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1.
Wang MZ Wu JQ Dennison JB Bridges AS Hall SD Kornbluth S Tidwell RR Smith PC Voyksner RD Paine MF Hall JE 《Proteomics》2008,8(20):4186-4196
The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable. 相似文献
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Lane CS Wang Y Betts R Griffiths WJ Patterson LH 《Molecular & cellular proteomics : MCP》2007,6(6):953-962
Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18O/16O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed. 相似文献
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Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data 总被引:2,自引:0,他引:2
Johansson C Samskog J Sundström L Wadensten H Björkesten L Flensburg J 《Proteomics》2006,6(16):4475-4485
The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels. 相似文献
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冬虫夏草不同发育时期蛋白质组iTRAQ质谱分析 总被引:3,自引:0,他引:3
本文选定冬虫夏草寄主昆虫幼虫(S1)、僵虫(S2)、子座初期(子座<1cm,S3)、子座早期(1cm<子座<3cm,S4)、成熟冬虫夏草(子座>7cm,S5)、商品冬虫夏草菌核(S6)、商品冬虫夏草子座(S7)及商品冬虫夏草(中期子座≈5cm,S8)8个样品,用定量蛋白质组学方法iTRAQ分析技术对冬虫夏草不同生长发育阶段的差异蛋白质组进行比较。共计获得9 924个不同肽段,鉴定到1 809个蛋白质,其中差异比值1.5倍以上,P<0.05蛋白个数506个,以商品冬虫夏草样本(S8)为参照,比较差异蛋白数量,寄主幼虫(S1)、僵虫(S2)、子座初期(S3)、子座早期(S4)、成熟冬虫夏草(S5)、商品冬虫夏草菌核(S6)及商品冬虫夏草子座(S7)阶段差异蛋白数分别为104、102、34、35、49、46和136个,说明昆虫幼虫、僵虫及子座与商品冬虫夏草样品差异显著。对鉴定蛋白质数据进行了主成分分析(principal component analysis,PCA),在第三主成分(component 3)上的显著差异,表明成熟冬虫夏草(S5)的蛋白组成不同于商品虫草,说明成熟虫草品质下降。层次聚类(hierarchy clustering)分析结果表明所有样品分为两支,僵虫(S2)和商品冬虫夏草菌核(S6)与寄主幼虫(S1)聚在了一个分支上,而不同发育阶段样品(S3、S4和S5)与商品虫草子座(S7)聚在一个分支上,说明商品冬虫夏草菌核中还残留有寄主昆虫蛋白。冬虫夏草菌核部位的蛋白到子座部位的蛋白呈现由寄主幼虫蛋白向真菌蛋白发育过渡的聚类关系。子集嵌套关系同步子实体生长发育阶段蛋白组成变化随时间的程序性变化的规律。K-均值(K-means)聚类和Gene Ontology(GO)注释分析,提供了冬虫夏草成熟过程中能量代谢通路的变化趋势以及与真菌侵染昆虫和有性生殖相关蛋白质信息。研究结果为理解寄主昆虫对冬虫夏草功能的潜在贡献、子实体形成和发育的分子机制提供借鉴,并为蛋白质组作为冬虫夏草质量标准提供了科学参考。 相似文献
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Jasminder Sahi Stacy S. Shord Celeste Lindley Stephen Ferguson Edward L. LeCluyse 《Journal of biochemical and molecular toxicology》2009,23(1):43-58
Cytochrome P450 2C9 (CYP2C9) expression is regulated by multiple nuclear receptors including the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). We compared coregulation of CYP2C9 with CYP2B6 and CYP3A4, prototypical target genes for human CAR and PXR using human hepatocyte cultures treated for three days with the PXR activators clotrimazole, rifampin, and ritonavir; the CAR/PXR activator phenobarbital (PB); and the CAR‐selective agonists CITCO, (6‐(4‐chlorophenyl)imidazo[2,1‐β][1,3]thiazole‐5‐carbaldehyde‐O‐(3,4‐dichlorobenzyl)oxime) and phenytoin. Clotrimazole, rifampin, ritonavir, phenytoin, and phenobarbital induced CYP2C9 consistent with previous findings for CYP3A4. We observed EC50 values of 519 μM (phenobarbital), 11 μM (phenytoin), and 0.75 μM (rifampin), similar to those for CYP3A4 induction. Avasimibe, a potent PXR activator, produced nearly identical concentration‐dependent CYP2C9 and CYP3A4 activity profiles and EC50 values. In 17 donors, rifampin increased mean basal CYP2C9 activity from 59 ± 43 to 143 ± 68 pmol/mg protein/min; fold induction ranged from 1.4‐ to 6.4‐fold. Enzyme activity and mRNA measurements after rifampin, CITCO and PB treatment demonstrated potency and efficacy consistent with CYP2C9 regulation being analogous to CYP3A4 rather than CYP2B6. We demonstrate that hepatic CYP2C9 is differentially regulated by agonists of CAR and PXR, and despite sharing common regulatory mechanisms with CYP3A4 and CYP2B6; this enzyme exhibits an induction profile more closely aligned with that of CYP3A4. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:43–58, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20264 相似文献
