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1.
A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450. 相似文献
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Murataliev MB Guzov VM Walker FA Feyereisen R 《Insect biochemistry and molecular biology》2008,38(11):1008-1015
The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost. 相似文献
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Ourlin JC Baader M Fraser D Halpert JR Meyer UA 《Archives of biochemistry and biophysics》2000,373(2):375-384
CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species. 相似文献
5.
N. A. Petushkova A. V. Lisitsa I. I. Karuzina V. G. Zgoda G. F. Sheremetyeva N. F. Samenkova I. P. Nikitin T. A. Sakharova A. T. Kopylov A. I. Archakov 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2008,2(1):47-54
Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged. 相似文献
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J Ma W Qu P E Scarborough K B Tomer C R Moomaw R Maronpot L S Davis M D Breyer D C Zeldin 《The Journal of biological chemistry》1999,274(25):17777-17788
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Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism. 总被引:10,自引:0,他引:10
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A Gaedigk M Blum R Gaedigk M Eichelbaum U A Meyer 《American journal of human genetics》1991,48(5):943-950
The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs. 相似文献
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Molecular cloning and expression of CYP6B8: a xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea 总被引:7,自引:0,他引:7
Xanthotoxin, a plant allelochemical, induces alpha-cypermethrin insecticide tolerance in Helicoverpa zea (corn earworm); inhibition of tolerance by piperonyl butoxide implicates cytochrome P450 monooxygenases (P450s) in the detoxification of this insecticide. To characterize the xanthotoxin-inducible P450 that might mediate alpha-cypermethrin tolerance in this species, a cDNA library prepared from xanthotoxin-induced H. zea fifth instar larvae was screened with cDNAs encoding furanocoumarin-metabolizing P450s from Papilio polyxenes (CYP6B1v2) and P. glaucus (CYP6B4v2) as well as a sequence-related P450 from Helicoverpa armigera (CYP6B2). One full-length cDNA isolated in this screening shares 51-99% amino acid identity with the CYP6B subfamily of P450s isolated from Papilio and Helicoverpa species and, thus, has been designated CYP6B8. All of these CYP6B subfamily members share a number of highly conserved domains, including substrate recognition site 1 (SRS 1) that is critical for xanthotoxin metabolism by CYP6B1v2 from Papilio polyxenes and coumarin metabolism by CYP2a5 from Mus musculus. Northern and RT-PCR analyses indicate that CYP6B8 expression is strongly induced by xanthotoxin and phenobarbital and negligibly induced by alpha-cypermethrin. 相似文献
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Runge D Köhler C Kostrubsky VE Jäger D Lehmann T Runge DM May U Stolz DB Strom SC Fleig WE Michalopoulos GK 《Biochemical and biophysical research communications》2000,273(1):333-341
Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly. 相似文献
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Ke N Baudry J Makris TM Schuler MA Sligar SG 《Archives of biochemistry and biophysics》2005,436(1):110-120
At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms. 相似文献
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Yu. D. Ivanov N. S. Bukharina T. O. Pleshakova P. A. Frantsuzov N. V. Krokhin V. S. Ziborov A. I. Archakov 《Biophysics》2011,56(5):892-896
An approach to measure the activity of single oligomers of the heme-containing enzyme cytochrome P450 CYP102A1 (CYP102A1)
by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single
CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the
same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends
on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit
amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 protein molecule was measured from the deformation
of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine
the integral and local Young’s modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101
(P450cam)-containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively. 相似文献
14.
Wiercinska P Lou Y Squires EJ 《Animal : an international journal of animal bioscience》2012,6(5):834-845
Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig. 相似文献
15.
We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system. 相似文献
16.
Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. 总被引:13,自引:3,他引:10
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We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis thaliana using a C4H cDNA from mung been as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-1 nmol-1 P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PAL1 and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PAL2 and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression. 相似文献
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A full-length cDNA encoding a new cytochrome P450, CYP6L1, was cloned from German cockroaches, Blattella germanica. CYP6L1 has an open reading frame of 1509 nucleotides with a deduced protein of 503 amino acids and molecular mass of 57 Kd. CYP6L1 is most similar to CYP6H1, a putative ecdysone 20-hydroxylase from Locusta migratoria. CYP6L1 mRNA was not detected in embryos nor nymphs, nor in adult females. CYP6L1 mRNA was detected only in the testes and accessory glands of male adult German cockroaches. Given that the testes and accessory glands are the most important components of the reproductive system in male insects, the expression of CYP6L1 mRNA exclusively in these tissues strongly suggests that CYP6L1 has a role in reproduction. Possible substrates for CYP6L1 are discussed. 相似文献
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Reduction of toxic metabolite formation of acetaminophen 总被引:5,自引:0,他引:5
Hazai E Vereczkey L Monostory K 《Biochemical and biophysical research communications》2002,291(4):1089-1094
Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation. 相似文献
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Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%). 相似文献