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1.
The role of cytochrome b5 in the p-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome b5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, and Triton X-100 in addition to cytochrome b5. The omission of cytochrome b5 from the complete system entirely abolished the activity. These results clearly show that cytochrome b5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome b5 in such a way that the second electron is transferred from cytochrome b5 and thus exhibits the demethylase activity.  相似文献   

2.
Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl-iso-propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and p-nitroanisole demethylase. In contrast, ethylmorpnine demethylase, benzpyrene-3-hydroxylase and ethoxyresofurin deethylase were not in all cases decreased in proportion to the loss of cytochrome P-450. After phenobarbital induction fluroxene and allyl-iso-propylacetamide degrade multiple forms of cytochrome P-450, but degrade in the greatest amounts the form(s) of cytochrome P-450 inducible by phenobarbital. After 3-methylcholanthrene induction fluroxene preferentially degrades cytochrome P-448, while allyl-iso-propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital.  相似文献   

3.
Total liver RNA has been isolated from male rats at different time points subsequent to a single injection of phenobarbital, and the level of cytochrome P-450 synthesis directed by these RNA preparations in a cell-free translation system has been determined. It is observed that the maximum in vitro synthesis of cytochrome P-450 occurs at 16 hours (3-fold above uninduced level) which is approximately 30 hours prior to the maximum induction of spectrophotometrically detectable cytochrome P-450 measured in liver homogenates. Thus, while cytochrome P-450 mRNA is involved in the induction process, its synthesis does not appear to be rate limiting. In addition, phenobarbital induced cytochrome P-450 is not synthesized in vitro in a form larger than that isolated from endoplasmic reticulum, but rather is also found to have a molecular weight of 50,000.  相似文献   

4.
The phenomenon of induction of the NADPH-specific hydroxylase system by sodium phenobarbital was used to determine the content of cytochrome b5 in each microsomal electron-transfer chain. It turned out that the specific activities of NADPH-dependent reductases and the cytochrome P-450 quantity were increased approximately 1.86 times and the activities of NADH-dependent reductases were somewhat decreased (0.89 times) in microsomes of induced rats. It is assumed that a subfraction of cytochrome b5 included in the NADPH-oxygenase complex is induced together with the other carriers of the chain. The second subfraction of the hemoprotein, in the course of induction, behaves as a typical component of the NADH-oxidizing complex. On the basis of the data obtained, the calculation was made, which showed that the NADPH-oxidation chain contains from 15 to 13 of the total microsomal cytochrome b5 pool.  相似文献   

5.
The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome P-450 was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome P-450 in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome P-450 content, of the presence of type I and type II substrates for cytochrome P-450. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome P-450. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome P-450 was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance (τR > 10?8s). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.  相似文献   

6.
In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome b5 or NADPH-cytochrome c reductase in vitro.  相似文献   

7.
Eight methylenedioxyphenyl (MDP) compounds were examined for their ability to induce cytochrome P450 (P450) in mouse liver. Induction by safrole, isosafrole, and dihydrosafrole was studied in both C57BL/6N (Ah-responsive) and DBA/2N (Ahnonresponsive) male mice after IP administration of 200 mg/kg/day MDP compound for 3 days. Hepatic P450 content, ethylmorphine N-demethylase, ethoxy-resorufin O-deethylase, and acetanilide hydroxylase activities were induced to the same extent in both strains of mice. Benzo(a)pyrene hydroxylase activity, however, was not induced in either C57 or DBA mice. The similarity of results in both strains of mice indicated induction of these P450 isozymes by these three MDP compounds is not mediated by the Ah receptor. Induction of P450 by butylbenzodioxole (n-butyl-BD), tertiarybutylbenzodioxole (t-butyl-BD), methylbenzodioxole (methyl-BD), nitrobenzodioxole (nitro-BD), and bromobenzodioxole (bromo-BD) was examined only in C57BL/6N mice. Methyl-BD, nitro-BD, and bromo-BD did not induce hepatic microsomal proteins or selected P450 monooxygenase activities. In contrast, n-butyl-BD, and t-butyl-BD induced P450 content, ethylmorphine N-demethylase, acetanilide hydroxylase, and ethoxyresorufin O-deethylase activities. Benzo(a)pyrene hydroxylase was not induced by any of the treatments. Induction of these P450 activities is consistent with induction of P450 IIB1 and P450 IA2, but not induction of P450 IA1. Western blot analysis with antibodies to P450 isozymes induced with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC) confirmed that both IIB1 and IA2 were induced, but that IA1 was not induced.  相似文献   

8.
Purified cytochrome P450SCC from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of P450SCC into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When P450SCC was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the P450SCC-containing liposomes showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, P450SCC was less stable than P450SCC in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal P450SCC. Liposomal P450SCC required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal P450SCC was subjected to p-chloromercuriphenyl sulfonic acid treatment. This reagent destroyed the liposomal P450SCC. These results suggest that the heme is located in the proximity of the p-chloromercuriphenyl sulfonic acid reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.  相似文献   

9.
Screening for new microbial cytochromes P450 to create a useful pool of oxygenating biocatalysts can be facilitated by applying multivalent inducers for it. The broad inducer spectrum for cytochrome P450 of Acinetobacter calcoaceticus EB 104 has been analyzed as a model for structure-function relationships governing the inducer recognition of hydrocarbon-oxidizing microbial cytochromes P450 with the aim to deduce structural requirements for the desired multivalent inducer. The cytochrome was induced by all compounds of adequate hydrophobicity that contain coplanar structural elements. It was concluded that due to the inherent low specificity of hydrophobic binding forces some few simple-structured hydrocarbons with a sterically flexible backbone can induce a variety of different hydrocarbon-oxidizing microbial cytochromes P450. This approach has been proved to be successfull by induction of cytochrome P450 with the help of n-hexane in Bacillus megaterium, in various strains of the genus Rhodococcus and in the yeast Candida apicola. Cytochrome P450 of Rhodococcus rhodochrous IMET 7278, that has partially been purified from n-hexane-induced cells, exhibited an induction profile differing to P450 of Acinetobacter.  相似文献   

10.
Several rate constants for one-electron reduction of cytochrome P450 are more rapid in the absence than in the presence of the specific substrate. The respective values for methyl viologen, nicotinamide adenine dinucleotide and the 1-methyl-4-(and -3-)carbamidopyridinium radicals are 2.6, 3.4, 6 and 35 × 107 M?1 s?1 without camphor, and 0.15, 0.1, 1.8 and 110 × 107 M?1 s?1 for the camphor complex. Hydrated electrons react with cytochrome P450 with a rate constant of 3.0 × 1010 M?1 s?1 whether camphor is bound or not, but little of the reduction takes place at the haem iron. No reduction of the haem iron by CO2?- or O2?- could be detected, whether camphor is bound or not.  相似文献   

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