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Tài liệu Báo cáo khoa học: Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc docx
... spectrum (Fig 6A), the correlation between 3-CH and 4-CH2 (2.53 p.p.m and 2.19 p.p.m.), and the correlation between 3-CH and 2-CH2 (1.97 p.p.m and 1.52 p.p.m.), are clearly intact In the TOCSY ... collection and interpretation of the NMR data Their spectra were recorded in CDCl3 and COSY spectra lacked fine detail HSQC and expanded COSY were not provided by Guryev et al [2] and, without ... from two independent samples shows that C23, and not C22, is hydroxylated Minor products and ⁄ or intermediates were identified by purifying them and determining the substrates from which they...
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Tài liệu Báo cáo khoa học: Cytochrome P450 Cyp4x1 is a major P450 protein in mouse brain doc
... was radiolabelled by random priming As shown in Fig 3A, there was expression of CYP4x1 RNA in brain, heart and kidney, and lesser expression in skeletal muscle and liver, and no detectable expression ... R53456 and AA337301 were obtained from HGMP (Hinxton, Cambridgeshire, UK), and sequenced on both strands The latter clone was digested with EcoRI and the 1.2-kb fragment was radiolabelled with random ... P450, and how this relates to physiological function Experimental procedures Animals and tissue Human cardiovascular system and 12-lane multitissue northern blots, human aorta cDNA library and...
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Tài liệu Báo cáo khóa học: Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism doc
... H117 and F484) and two in substrate specificity (F116 and F484) This was achieved after sequence alignment analyses and site-directed mutagenesis [32] These amino acids belong to the SRS1 and SRS6 ... presented for each metabolite and variant Four specific activities are significantly different from the control, namely dicofol production by CYP6A2vS and DDA, and DDD and dicofol production by CYP6A2vSVL ... CYP6A2wt, CYP6A2vS and CYP6A2vSVL, the ratio dicofol : DDA (specific activities) is 70.00, 251.50 and 28.62, respectively, and the ratio DDD/DDA (specific activities) is 43.00, 62.25 and 11.90, respectively...
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Tài liệu Báo cáo khoa học: Models and mechanisms of O-O bond activation by cytochrome P450 A critical assessment of the potential role of multiple active intermediates in oxidative catalysis doc
... and CYP2C3 [18] to yield hexacoordinate low-spin complexes has been shown to be characterized by absorption bands around 420 and 557 nm in the absolute spectra and broad maxima at about 440 and ... Boussac, A., Hildebrandt, P & Girer, J.J (2000) FeIII-hydroperoxo and peroxo complexes with aminopyridyl ligands and the resonance Raman spectroscopic identication of the Fe-O and O-O stretching ... equimolar combination of a- and b-subunits, each a-subunit bearing an Fe2S2 cluster and a mononuclear iron site [208] Two histidines and one bidentate aspartate ligand, the socalled ễ2-His-1-carboxylate...
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Báo cáo khoa học: Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants potx
... long chain fatty acids by recombinant CYP4F8 and CYP4F12 and catalytic importance of Tyr-125 and Gly-328 of CYP4F8 Arch Biochem Biophys 441, 174–181 19 Kandel S, Sauveplane V, Olry A, Diss L, Benveniste ... (53%), x-2 (15%), x-3 (8%), x-4 (18%) and x-5 (6%) For substrate oxidation, we determined, by kinetic studies, Km,app and Vmax,app values of 172 ± 13 lm and 117 ± nmolÆmin)1Ænmol)1 P450, respectively ... acid, and was indeed identified as 9,10-epoxystearic acid by GC ⁄ MS analysis (Fig S4) For substrate oxidation, we determined, by kinetic studies, Km,app and Vmax,app values of 84 ± 23 lm and 26...
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Báo cáo khoa học: Inter-flavin electron transfer in cytochrome P450 reductase – effects of solvent and pH identify hidden complexity in mechanism potx
... values of kfast (up phase) are $ 20 and $ 3ặs)1 in H2O and D2O, respectively The mean values of kslow (down phase) are $ 2.1 0.4 and $ 1.5 0.2ặs-1 in H2O and D2O, respectively The values obtained ... rate constants for the up phase and down phase in H2O and D2O The symbols are the same as those in (B) Figure S5 presents the ratio of kfast and kslow in H2O and D2O as a function of the pH value ... amounts of CPR and NADPH showed three up-phases at 600 nm and were analysed using a triple-exponential equation yielding the three rate constants k1, k2 and k3 with the amplitudes De1, De2 and De3,...
