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1.
Soluble cytochrome P-450 prepared from the microsomal fraction of abdomen homogenates of an insecticide resistant strain (Rutgers) and a susceptible strain (NAIDM) of the house fly, Musca domestica L., was characterized by spectral and electrophoretic methods. Six chromatographically distinct fractions were obtained after chromatography on DEAE-cellulose and hydroxylapatite. Examination of the six fractions by difference spectrophotometry indicated that the wave lengths for maximum absorption of the cytochrome P-450-carbon monoxide complexes were at 450, 451, and 452 nm for the NAIDM fractions and at 449, 450, and 451 nm for the Rutgers fractions. The type II binding spectra of the cytochrome P-450 in each fraction were measured with n-octylamine. Several of these resembled spectra which, in studies of hepatic cytochrome P-450, have been shown to be due to the presence of the high spin form of this hemoprotein. Four of the fractions from the resistant strain were of this type compared to one from the susceptible strain. Electrophoresis experiments indicated that there were at least three hemoproteins in the 40,000–60,000 molecular weight range in the fractions from the resistant strain while four could be detected in those from the susceptible strain. The specific aldrin epoxidase activity of the most active Rutgers fractions was considerably higher than that of similar fractions from the NAIDM microsomes in reconstitution experiments.  相似文献   

2.
Microsomes prepared from the abdomens of the flesh fly (Sarcophaga bullata, Parker) and the blow fly (Phormia regina (Meigen)) contain approximately one-fifth and one-eighth as much cytochrome P-450, respectively, as those prepared from house fly (Musca domestica, L.) abdomens. These values correlate well with the microsomal aldrin epoxidase activity of the three species and with their respective susceptibilities to the insecticide, propoxur. When the microsomes of the flesh fly and the blow fly are solubilized by treatment with deoxycholate and resolved by ion-exchange chromatography on DEAE-cellulose and hydroxylapatite, four chromatographically distinct fractions containing cytochrome P-450 are obtained. Spectrophotometric assays of the cytochrome P-450 in these fractions indicate purifications of two-to sixfold for the flesh fly hemoprotein and two-to eightfold for that of the blow fly. SDS-Polyacrylamide gel electrophoresis of the four column fractions from the flesh fly microsomes indicates that six hemoproteins in the 40,000–60,000 molecular weight range are present. In similar experiments with blow fly fractions containing approximately the same amount of cytochrome P-450 no high molecular weight hemoproteins could be detected. This result is interpreted, with other evidence, as an indication of the greater instability of the blow fly hemoprotein. The results indicate that multiple forms of cytochrome P-450 are present in both species but there is insufficient data on which to estimate the number of such forms.  相似文献   

3.
The polysubstrate monooxygenases (PSMO or cytochrome P-450) of house fly larvae were studied at the mature larval or “clear gut” stage. Fat body and gut tissues were most efficient in the conversion of aldrin to dieldrin. Microsomal fractions of larval homogenates had the highest PSMO activities, with lower PSMO activities also found associated with mitochondrial fractions. Microsomes from Rutgers (resistant) larvae had higher levels of NADPH:cytochrome c reductase (2×), cytochrome P-450 (2×), aldrin (4×), and heptachlor (9×) epoxidases than microsomes from CSMA (susceptible) larvae. Cytochrome P-450 of Rutgers larvae had an absorption maximum at 449 nm, 2 nm lower than the cytochrome P-450 of CSMA larvae. n-Octylamine spectra showed that the level of high-spin cytochrome P-450 was higher in Rutgers larvae. NADPH:cytochrome c reductase, cytochrome P-450, and aldrin epoxidase were induced by phenobarbital, and Rutgers larvae were shown to be more sensitive to this inducer than CSMA larvae. Induction of larval PSMO by phenobarbital did not affect the expression or the inducibility of PSMO in adults.  相似文献   

4.
Development and phenobarbital (PB) induction of microsomal cytochrome P-450, NADPH-cytochrome c (P-450) reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of female black blow flies (Phormia regina, Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O-demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O-demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P-450 and 5-fold for NADPH-cytochrome c (P-450) reductase. These data suggest that PB induces several P-450's in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.  相似文献   

5.
Development and phenobarbital (PB) induction of microsomal cytochrome P-450, cytochrome P-450 reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P-450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min?1 mg protein?1) in all enzymes returned to control values in 24 hr while metabolic capacity (product min?1 insect?1) achieved control values within 48 hr. The changes in turnover numbers (pmol product min?1 pmol P-450?1), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.  相似文献   

