UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A comparative study of the ability of phenobarbital, testosterone and their combination to induce the liver microsomal monooxygenase system after 9-day administration of these compounds to intact male and female rats was carried out. It was shown that administration of testosterone does not increase the level of cytochromes P450 and b5 in the livers of male and female rats. However, after a combined administration of the two compounds testosterone significantly enhances the inducing effects of phenobarbital (i. e. superinduction) in female rats; no such effect was observed in the livers of male rats. The rates of oxidation of hexobarbital, ethylmorphine and testosterone by liver microsomes are also increased after a combined administration of the two inducers. However, the additive effects of the two substances on substrate oxidation are observed when the latter was calculated per mole of cytochrome P450. An administration of testosterone to male rats does not result in an increase of the rate of hexobarbital and testosterone oxidation by isolated liver microsomes.
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PMID:[Induction of the microsomal monooxygenase system of rat liver by combined administration of testosterone and phenobarbital]. 50 68

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.
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PMID:P450BM-3: reduction by NADPH and sodium dithionite. 156 20

The cDNA containing the full coding sequence of human NADPH-P450 oxidoreductase was isolated and completely sequenced. The cDNA contained 2398 base pairs, including 9 and 358 base pairs of 5' and 3' noncoding sequences, respectively. The human NADPH-P450 oxidoreductase protein deduced from the cDNA has 677 amino acids, with a calculated molecular weight of 76,656. The cDNA nucleotide and deduced amino acid sequences displayed 83 and 92% similarities, respectively, with those of the rat NADPH-P450 oxidoreductase. By use of somatic cell hybrids, the NADPH-P450 oxidoreductase gene was regionally localized to human chromosome 7 (7p15-q35). The levels of NADPH-P450 oxidoreductase protein and mRNA were analyzed in 13 human liver specimens and less than 3-fold variation was found among the different livers. The NADPH-P450 oxidoreductase cDNA was inserted into vaccinia virus and expressed in cell culture. The cDNA-expressed enzyme was active in reducing the electron acceptor cytochrome c. In addition, the NADPH-P450 oxidoreductase stimulated the enzymatic activity of vaccinia virus-expressed human P3(450) when both recombinant viruses were used to coinfect human cells in culture. An approximate equal mole level of NADPH-P450 oxidoreductase and P3(450) was required to achieve maximal activity for both ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase.
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PMID:Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7. 250 55

The 7-ethoxycoumarin O-deethylase activity of rat cytochrome P450 (P450) 2B1 was inactivated by 9-ethynylphenanthrene (9EPh) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. At 20 degrees C, the extrapolated maximal rate constant of inactivation (kinactivation) was 0.45 min-1 and the inactivator concentration required for half-maximal inactivation (KI) was 138 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis demonstrated that [2'-3H]-9EPh was irreversibly bound to the protein moiety of P450 2B1 and the stoichiometry of binding was determined to be 0.82 mol of inactivator bound per mole of P450 2B1. A radiolabeled peptide of approximately 3.0 kDa was identified by autoradiography after Tricine SDS-PAGE analysis of the peptides generated from a cyanogen bromide cleavage of [2'-3H]9EPh-inactivated P450 2B1. After HPLC separation of these peptides, the fraction containing the most radioactivity was analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and peaks at m/z 2720.9 and 2939.9 were detected. The lower mass peak represents the molecular ion (MH+) for the peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the MH+ of the modified peptide (theoretical 2940.5). The difference in mass (approximately 219) would correspond to the addition of a phenanthrylacetyl group to the peptide. When the fraction containing the modified and unmodified peptides was further digested with pepsin and reanalyzed by MALDI-MS, the site of attachment could be assigned to one of the amino acids contained in the peptide Phe297 to Leu307.
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PMID:Mechanism-based inactivation of cytochrome P450 2B1 by 9-ethynylphenanthrene. 748 91

