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

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the Km and an 80% decrease in kcat/Km (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.
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PMID:Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450. 192 97

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

In common with a diverse group of low-molecular-weight volatile substrates, dichloromethane (DCM; methylene chloride) is a high-affinity, low-capacity substrate for oxidation by several cytochrome P450 isoenzymes in vivo. DCM oxidation, catalyzed primarily by the 2E1 and 2B1 cytochrome P450 isoforms, yields carbon monoxide (CO) and carbon dioxide. We have studied the characteristics of DCM oxidation in vivo by examining the metabolism of DCM and of both deuterated forms ([2H2]-DCM and [2H]DCM) in female B6C3F1 mice with gas uptake methods. Gas uptake and CO production curves were analyzed by physiologically based pharmacokinetic (PBPK) modeling techniques, permitting differentiation of isotope effects on specific metabolic parameters from those associated with blood flow or diffusion limitations in vivo. A marked isotope effect was observed on the moles of CO produced per mole of DCM oxidized (0.76 +/- 0.06, 0.33 +/- 0.006, and 0.31 +/- 0.07, with DCM, [2H]DCM, and [2H2]DCM, respectively). Based on these ratios, the calculated kH/kD ratio for the rate constant of disproportionation of the putative formyl chloride intermediate was about 7, indicating a significant role of C-H bond breaking in this reaction. Deuterium substitution altered the apparent Km for metabolism; there was 14-fold increase in the apparent Km between DCM and [2H2]DCM (6.5 +/- 0.69 to 97 +/- 3.5 microM) with little effect on Km with [2H]DCM (14.4 +/- 0.015 microM). Vmax was not greatly affected by deuteration (151 +/- 1.2, 116 +/- 0.82, and 149 +/- 2.3 mumol/hr/kg with DCM, [2H]DCM, and [2H2]DCM, respectively). Two kinetic mechanisms are discussed, both of which are consistent with these observations. One, a conventional cytochrome P450 mechanism has a rate-limiting product-release step after the isotopically sensitive step; a second, more like a peroxidase mechanism, has a flux-limiting oxygen activation step followed by a second-order reaction between an activated oxygen-enzyme complex and DCM. Regardless of the correct mechanism, the in vivo kinetic constants for oxidation of DCM are complex and represent more than simple rate-limiting bond-breaking (Vmax) and enzyme-substrate binding (Km). Current PBPK models for metabolism of these volatiles may have to be restructured to account for this unusual kinetic mechanism.
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PMID:Gas uptake studies of deuterium isotope effects on dichloromethane metabolism in female B6C3F1 mice in vivo. 807 49

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

The 7-ethoxycoumarin O-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The extrapolated KI and kinactivation were 0.08 microM and 0.83 min-1, respectively. The loss of 7-ethoxycoumarin O-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that [3H]2EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the [3H]2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this amino-terminally blocked peptide. These results in conjunction with the results from the cleavage of the intact [3H]2EN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(vinylidene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T.L. (1988) Pharm. Res. 5, 67-75] that contains a highly conserved threonine residue involved in oxygen binding.
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PMID:Mechanism-based inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene: identification of an active-site peptide. 837 44

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

Langmuir-Blodgett films prepared from cytochrome P450scc and its complex with adrenodoxin have been prepared and studied. Adrenodoxin was preliminarily selectively modified with fluorescein isothiocyanate and the effect of this modification on the interaction with cytochrome P450scc was studied. Using selectively modified adrenodoxin the ratio of the proteins in the film was found to be 1 mole of adrenodoxin per 2 moles of cytochrome P450scc. Langmuir-Blodgett films were also prepared from adrenodoxin-reductase and it was shown that this flavoprotein is transferred to the substrate as an apo-protein. It is also shown that the adrenodoxin-binding region of cytochrome P450scc is exposed to the subphase under all pressures in the interval studied. The relationship between the orientation of cytochrome P450scc-adrenodoxin complex in monolayers on the water-air interface and the pressure produced at the interface at the moment of monolayer formation was found. Our data are in excellent accordance with ideas on the molecular organization of cytochrome P450scc in the inner adrenocortical membrane and allows the use of this approach to model membrane structures containing cytochrome P450.
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PMID:Langmuir-Blodgett films of cytochrome P450scc and its complex with adrenodoxin. 928 46

The metabolism of amitriptyline was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6 and 2E1. CYP 2C19 was the most important enzyme with regard to the demethylation of amitriptyline, the quantitatively most important metabolic pathway. CYP 1A2, 3A4, 2C9 and CYP 2D6 also participated in the demethylation of amitriptyline. CYP 2D6 was the sole enzyme mediating the hydroxylation of amitriptyline, and (E)-10-OH-amitriptyline was exclusively produced. CYP 2E1 did not metabolize amitriptyline. Concerning the quantitative relations, CYP 2C19 and 2D6 exhibited high affinities with Km values in the range of 5-13 mumol/l, whereas the affinities of 1A2, 3A4 and 2C9 were somewhat lower with Km values ranging from 74 to 92 mumol/l. CYP 2C19 displayed the highest reaction capacity per mole with Vmax equal to 475 mol h-1 (mol CYP)-1. The other enzymes had Vmax values in the range of 90-145 mol h-1 (mol CYP)-1. Allowing for the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that, at therapeutic doses, on average about 60% of the metabolism depended on CYP 2C19. At toxic doses, CYP 2C19 is expected to be saturated, and CYP 3A4 may now play a dominant role in the metabolism.
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PMID:Metabolism of the tricyclic antidepressant amitriptyline by cDNA-expressed human cytochrome P450 enzymes. 939 33

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