6.
The low mixed-function oxidase activity of house fly microsomes has been associated with low cytochrome P-450 content and NADPH-cytochrome c reductase activity. The microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity could be decreased by the addition of catechol and increased by the addition of cyanide to the homogenates. Similar results were obtained with rat liver microsomes treated with tyrosinase and catechol. During the inactivation of rat liver microsomal enzymes by tyrosinase and catechol, crosslinking of microsomal proteins occurred. These results suggest that the instability of house fly microsomal mixed-function oxidase may be due in part to the action of contaminating tyrosinase on endogenous substrates.  相似文献   

7.
In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl-N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)-N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)-N′-methylurea.  相似文献   

8.
Induction of the microsomal oxidase system by dietary phenobarbital and β-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eucalliphora lilica (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P-450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P-450 and aldrin epoxidase caused by β-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomal oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity.  相似文献   

9.
The fluorescent insect growth regulator 5[[[5-(dimethylamino)-1-naphthalenyl]amino]-1,3-benzodioxole (DNSAB) forms a metabolite complex with house-fly microsomal cytochrome P-450. Formation of the metabolite complex is dependent on the presence of NADPH and O2; NADH supports the reaction at a reduced rate. The presence of antibodies to house-fly cytochrome c (P-450) reductase in reaction mixtures inhibits the complex formation, indicating that the reductase is necessary for transfer of electrons from NADPH to cytochrome P-450 to complete the reaction. In the oxidized form, the metabolite complex has a single absorbance maximum at 431 nm, whereas the reduced form has two absorbance maxima at 426 (major) and 455 nm (minor). The pH of the media affects the extinction of the 426- and 455-nm Soret bands; increased pH decreases the extinction of the 426-nm band and increases the extinction of 455-nm band. Formation of the DNSAB metabolite-cytochrome P-450 complex decreases the amount of CO-reactive cytochrome P-450 by 24%. The metabolite complex is not dissociable by treatment with ferricyanide or by using centrifugation techniques. Dissociation is accomplished by addition of DNSAB to the oxidized metabolite complex. Kinetic analysis of the complex formation gives apparent Km and Vmax values at 2.55 ± 1.0 μM and 1.1 ± 0.4 × 10?2 ΔA min?1 nmol?1 cytochrome P-450, respectively. Addition of juvenile hormone [(E,E)-cis-methyl-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate; JH] to the reaction medium competitively inhibits the formation of the metabolite complex giving an inhibition constant of 16 μM. DNSAB synergized the lethal effects of JH against Aedes aegypti larvae threefold; however, JH did not synergize DNSAB. These data suggest that DNSAB may acquire its hormonal qualities by complexing a species of cytochrome P-450 that metabolizes JH, thereby prolonging the in vivo lifetime of this hormone.  相似文献   

10.
Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b5, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).  相似文献   

11.
The interaction of chlordecone (decachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]-pentalene-2-one, Kepone) with hepatic microsomal cytochrome P-450 was studied. Chlordecone binds to cytochrome P-450 to produce a Type I spectral change the magnitude of which is dependent upon the chemical pretreatment of the animal. In kinetic studies of chlordecone was found to be a competitive inhibitor of aminopyrine N-demethylase and p-nitroanisole O-demethylase and a noncompetitive inhibitor of aniline p-hydroxylase.  相似文献   

12.
Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b5, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the 430455-nm peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.  相似文献   

13.
The effects of chlordecone treatment on the hepatic microsomal monooxygenase system of male rats were investigated. Chlordecone increased the microsomal content of cytochrome P-450, NADPH-cytochrome P-450 (c) reductase and, to a lesser extent, cytochrome b5 in a time- and dose-dependent manner. The content of NADH-cytochrome b5 (c) reductase was reduced. The turnover of seven substrates was studied in detail and, with the exception of aniline, was found to be increased between 1.3- and 2.2-fold. The apparent Km's for these substrates were increased 2.1- to 16.7-fold. In addition, zoxazolamine paralysis time was reduced as a result of chlordecone treatment. These kinetic changes are explained on the basis of alterations in the cytochrome P-450 pool together with residual chlordecone acting as an inhibitor of substrate turnover. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein pattern of microsomes isolated from chlordecone-treated rats more closely resembled that of microsomes isolated from untreated rats than that of microsomes isolated following phenobarbital or 3-methylcholanthrene treatment.  相似文献   

14.
A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a Vmax of 7.19 mol/mol P-450/min and a Km of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of O2CO2 onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/106 cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a Vmax of 5.11 mol/mol P-450/min and a Km of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.  相似文献   

