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Expression of house fly cytochrome P-450lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450lpr was expressed in both male and female adult house flies. P-450lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450lpr.  相似文献   

3.
D. melanogaster development was markedly retarded and its survival decreased by larvae treatment with compounds being strong inducers of the cytochrome P-450 2B in mammals— phenobarbital (PB*), perfluorodecaline (PFD), transstilbene oxide (TSO), and triphenyldioxane (TPD). At the same time, the weak inducer hexobarbital or the selective cytochrome P-450 inducer in mice but not in rats 1,4-bis[2-(dichloropyridyl-oxy)]-benzene (DPB) did not affect the larvae development. The cytochrome P-450 1A1 inducers benzo(a)anthracene (BA) and β-naphtoflavone (BNF) were also not effective. The toxicity of phenobarbital was shown to be decreased by the cytochrome P-450 inhibitor piperonyl butoxide by adding 20-hydroxyecdysone or by treatment with aminophylline—the indirect enhancer of ecdysone production in the larval prothoracic gland. The hypothesis of the moulting hormone degradation as the cause of elevated larvae mortality resulting from the induced high mixed function oxidase activity has been discussed.  相似文献   

4.
Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO2=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO2 of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.Abbreviation cyt cytochrome  相似文献   

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The cytochrome P-450 (P-450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P-450b,e and P-450c,d to brain P-450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P-450b and P-450c, respectively). P-450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p-chloroamphetamine-coupled Sepharose. The yield of P-450 from whole brain was 90 +/- 19 pmol/g of tissue, which is approximately 1% of the level in liver microsomes from control rats. The amount of P-450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P-450 content, with yields of up to 400 pmol/g whereas the substantia nigra yielded only 16-20 pmol/g. Immunohistochemical studies with anti-P-450b and anti-P-450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti-P-450c. In the cerebellum, both anti-P-450b and anti-P-450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti-P-450b gave weak staining of certain astroglia, but with anti-P-450c, there was intense staining of neuronal somata.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
Bentazon and sulfonylureas have been used for selective control of broadleaf weeds and sedges in rice fields for more than 20 years. A bentazon and sulfonylurea susceptible mutant, bel, was previously identified for the purpose of allowing these herbicides to be used for removing false hybrids from hybrid rice. While this mutation has been used successfully in rice breeding, the genetic nature of bel is not known. Using 1,776 susceptible plants from a population of 10,000 F2 individuals, we constructed a fine map for the Bel locus and delimited it to a 36-kb DNA fragment between two restriction fragment length polymorphism markers, L5 and P17. Bioinformatic analysis indicated that there are five genes within this interval, an ethylene-responsive OsER33 gene and four tandem repeats of cytochrome P450 genes designated as CYP81A5, CYP81A6, CYP81A7, and CYP81A8. Comparative sequencing could not find any differences in the coding regions of the OsER33, CYP81A5, CYP81A7, and CYP81A8 genes between the mutant bel and its wild-type progenitor W6154S, but did identify a single base guanine deletion at position +1,332 bp downstream from the translation start codon of CYP81A6. This deletion introduces a premature stop codon and leads to the loss of the heme-binding motif, which is essential for cytochrome P450 function because it contains an absolutely conserved cysteine that serves as the fifth ligand to the heme iron. CYP81A6 presumably functions as a hydroxylase for the detoxification of bentazon and sulfonylurea herbicides in rice. A gene-specific cleaved amplified polymorphic sequence marker and tightly linked flanking markers were developed that will be very useful for selection of the bel allele when transferred to photoperiod-/thermo-sensitive genic male sterility and CMS lines in hybrid rice breeding programs.  相似文献   

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The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.  相似文献   

10.
The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.  相似文献   

11.
Indirect evidence of the participation of cytochrome P-450 (P-450) in the microsomal N-oxygenation of secondary and tertiary nitrogen functions is presented by studies employing diagnostic modifiers of the hemoprotein system as well as antibodies directed toward the diverse P-450 isoforms and NADPH-cytochrome P-450 reductase. Experiments with recombinant hemoproteins or P-450 isozymes directly purified from the tissues of various animal species support the results obtained by the inhibitor assays. Although the intermediacy of aminium radicals is thought to be restrictive to P-450-catalyzed N-oxygenation of secondary and tertiary amine groups bearing accessible hydrogens on the α-carbon, numerous exceptions to this rule are documented. It is proposed that aminium radicals partition between oxygen rebound and α-hydrogen abstraction to yield a finite level of N-oxygenated product in all P-450-mediated amine oxidations, the partition ratio depending on the amine structure and particular P-450 isozyme operative. In some instances, N-oxygenation appears to proceed by peroxidatic mechanisms. The relative contribution of P-450 to the N-oxygenation of secondary and tertiary amines in crude preparations or live animals, where competition with the flavin-containing monooxygenase (FMO) occurs, seems to be a function of the relative amounts and catalytic capacities of the two enzyme systems. Both parameters are species and tissue dependent. Accordingly, the extent to which P-450 contributes to total N-oxidative turnover of the amine substrates varies from minor to major. © 1996 John Wiley & Sons, Inc.  相似文献   

12.
NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's Vmax values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.  相似文献   

