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1.
Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a 总被引:8,自引:0,他引:8
D R Koop 《Molecular pharmacology》1986,29(4):399-404
The hydroxylation of p-nitrophenol to 4-nitrocatechol was investigated using rabbit hepatic microsomes and six purified isozymes of cytochrome P-450. The microsomal activity was maximal at pH 6.8 and at 100 microM p-nitrophenol. At higher substrate concentrations inhibition was observed. At pH 6.8 and 100 microM p-nitrophenol, isozyme 3a exhibited the highest activity of the purified isozymes: 3.4-fold more active than isozyme 6, and 8-fold more active than isozymes 2 and 4. The isozyme 3a-catalyzed hydroxylation reaction was stimulated 2.4-fold by the addition of a 4:1 ratio of cytochrome b5/P-450. At optimal concentrations of cytochrome b5, isozyme 3a was 8- to 9-fold more active than isozymes 2 and 6 and 20-fold more active than isozyme 4. Under the same conditions, isozyme 3a-catalyzed butanol oxidation was inhibited 40%. Antibodies to isozyme 3a inhibited greater than 95% of the p-nitrophenol hydroxylase activity of microsomes from untreated or from ethanol- or acetone-treated rabbits. The microsomal hydroxylase activity was linearly correlated with the microsomal concentration of isozyme 3a (correlation coefficient of 0.94) and had an intercept near zero. The results from reconstitution, antibody inhibition, and correlation experiments indicate that isozyme 3a is the principal catalyst of rabbit microsomal p-nitrophenol hydroxylation. The ability of the ethanol-inducible isozyme to catalyze catechol formation may be important in the ethanol-enhanced toxicity of aromatic compounds such as benzene. 相似文献
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3.
Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1) 总被引:15,自引:0,他引:15
The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed. 相似文献
4.
New Zealand White rabbits were treated with rifampicin at a dose of 50 mg/kg for 4 days. The total amount of microsomal hepatic cytochrome P-450 was not modified in treated, with respect to control, animals. However, further studies involving SDS-PAGE analysis, monooxygenase activity measurements and radial immunodiffusion assays indicated that rifampicin strongly affects the level of two P-450 isoenzymes. An LM3 form was induced; this form, apparently associated with erythromycine demethylase activity and hydroxylation of progesterone preferentially in position 6β, was shown to be immunologically and functionally different from LM3a and LM3b. On the other hand, an LM4 form typically induced by β-naphthoflavone, was repressed. The concomitant inductive/repressive effect of rifampicine on two cytochrome P-450 isoenzymes makes this drug a very atypical inducer, at least in the rabbit. 相似文献
5.
P-450-male is a male specific form of cytochrome P-450 in rat liver microsomes. Cytochrome P-450 crossreactive with anti-P-450-male antibodies was purified to an electrophoretical homogeneity from liver microsomes of male beagle dogs. The specific content of the purified cytochrome P-450 (P-450-D1) was 16.9 nmol/mg protein. The apparent monomeric molecular weight of P-450-D1 was 48,000, which was smaller than P-450-male (51,000). P-450-D1 showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities with some limited exceptions: P-450-D1 did not catalyze 2 alpha-hydroxylation of testosterone and progesterone and catalyzed 21-hydroxylation of progesterone. Based on these results, we propose that P-450-D1 is a form of cytochrome P-450 in the same gene subfamily as P-450-male. 相似文献
6.
Y Oda S Imaoka Y Nakahira A Asada M Fujimori S Fujita Y Funae 《Biochemical pharmacology》1989,38(24):4439-4444
The metabolism of lidocaine was studied using rat liver microsomes or a reconstituted lidocaine monooxygenase system with one of eight forms of cytochrome P-450 purified from liver microsomes from untreated- (P450 UT-2 and UT-5), phenobarbital- (P450 PB-1, PB-2, PB-4, and PB-5) or 3-methylcholanthrene- (P450 MC-1 and MC-5) treated rats. A reverse phase high-performance liquid chromatography system capable of simultaneously assaying four major lidocaine metabolites, namely, monoethylglycinexylidide (MEGX), 3-hydroxylidocaine (3-OH LID), methylhydroxylidocaine (Me-OH LID) and glycinexylidide (GX), was employed to determine the rate of formation of each metabolite. Untreated microsomes generated MEGX, Me-OH LID, and 3-OH LID, but the formation of GX was not detected. In male rat liver microsomes, MEGX was the major metabolite of lidocaine when a concentration of 1 mM was employed. The formation of MEGX and Me-OH LID was increased significantly (P less than 0.01) by microsomes from phenobarbital-treated rats, and the formation of 3-OH LID was increased with 3-methylcholanthrene. The study with the reconstituted system with purified cytochrome P-450 isozymes revealed that all eight forms of cytochrome P-450 used have an ability to N-deethylate lidocaine to form MEGX. Among these isozymes, cytochrome P450 PB-4 and P450 UT-2 showed a higher turnover number for the formation of MEGX. Me-OH LID was formed exclusively by P450 PB-5, and 3-OH LID exclusively by P450 MC-1. Selectivity of cytochrome P450 PB-5 for aromatic methyl hydroxylation of lidocaine was confirmed by an inhibition study; formation of Me-OH LID by microsomes of rats treated with phenobarbital was inhibited completely by antibody against P450 PB-5. It was concluded that different cytochrome P-450 isozymes metabolize lidocaine with a different rate and different position selectivities. Since a specific substrate of cytochrome P450 PB-5 (P-450e) is not known, lidocaine may be a useful substrate for the identification of P450 PB-5. 相似文献
7.