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Dimethylcyclosiloxanes (DMCS) are components of silicone gel containing implants and are known inducers of human drug metabolizing enzymes. The effects of the major DMCS, octamethyltetracyclosiloxane (D4) on cytochrome P450 (CYP) induction were examined in young adult, mature, and pregnant female Sprague-Dawley rats. Also, the ability of D4 administered to pregnant dams to affect CYP expression in fetal liver was examined. Female young, mature, and pregnant Sprague-Dawley rats were administered 0, 5, 20, and 100 mg/kg D4 daily by gavage for 8 days. Liver microsomal CYP (CYP2B, CYP3A, CYP1A) concentrations were evaluated by Western blots using specific antisera, and CYP activities were assayed using CYP selective assays. D4 treatment resulted in a significant induction of CYP2B and CYP3A isoforms. CYP induction was dose and age dependent. A comparison of the inducibility of CYP3A protein by D4 in rats from different age groups showed that the degree of increase was the highest in the pregnant rats at doses of 20 mg/kg D4 or higher. The mature rats had a lesser degree of responsiveness than did the young rats at the dose of 100 mg/ kg D4. Significant increases in CYP2B immunoreactive protein concentrations were observed in young and mature rats given D4 at doses >5 mg/kg and in pregnant rats at doses >20 mg/kg. Maximal CYP2B induction detected with blotting was more than 90-fold in mature rats; however, no significant changes were detected in CYP1A expression. There was a 20% increase of liver to body weight ratio in the mature rats treated with 100 mg/kg D4. D4 has different inductive properties in female rats of different ages and reproductive status. Also, D4 administered to the pregnant dam is capable of inducing CYP expression in fetal liver as well as decreasing fetal body weight. 相似文献
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定量蛋白质组研究是蛋白质组研究的热点和难点,而液相色谱质谱技术已经被广泛地应用于蛋白质的定性和定量研究.该研究建立和优化了一种基于液相色谱质谱联用技术的蛋白质组非标记定量方法,并对两种肽段质谱检测计数的归一化算法进行了比较,结果发现ASC法要优于Rsc法.最后,将建立的方法应用于肝癌细胞模型HepG2和HepG2-HBx细胞系的差异蛋白质组表达研究.质谱鉴定结果用聚类分析软件cluster3.0进行分析,最后鉴定出107个重叠蛋白,其中9个蛋白质表达上调(Ratio>1.75),6个蛋白质表达下调(Ratio<0.5),这些蛋白质均与肝癌发生和恶化密切相关.结果表明,该技术操作简单、方便,具有较高的灵敏度和动态范围,利用该方法进行差异蛋白质组研究和发现生物标志物在理论和临床上具有十分重要的意义. 相似文献
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Low molecular weight dimethylcyclosiloxanes (DMCS) are important precursors in the synthesis of polydimethysiloxane polymers widely used in industry, and in medical and personal care products. The objective of this study was to characterize the ability of two DMCS, octamethylcyclosiloxane (D4) and decamethylcyclopentasiloxane (D5) to induce drug metabolizing enzymes in rats. Male and female Sprague-Dawley rats were administered 1, 5, 20, or 100 mg/kg D4 or D5 in corn oil daily by gavage for 4 days. Changes in the levels of activity and/or immunoreactivity of CYP1A1/2, CYP2B1/2, CYP3A1/2 and NADPH cytochrome P450 reductase in liver microsomes were examined. Significant increases were observed in the liver to body weight ratio in female rats administered either D4 or D5 at doses > or = 20 mg/kg. Increases in the liver to body weight ratio were observed in male rats treated with > or = 100 mg/kg D5 but not with D4. Relatively large increases in CYP2B1/2 enzymatic activity and immunoreactive protein were observed with increasing concentrations of both D4 and D5. Significant increases in 7-pentoxyresorufin O-depentylase (PROD) activity were also detected in male and female rats given D4 at doses > or = 5 mg/kg. D5 increased PROD activity in male rats at doses > or = 20 mg/kg and in female rats at doses > or = 5 mg/kg. 7-Ethoxyresorufin O-deethylase (EROD) activity was increased in both male and female rats receiving > or = 20 mg/kg D4 or > or = 5 mg/kg D5; however, no changes were detected in CYP1A1/2 immunoreactive protein in rats of either sex. D4 and D5 caused significant increases in CYP3A1/2 immunoreactive protein in only male rats treated with 100 mg/kg of either compound. However, significant increases were detected in CYP3A1/2 immunoreactive protein in female rats at D4 doses > or = 20 mg/kg and D5 doses > or = 5 mg/kg. Induction of NADPH cytochrome P-450 reductase immunoreactive protein was observed with D4 in female rats