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of rat cytochrome P450 (P450) 2B1 with 1.3 mol of adduct bound per mole of P450 inactivated [Roberts, E.S., Hopkins, N.E., Alworth, W.L., & Hollenberg, P.F. (1993) Chem. Res. Toxicol. 6, 470-479]. Further studies have shown that 2EN is also an efficient mechanism-based inactivator of the 7-ethoxycoumarin O-deethylase activity of rabbit P450 2B4 with 0.83 mol of adduct bound per mole of P450. Cleavage of [3H]2EN-inactivated 2B1 with cyanogen bromide, separation of the peptides by HPLC, and further purification of the radiolabeled fraction by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) led to the identification by autoradiography of a radiolabeled peptide (M(r) approximately 3000). Amino acid sequence analysis of the first 12 N-terminal residues revealed the sequence ISLLSLFFAGTE corresponding to positions 290-301 in the protein. When the radiolabeled fraction from the HPLC separation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), peaks at m/z 2722.5 and 2890.6 were detected. The lower mass peak corresponds to the molecular ion (average mass) of the cyanogen bromide peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the same peptide with a bound 2-naphthylacetyl group (theoretical 2890.4). When [3H]2EN-inactivated 2B4 was treated with cyanogen bromide, the peptides were separated by HPLC, and the fractions were analyzed by Tricine-SDS-PAGE, two radiolabeled peptides (M(r) = 5000 and 8000) were identified by autoradiography. Amino acid sequence analysis of the first 11 residues revealed identical N-termini with the sequence EKDKSDPSSEF corresponding to positions 273-283.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of active-site peptides from 3H-labeled 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 using amino acid sequencing and mass spectrometry. 814 77

Chemical modification of cytochrome P450 was used to study the involvement of lysine and arginine residues in the interaction between cytochrome P450 and NADPH-cytochrome P450 reductase. Acetylation of 2.2 and 8.5 mol of lysine/mole of P450 by acetic anhydride led to 38.7 and 95% reductions, respectively, in benzphetamine demethylation activity by NADPH-dependent reconstituted P450/reductase complex, while modification of up to 8.5 mol of lysine/mol of P450 did not inhibit cumene hydroperoxide-supported P450-dependent benzphetamine demethylation. Acetylation of lysine residues by acetic anhydride does not grossly disturb the P450 protein conformation as revealed by absolute, CO-difference and fluorescence spectral studies. Modification of P4502B1 by acetic anhydride did not affect its substrate binding ability either. Lysine residues of P4502B1 putatively involved in the interaction with reductase have been identified by radiolabeling of lysine residues with [14C]acetic anhydride followed by trypsin digestion, HPLC separation, and amino acid microsequencing. Radiolabeled lysines occur at positions 251, 384, 422, 433, and 473. Modification of arginine residues in P4502B1 with phenylglyoxal and 2,3-butanedione seemed to have no significant effect on the benzphetamine demethylation activity of P4502B1 either reconstituted with reductase and NADPH or supported by cumene hydroperoxide. Studies of incorporation of [14C]phenylglyoxal showed no concentration- or time-dependent incorporation of phenylglyoxal into the P4502B1. These results support the hypothesis of a predominant role of lysine residues of P450 in the electrostatic interaction with NADPH-cytochrome P450 reductase.
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PMID:Role of lysine and arginine residues of cytochrome P450 in the interaction between cytochrome P4502B1 and NADPH-cytochrome P450 reductase. 832 89

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.
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PMID:Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean. 846 4

The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2. During reduction with NADPH, an absorbance increase characteristic of the formation of a flavin-pyridine nucleotide charge-transfer complex was observed only during the addition of the second mole of NADPH per mole of BMR. On the basis of these results, we conclude that the midpoint reduction potential for the FMN semiquinone/FMNH2 couple is more positive than that for FMN/FMN semiquinone. The kinetics of reduction of BMR with NADPH were studied by stopped-flow spectrophotometry. With a 1:1 ratio of NADPH to BMR, the absorbance changes can be fit to five consecutive first order reactions with rate constants of 350 s-1, 130 s-1, 27 s-1, 2.3 s-1, and 0.05 s-1. These reactions are most probably the following: (a) complex formation between BMR and NADPH; (b) reduction of FAD with formation of the NADP(+)-FADH- charge-transfer complex; (c) transfer of the first electron from FADH- to FMN to form an anionic, red FMN semiquinone leaving the FAD as the neutral, blue semiquinone. Precise identification of intermediates beyond this point is difficult. In the presence of a 10-fold molar excess of NADPH, the absorbance changes and rate constants are somewhat different due to the formation of several additional reduced species of BMR. The rate of the first step increases, confirming that this is the formation of the NADPH-BMR complex. Our results indicate that the kinetic and thermodynamic control of the flavins in BMR is significantly different from that in microsomal P450 reductase. The low potential of the anionic FMN semiquinone can be utilized to reduce the P450 heme. When the anionic semiquinone becomes protonated, its potential becomes more positive and it is readily reduced to FMNH2, which is not capable of reducing P450.
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PMID:Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. 867 31

We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.
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PMID:Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans. 973 72

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