A monoclonal antibody raised to rat liver cytochrome P-448 (form C) which recognises an epitope common to many other forms of cytochrome P-450 总被引:1,自引:0,他引:1
A R Boobis J McQuade D Sesardic R T Robson C Hayward E A Lock C R Elcombe M S Rose D S Davies 《Biochemical pharmacology》1985,34(10):1671-1681
A murine monoclonal antibody has been raised against a partially purified preparation of hepatic cytochrome P-448 (form c) from beta-naphthoflavone-treated rats. The monoclonal origin of the antibody was established by limiting dilution culture and isoelectricfocusing. The antibody has been designated 3/4/2. It reacts with apparently homogeneous cytochrome P-448 from rat liver in solid phase assay. It also cross reacts with a number of other cytochromes P-450, from rat and rabbit. In addition, a positive reaction was obtained with microsomal fractions from a variety of species, including man. None of the species tested was negative. The antibody does not react appreciably with purified haemoproteins other than cytochromes P-450. Antibody 3/4/2 is not inhibitory, either in reconstituted systems or with intact microsomal fraction. However, evidence was obtained that the antibody does cause some perturbation of the tertiary structure of the apoprotein at or near the haem. 相似文献
8.
When guinea pigs were treated with phenobarbital (PB), the specific activity of liver microsomal nicotine oxidase increased by 42%. PB-inducible cytochrome P-450 (PB-P-450) was purified to homogeneity from liver microsomes of PB-treated guinea pigs. Purified PB-P-450 catalyzed nicotine oxidation when reconstituted with NADPH-P-450 reductase and phospholipid system. Antibody prepared against the purified PB-P-450 formed single precipitation lines with both purified PB-P-450 and microsomal components in livers of PB-treated guinea pigs, and both precipitation lines fused. The antibody against PB-P-450 strongly inhibited nicotine oxidation in the reconstituted system. The antibody also inhibited liver microsomal nicotine oxidase activities in PB-treated and untreated guinea pigs by about 30% and less than 5% respectively. About 45% of total P-450 in liver microsomes of PB-treated guinea pigs was precipitated by the antibody. These results show that PB-P-450 participates in liver microsomal nicotine oxidation in PB-treated guinea pigs but not in untreated control animals. 相似文献
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Sang S. Park Tadahiko Fujino Haruko Miller Frederick P. Guengerich Harry V. Gelboin 《Biochemical pharmacology》1984,33(13):2071-2081
Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities of PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), β-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-α-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immunoprecipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450. 相似文献
11.
Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat 总被引:10,自引:0,他引:10
S A Wrighton C Campanile P E Thomas S L Maines P B Watkins G Parker G Mendez-Picon M Haniu J E Shively W Levin 《Molecular pharmacology》1986,29(4):405-410
The rat 3-methylcholanthrene-inducible family of liver cytochromes P-450 contains two proteins (P-450c and P-450d) that are immunochemically related, possess 68% total sequence homology, and are induced by a number of toxic or carcinogenic compounds. To determine whether equivalent isozymes of hepatic cytochrome P-450 are expressed in humans, as they are in several mammalian species, we performed immunoblot analyses on microsomes prepared from 14 human liver specimens and found that each one contained a 52.5-kDa protein (termed HLd) that reacted with antibodies specific for rat P-450d. In addition, one specimen contained a 54-kDa protein (termed HLc) that reacted with antibodies specific for rat P-450c. HLd was purified through the use of immunoaffinity chromatography and was found to be 56% homologous to rat P-450d and 61% homologous to the equivalent isozyme in the rabbit (P-450 LM4) through their first 18 NH2-terminal amino acids. Finally, levels of immunoreactive HLd varied more than 10-fold among these patients but were unrelated to the patients' drug treatments, smoking habits, or amount of immunoreactive HLp, a human liver cytochrome P-450 related to the glucocorticoid-inducible family of rat cytochromes P-450. We conclude that, in man, there is a cytochrome P-450 family composed of two isozymes (HLc and HLd) that are immunochemically and structurally related to the 3-methylcholanthrene-inducible family observed in several other species. 相似文献
12.