and in both male and female rats with D5. Induction of CYP2B/1/2, CYP3A1/2 and NADPH cytochrome P450 reductase was observed in rats treated with 50 mg/kg phenobarbital by intraperitoneal injection. Maximal CYP2B induction detected with D4 was approximately 50% of the increase observed with phenobarbital. In summary, D4 and D5 induced CYP2B1/2 in adult rat liver in a manner similar to that observed with phenobarbital; however, differences were observed between D4 and D5 in their ability to induce CYP3A1/2 and NADPH cytochrome P450 reductase. Female rats were more sensitive to the inductive properties of low doses of both DMCS than male rats whereas male rats were more responsive to phenobarbital induction. 相似文献
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Uno S Dragin N Miller ML Dalton TP Gonzalez FJ Nebert DW 《Free radical biology & medicine》2008,44(4):570-583
The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal. 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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Jaspreet S. Sidhu Craig B. Marcus Andrew Parkinson Curtis J. Omiecinski 《Journal of biochemical and molecular toxicology》1998,12(5):253-262
A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n -alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0–500 μ M of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n -alkyl side-chain length (C0–C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 μ M concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain–substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 253–262, 1998 相似文献
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Smith MP Wood SL Zougman A Ho JT Peng J Jackson D Cairns DA Lewington AJ Selby PJ Banks RE 《Proteomics》2011,11(11):2222-2235
Immunodepletion of clinical fluids to overcome the dominance by a few very abundant proteins has been explored but studies are few, commonly examining only limited aspects with one analytical platform. We have systematically compared immunodepletion of 6, 14, or 20 proteins using serum from renal transplant patients, analysing reproducibility, depth of coverage, efficiency, and specificity using 2-D DIGE ('top-down') and LC-MS/MS ('bottom-up'). A progressive increase in protein number (≥2 unique peptides) was found from 159 in unfractionated serum to 301 following 20 protein depletion using a relatively high-throughput 1-D-LC-MS/MS approach, including known biomarkers and moderate-lower abundance proteins such as NGAL and cytokine/growth factor receptors. On the contrary, readout by 2-D DIGE demonstrated good reproducibility of immunodepletion, but additional proteins seen tended to be isoforms of existing proteins. Depletion of 14 or 20 proteins followed by LC-MS/MS showed excellent reproducibility of proteins detected and a significant overlap between columns. Using label-free analysis, greater run-to-run variability was seen with the Prot20 column compared with the MARS14 column (median %CVs of 30.9 versus 18.2%, respectively) and a corresponding wider precision profile for the Prot20. These results illustrate the potential of immunodepletion followed by 1-D nano-LC-LTQ Orbitrap Velos analysis in a moderate through-put biomarker discovery process. 相似文献
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Matsunaga T Maruyama M Harada E Katsuyama Y Sugihara N Ise H Negishi N Ikeda U Ohmori S 《Biochemical and biophysical research communications》2004,318(2):428-434
CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR. 相似文献
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Wang-Buhler JL Lee SJ Chung WG Stevens JF Tseng HP Hseu TH Hu CH Westerfield M Yang YH Miranda CL Buhler DR 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2005,140(2):207-219
A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate. 相似文献
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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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