13.
The cytochrome P-450 family of enzymes is the primary means of foreign compound detoxification in virtually all organisms. Cytochrome P-450s have been strongly implicated in the metabolism of cactus alkaloids, and consequently, the observed patterns of host plant utilization by cactophilic species of Drosophila in the Sonoran Desert. The current study looked for evidence of alkaloid-metabolizing P-450 enzymes in a non-cactophilic species, D. melanogaster. The results of in vitro metabolism assays indicate the presence of a phenobarbital-inducible P-450 in adult D. melanogaster which is capable of metabolizing alkaloids. P-450 quantification data suggest that the enhanced level of metabolism is not the result of an overall increase in total P-450 content. Results from larval viability and adult longevity studies indicate that D. melanogaster's in vitro activity does not produce an enhanced in vivo tolerance of alkaloids.  相似文献   

14.
K N Myasoedova  P Berndt 《FEBS letters》1990,270(1-2):177-180
Subunit interactions in the purified hexameric cytochrome P-450LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.  相似文献   

15.
Summary The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda+ isolates) hybridized to PdaT9, while only one Pda isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6–2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.  相似文献   

16.
B K Fung  H K Yamane  I M Ota  S Clarke 《FEBS letters》1990,260(2):313-317
Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apoand reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.  相似文献   

17.
Molecular dimensions and molecular orbital calculations of the electronic structures of 56 substrates, inhibitors and inducers of the cytochromes P-448 and other families of the cytochromes P-450 are reported. Substrates of the cytochromes P-448 are shown to be planar molecules with relatively large values of area/depth2, and to have electronic structures with relatively low values for ΔE, the difference in energy between the frontier orbitals (E(LEMO) − E(HOMO)). Substrates of other families of the cytochromes P-450 are globular molecules, with relatively low values of area/depth2 and relatively high values of ΔE. Molecular orbital calculations of the active oxygen species, singlet oxygen and superoxy anion, have also been made. Singlet oxygen is a poor electron donor (low values of E(HOMO)) but a good electron acceptor (low values of E(LEMO)), whereas superoxy anion is a good electron donor and a poor electron acceptor. Cytochrome P-448 substrates, which are good electron donors, would preferentially accept singlet oxygen, a good electron acceptor; substrates of the other families of cytochrome P-450, which are less effective electron donors, would preferentially accept superoxy anion, a good electron donor, although substrates of both cytochromes P-448 and other P-450s may accept both species of active oxygen. Together with recent published evidence, these data provide a greater understanding of the mode of activation of oxygen by the various families of the cytochromes P-450, and to the insertion of active oxygen into the substrates. Mechanisms are proposed for the oxygenation of substrates, namely, epoxidation involving singlet oxygen and hydroxylation by superoxy anion. Finally, a detailed explanation of the cytochrome P-450 cycle is discussed, and mechanisms of the different types of oxidative metabolism are presented.  相似文献   

18.
Flavonoids represent a group of phytochemicals exhibiting a wide range of biological activities arising mainly from their antioxidant properties and ability to modulate several enzymes or cell receptors. Flavonoids have been recognized to exert anti-bacterial and anti-viral activity, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, hepatoprotective, cytostatic, apoptotic, estrogenic and anti-estrogenic properties. However, not all flavonoids and their actions are necessarily beneficial. Some flavonoids have mutagenic and/or prooxidant effects and can also interfere with essential biochemical pathways. Among the proteins that interact with flavonoids, cytochromes P450 (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens) and endogenous substrates (e.g. steroids), play a prominent role. Flavonoid compounds influence these enzymes in several ways: flavonoids induce the expression of several CYPs and modulate (inhibit or stimulate) their metabolic activity. In addition, some CYPs participate in metabolism of flavonoids. Flavonoids enhance activation of carcinogens and/or influence the metabolism of drugs via induction of specific CYPs. On the other hand, inhibition of CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP-mediated reactions can be beneficial properties of various flavonoids. Flavonoids show an estrogenic or anti-estrogenic activity owing to the structural similarity with the estrogen skeleton. Mimicking natural estrogens, they bind to estrogen receptor and modulate its activity. They also block CYP19, a crucial enzyme involved in estrogen biosynthesis. Flavonoids in human diet may reduce the risk of various cancers, especially hormone-dependent breast and prostate cancers, as well preventing menopausal symptoms. For these reasons the structure-function relationship of flavonoids is extensively studied to provide an inspiration for a rational drug and/or chemopreventive agent design of future pharmaceuticals.  相似文献   

19.
The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-14C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (–)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of 18O2, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.Abbreviations CD circular dichroism - DMF N,N-dimethyl-formamide - GC/MS gas chromatography/mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - ODS octadecylsilane - PAH polycyclic aromatic hydrocarbon - TLC thin-layer chromatography - TMS tetramethylsilane - UV ultraviolet  相似文献   

20.
Cunninghamella elegans degraded tributyltin (TBT) at 20 mg l–1 when grown in Sabouraud medium. Above this concentration, growth was inhibited. After 7 d 70% TBT (added at 10 mg l–1) was converted to less toxic derivatives: dibutyltin and monobutyltin. TBT metabolism was totally blocked by cytochrome P-450 inhibitors, metyrapone and proadifen. Only in medium with 1-aminobenzotriazole, was dibutyltin (0.42 mg l–1) found after 7 d of culturing. It is postulated that the significant resistance of C. elegans to TBT is associated with the capacity of the fungus to metabolise TBT.  相似文献   

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