows 55% identity to the sequence of rabbit lung monooxygenase derived from the cDNA data. The putative FAD and NADP binding segments around residues 9 and 190 are conserved in all three forms. Three variable segments can also be identified in these isoforms. These are residues 308 to 321, residues 408 to 421, and the membrane binding domain, residues 505 to 533. A comparison of the presently limited amino acid sequence data of flavin-containing monooxygenases (FMOs) implies that a particular FMO in different mammalian species may be very similar, but isozymes within a species may exhibit more extensive variability with respect to homology and catalytic activity. This study documents the structural diversity of a second hepatic FMO from rabbit liver and establishes this class of drug-metabolizing enzymes as a family of related proteins.
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PMID:Multiple forms of liver microsomal flavin-containing monooxygenases: complete covalent structure of form 2. 189 80

The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase. 189 33

Treatment of rat liver microsomes with alkaline phosphatase results in a loss in the FMN but not the FAD flavin prosthetic group of NADPH-cytochrome P-450 reductase (Taniguchi, H. and Pyerin, W. (1987) Biochim. Biophys. Acta 912, 295-307). Experiments were carried out to evaluate the effect of preventing electron transfer from the FADH2 to FMN component of the reductase, and subsequent mixed function oxidase activity, on reduction of ferric chelates, production of H2O2, and the generation of .OH-like species by microsomes. Treatment with alkaline phosphatase was confirmed to decrease NADPH-cytochrome c, but not NADPH-ferricyanide, reductase activity by microsomes and by purified NADPH cytochrome P-450 reductase. The oxidation of hydroxyl radical scavenging agents by microsomes and reductase was decreased by the alkaline phosphatase treatment in accordance with the decline in cytochrome c reductase activity. This decrease in hydroxyl radical production occurred in the presence of various ferric chelate catalysts. Rates of microsomal reduction of the ferric chelates were also inhibited after alkaline phosphatase treatment. Production of H2O2 was decreased in accordance to the fall in cytochrome c reductase activity and .OH production. Rates of H2O2 production appeared to be rate-limiting for the overall generation of .OH as the addition of an external H2O2-generating system stimulated .OH production as well as prevented the decline in .OH production caused by the alkaline phosphatase treatment. These results suggest that both the FAD and FMN flavin prosthetic groups of the reductase contribute towards the reduction of various ferric chelates. However, loss of the FMN component and activities dependent on electron transfer from this prosthetic group result in a decrease in H2O2 production, which appears to be responsible for the decline in the generation of .OH-like species by microsomes after treatment with alkaline phosphatase.
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PMID:Inhibition of the oxidation of hydroxyl radical scavenging agents after alkaline phosphatase treatment of rat liver microsomes. 190 77

Bacillus megaterium cytochrome P-450BM-3 and its two functional domains, the heme and flavin domains, have been purified and characterized using an Escherichia coli expression system. Recombinant P-450BM-3 behaves both spectrally and enzymatically the same as the enzyme produced from the natural host, B. megaterium, and another E. coli system recently described (Bouddupalli, S. S., Estabrook, R. W., and Peterson, J. A. (1990) J. Biol. Chem. 265, 4233-4239). Reduction of the flavins in P-450BM-3 domain with NADPH appears to be very similar to microsomal P-450 reductases where two reducing equivalents are consumed to fully reduce the FMN while the FAD is converted to the semiquinone in an one electron reduction. NADPH reduction of the heme occurs only in the presence of substrate suggesting, by analogy with the cytochrome P-450CAM system, a possible increase in iron redox potential of the heme upon substrate binding which facilitates electron transfer from the flavins to the heme. The flavin domain retains a high level of cytochrome c reductase activity and also reacts with NADPH to give a 3-electron reduced product. The heme domain retains the ability to bind substrate and generates the characteristic 450-nm absorption band upon reduction in the presence of CO. The heme domain has been crystallized and a preliminary set of x-ray diffraction data obtained.
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PMID:Characterization of recombinant Bacillus megaterium cytochrome P-450 BM-3 and its two functional domains. 190 73


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