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Báo cáo khoa học: D88A mutant of cytochrome P450nor provides kinetic evidence for direct complex formation with electron donor NADH ppt
... NAD(P)H There are many Arg and Lys residues inside and outside the haem-pocket of P450nor We have shown that this positive charge cluster plays a crucial role in attracting and binding to the negatively ... (Michaelis intermediate) formation between P450nor and NADH, and a subsequent catalytic (electron transfer) reaction (Eqn 5) On the other hand, the possibility cannot be ruled out yet that the ... P450nor with NADH Materials and methods Mutagenesis and expression plasmids The construction of each mutant of P450nor of F oxysporum was carried out according to a standard method [13] Each recombinant...
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Báo cáo khoa học: Testosterone 1b-hydroxylation by human cytochrome P450 3A4 pdf
... Germany/Spark Holland, Emmen, the Netherlands) Peaks were automatically trapped on · 10 mm SPE cartridges filled with Hysphere GP, a cross-linked polystyrene-divinylbenzyl copolymer (Spark Holland) After ... known because of their coelution with standards in this and previous work However, the peak eluted at 12.1 did not correspond to any of the available standards in our collection (2a-, 2b-, 6a-, ... human of the testis hormone, testosterone, into the urinary androgen, androsterone J Biol Chem 130, 285–295 Ryan, K.J (1958) Conversion of androstenedione to estrone by placental microsomes Biochim...
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Báo cáo khoa học: Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacteriumsp. strain HE5 ppt
... 1148–1159 ª 2005 FEBS B Sielaff and J R Andreesen more bulky and ⁄ or hydrophobic compounds However, it could not be excluded that morpholine is a natural substrate and, thus, converted by P450mor ... electron-transfer protein and P450mor, which acts as a mono-oxygenase FdRmor has already been cloned, purified and characterized as a NADH-dependent, FAD-containing protein and shown to be structurally ... was an absolute requirement for Fdmor, as no reduction was observed with NADH and FdRmor alone 1150 B Sielaff and J R Andreesen Fig NBT reduction by the FdRmor ⁄ Fdmor couple showing dependence...
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Báo cáo khoa học: The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism docx
... comparison with Fig and consisted of 85% methanol and 0.1% acetic acid from to 25 min, followed by linear gradient to 100% methanol and 0.1% acetic acid from 25 to 35 min; and 100% methanol and 0.1% acetic ... tumorostatic and anticarcinogenic properties, affecting proliferation, differentiation and apoptosis in cells of different lineages, and protecting DNA against oxidative damage [1–4] Besides the liver and ... time and mass spectrum matching the corresponding standard None of the major peaks showed detectable amounts of species with m ⁄ z at 417, 399 and 381 that represent dihydroxyvitamin D3 and products...
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Báo cáo Y học: Baboon cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17) Characterization of the adrenal microsomal and cloned enzymes docx
... human and baboon CYP17 lie in the predicted substrate access and binding regions which include helices F and G and the F-G loop The differences between the two species in the F and G helix and ... DNA was expressed in nonsteroidogenic cells and the Km and V values of the expressed enzyme were determined MATERIALS AND METHODS Animals Adrenal glands and blood were obtained from normal adult ... investigated thus far, and may contribute to a further understanding of the hydroxylase and lyase activities of the enzyme in relation to substrate binding and orientation PROG and 17-OHPROG metabolism...
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Báo cáo Y học: Substrates modulate the rate-determining step for CO binding in cytochrome P450cam (CYP101) A high-pressure stopped-flow study pdf
... kinetics while the substrates norcamphor and adamantane and the substrate-free protein have positive activation volumes and fast rebinding kinetics Stopped-flow and flash photolysis studies should give ... activation volume and a fast CO binding (substrate-free, norcamphor and norbornane) The other group shows a negative activation volume and slow CO binding kinetics (1R-camphor and camphane) (b) ... between its keto group and the hydroxyl group of the amino-acid residue Tyr-96, and (b) by hydrophobic contacts of its methyl groups C-8, C-9 to Val295 and Asp297 in the b3 sheet, and of the methyl...
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Báo cáo khoa học: Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation pdf
... assays with d0 and perdeuterated (at the carbon being oxidized) substrates and comparing the v versus S plots (Figs and 3, and Tables and 3) The patterns were very similar for 7-OMe and 7-OEt coumarin, ... (3-Hydroxylation has also been reported in human [30] and other [31] liver microsomes, but rates were not reported.) As shown in Figs and and Tables and 3, deuteration of the alkoxy group had little ... (2001) Analysis and characterization of enzymes and nucleic acids In Principles and Methods of Toxicology (Hayes, AW, ed.), pp 1625–1687 Taylor & Francis, Philadelphia, PA, USA 35 Sandhu P, Guo...