15.
The capacity of several methylenedioxyphenyl insecticide synergists to generate metabolite complexes with cytochrome P-450 was studied in midgut tissues of the Southern armyworm (Spodoptera eridania). Examination of the NADH-reduced versus oxidized spectra from methylene-dioxyphenyl-induced midgut indicated that isosafrole, dihydrosafrole, and 4-ethoxy-1,2-methylenedioxybenzene generated metabolite complexes with a principal absorbance maximum at 427 nm and smaller absorbance maxima near 460 and 556 nm. Further studies with 2-n-heptylbenzimidazole showed that the complex between insect cytochrome P-450 and dihydrosafrole was unusually resistant to displacement. Initial rates of complex displacement in insect microsomes were found to be approximately an order of magnitude slower than those of the corresponding complexes in rat hepatic microsomes. Nevertheless, with the exception of the dihydrosafrole complex in insect microsomes, the “time to half-maximal displacement” parameter was found to be very similar for each complex. These findings indicate that the formation of dissociable complexes between cytochrome P-450 and the methylenedioxyphenyl metabolite occurs in both insect midgut and rat hepatic microsomes after in vivo exposure. From the present study it would appear that dihydrosafrole may constitute a useful probe to distinguish binding sites within insect and mammalian cytochrome P-450.  相似文献   

16.
Cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in the postmitochondrial centrifugal fraction, microsomes, and supernatant of rat liver, lungs, kidneys, and testes. Liver microsomes contained the highest P-450 levels and A-esterase activity. B-esterase activity was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in rat lung and lowest in the liver (per mg protein). Glutathione S-aryl transferase activity was highest in the liver. The in vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed many differences. Carbaryl metabolism was greater in the liver microsomal fractions than in any other preparation. 1-Naphthol was the major metabolite in all tissue fractions. Although very little metabolism of phosphamidon occurred in the rat, metabolism in the rat liver postmitochondrial fraction was slightly higher with respect to the production of metabolites than in the supernatant and microsomes combined. Chlorotoluron was not metabolized by any of the tissue fractions of the rat. At least a low level of activity toward some compounds was observed in all tissues, but this study confirmed that the liver was the most active metabolizing tissue as well as having the highest levels of enzymatic activity usually associated with pesticide metabolism.  相似文献   

17.
The spectral interactions of 45 insecticide synergists and related compounds with oxidized and reduced cytochrome P-450 from microsomes of insecticide-resistant and -susceptible house flies were investigated. The type III interaction typical of piperonyl butoxide was the most common spectral interaction for the compounds studied. In addition to this, several other varients of the type III interaction were noted. In general these responses with house fly microsomes were similar to those reported for mammals, although some minor species and strain differences were observed. The cytochrome P-450 from susceptible house flies, although reported previously not to exhibit type I difference spectra with many xenobiotics, was found to elicit this spectral response with several methylenedioxyphenyl compounds.  相似文献   

18.
Aldrin epoxidase of the earthworm, Lumbricus terrestris L., has been shown to occur mainly in the intestine and seemed to increase in activity with the development of the animal. The microsomal fraction was identified by electron microscopy as the locus of the epoxidase. Although sesamex was not inhibitory, inhibition of expoxidase by carbon monoxide suggested the involvement of cytochrome P-450. However, the carbon monoxide difference spectrum of the microsomes was dominated by a material which was spectroscopically identical to earthworm hemoglobin.  相似文献   

19.
An active microsomal oxidase system was found in various tissues of the mole cricket adults, Scapteriscus acletus Rehn and Hebard and Scapteriscus vicinus Scudder. Aldrin epoxidase activity was mainly located in the midgut and Malpighian tubules. p-Nitroanisole O-demethylase and p-chloro-N-methylaniline N-demethylase activities were also detected in these two tissues. The overall oxidase activities in the two species were generally similar. Seasonal variation in epoxidase activity was evident in the two species with the spring populations having more activity than the fall populations. Spectral characterization of the cytochrome P-450 revealed that the enzyme was not of “resistant type” in both species. S. vicinus was four- to fivefold more susceptible to the insecticide propoxur than S. acletus.  相似文献   

20.
The midgut microsomal aldrin epoxidase of variegated cutworm larvae (Peridroma saucia, Hübner) fed bean or peppermint leaves was up to 10 and 45 times more active, respectively, than that of larvae fed a basic control diet. Large increases in oxidase activity and cytochrome P-450 levels also occurred in larvae fed mint plant constituents such as menthol menthone, α-pinene, and β-pinene. Mint-fed larvae were more tolerant of the insecticide, carbaryl, than bean-fed larvae.  相似文献   

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