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Báo cáo khoa học: The first cytochrome P450 in ferns Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare doc
... subcellular fraction This protein was detected and measured using difference spectroscopy [23,24], and the amount was dependent on the dose of E applied to the calli and the incubation time This showed ... concentrations were applied to calli and incubated for 24 h Microsomes were prepared and their P450 content was measured (B) We applied 60 nmol E per 100 mg to the calli and P450 was measured at different ... (B) 8.6 nmol and (C) 86 nmol EÆ100 mg calli)1 for different times, and after methanolic extraction, ecdysteroids were determined by HPLC The amount of 20E is represented by rhombus, and the bioconversion...
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Báo cáo khoa học: Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locus Potencial role in the glucocorticoid response ppt
... analysis of C/EBPa and CYP3A1 mRNA and protein accumulation in the liver Our in vitro and the in vivo results indicate that C/EBPa is an activator of the CYP3A1 gene promoter and suggest a role ... lg of CYP3A1T81Luc plasmid and different amounts of each expression vector Cells were inoculated in 24-well plates and maintained for 48 h These cells were harvested and lysed in reporter lysis ... Electrophoretic mobility-shift assay (EMSA) Double-stranded DNA probes and competitors were generated by annealing from the following complementary single-stranded oligonucleotides: C/EBP consensus oligonucleotide...
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Báo cáo khoa học: Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1 ppt
... binding and metabolism as well as coupling of the reaction was described previously [9] Modifications of I371, especially I371F, produced a dramatic loss in binding and activity with CA and all ... from spearmint and CYP71D15 from peppermint for the synthesis of carvone and menthol, respectively [28] In the mammalian CYP2B family, residues 294 and 363 are equivalent as N302 and I371, respectively ... residues (P360 and P364) The resulting topology orients three leucine side chains toward the active site (L358, L359 and L363) In P450CAM [46] and P450TERP [47], SRS is a b-strand partially involved...
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Báo cáo khoa học: Metabolism of N-methyl-amide by cytochrome P450s Formation and characterization of highly stable carbinol-amide intermediate pdf
... quantication and isolation of metabolites starting with both natural and 14C-radiolabeled substrates using HPLC; (b) structural identication of metabolites by HPLC MS and MS-MS, and NMR spectroscopy; ... 2.0 to 1.6 and 0.18 for pH values of 6.8, 7.4 and 8.2, respectively Preliminary attempts to determine the structure of M1 and M2 using routine protocols revealed that: (a) isolated M1 and M2 both ... Da and identical MS fragmentation schemes; (b) HPLC analysis of the separated and puried metabolites M1 and M2 led to a single peak corresponding to M2 that ts with the HPLC retention time and...
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Báo cáo khoa học: Cytochrome P450 metabolizing fatty acids in plants: characterization and physiological roles pptx
... [47] and by FA derivatives such as prostaglandins Clofibrate produces similar effects in plants and animals [8,45] Furthermore, there are evident structural analogies between prostaglandins and ... species and acted on distinct substrates in a variety of biochemical pathways EST and genome sequencing projects demonstrated that P450s acting on FAs belonged to multigenic families and analysis ... only one CYP703 [37] CYP704B1 and CYP704B2 belong to a conserved and ancient P450 family in moss and seed plants This suggests that reactions catalysed by CYP703 and by members of the CYP704 subfamily...
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Báo cáo khoa học: The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism pdf
... fungal and bacterial sterol oxidases (CYP51) This branch gave rise to the plant obtusifoliol 14 demethylase (CYP51) and brassinolide hydroxylases (CYP85 and CYP90 families) Microbial P450s and fatty ... animals and fungi, but also occur in certain bacteria Also the well-described mammal prostaglandins and leukotrienes belong to the oxylipin family Although oxylipin synthesis in mammals and plants ... magnitude lower [61] The self-sufficient and soluble properties simplify the enzyme’s overexpression and purification, and turn it into an ideal model for spectroscopic and structural studies The derived...
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Báo cáo khoa học: An alternative pathway of vitamin D2metabolism Cytochrome P450scc (CYP11A1)-mediated conversion to 20-hydroxyvitamin D2and 17,20-dihydroxyvitamin D2 pot
... KCl, mm MgSO4, and 0.2 mm EDTA The reactions were started by adding NADPH (0.5 mm) and isocitrate (5 mm) to the samples, and after 90 mixtures were extracted with methylene chloride and the organic ... rotor was 40 lL, and all spectra were acquired at a temperature of 294 K with a spinning rate of 2500 Hz Proton 1D NMR, COSY and HSQC spectra were acquired and processed with standard parameters ... consisted of 85% methanol and 0.1% acetic acid from to 25 min, followed by a linear gradient to 100% methanol and 0.1% acetic acid from 25 to 35 min, and 100% methanol and 0.1% acetic acid from...
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