W C Gelderblom P Swart P S Kramer 《Xenobiotica; the fate of foreign compounds in biological systems》1988,18(9):1005-1014
1. The spectral interaction of a mutagenic fungal metabolite, fusarin C, with rat liver microsomal cytochrome P-450 was investigated using a method which determines competitive inhibition between substrates eliciting the same type of spectral change. The strong u.v. absorption of fusarin C in the region where the spectral changes of cytochrome P-450 are monitored prevented direct binding studies. 2. The conversion of fusarin C to fusarin PM1 by the microsomal carboxylesterase was effectively inhibited by either decreasing the temperature during the binding studies or by addition of NaF (20 mM) during the enzymic inhibition investigations. 3. Fusarin C competitively inhibited the binding of the type II substrate aniline, yet the enzymic hydroxylation reaction of aniline was inhibited in a non-competitive manner. 4. Although the C-13/C-14 epoxide group of fusarin C is necessary for mutagenicity, an additional metabolic step is required. The present data indicated that fusarin C may interact with cytochrome P-450 in a similar way to aniline. 相似文献
13.
A W Wright D J Winzor P E Reilly 《Xenobiotica; the fate of foreign compounds in biological systems》1991,21(2):193-203
1. Cimetidine pretreatment of male Sprague-Dawley rats caused a significant increase in the specific content of total hepatic cytochrome P-450, supporting the hypothesis that this H2-receptor antagonist has monooxygenase induction effects. 2. Quantitative ultrastructural studies of liver of cimetidine-pretreated animals also supported this hypothesis in showing a significant proliferation of smooth endoplasmic reticulum. These ultrastructural changes were qualitatively similar to those produced by treatment of rats with phenobarbital, a well-characterized monooxygenase-inducing agent whose effects were studied for comparative purposes. 3. Competitive inhibition of metoprolol alpha-hydroxylation by cimetidine in liver microsomes prepared from untreated animals (Ki = 18.8 microM) was also demonstrated. 4. These results allowed testing of the hypothesis (Burnet et al. 1986) that inhibition of a defined monooxygenase should lead to induction of the synthesis of the relevant cytochrome P-450 isozyme. 5. The finding that metoprolol alpha-hydroxylase activity of liver microsomes was lowered, not elevated, by pretreatment of animals with cimetidine argues against the concept of a causal link between monooxygenase inhibition and induction. 相似文献
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Monoclonal antibodies against human liver cytochrome P-450 总被引:7,自引:0,他引:7
Monoclonal hybridomas which produce antibodies against human liver microsomal cytochrome P-450 were developed. Three similar hybridomas produced antibodies which recognized an epitope specific to a family of human P-450 isozymes (P-450(5)). This epitope was also present on cytochrome P-450 PCN-E (pregnenolone-16 alpha-carbonitrile induced) from rat liver microsomes, but this isozyme differed from the human P-450(5) by its molecular weight. These antibodies enabled us to quantify cytochrome P-450(5) in human liver microsomes and to demonstrate an important quantitative polymorphism in the human liver monooxygenase system. 相似文献
16.
J R Foster C R Elcombe A R Boobis D S Davies D Sesardic J McQuade R T Robson C Hayward E A Lock 《Biochemical pharmacology》1986,35(24):4543-4554
The cellular distribution of cytochrome P-450 has been studied in the liver and a number of extrahepatic tissues in the rat by immunocytochemistry, using an antibody raised against cytochrome P-450 form c. Immunoreactive cytochrome P-450, most probably form c, was found in the proximal tubules of the kidney, in the Clara cells of the lung, and in the olfactory epithelium and Bowman's glands of the olfactory tissue, in addition to its location in the liver. Immunoreactive cytochrome P-450 was not found in the small intestine, the testes or the adrenal gland, although these organs are known to contain isoenzymes of cytochrome P-450. The use of antibody titration enabled the effects of phenobarbitone, beta-naphthoflavone and clofibrate on the content and distribution of immunoreactive cytochrome P-450 to be studied in both the liver and in the other organs discussed. Phenobarbitone induces epitope-specific cytochrome P-450 in the centrilobular cells of the liver but has no effect in any of the other tissues studied. Clofibrate is without effect on the levels of immunoreactive cytochrome P-450 in any of the tissues studied. In contrast, beta-naphthoflavone induces immunoreactive cytochrome P-450 in the periportal region of the liver, and also in the Clara cells of the lung, in the enterocytes of the small intestine and in the proximal tubules of the kidney. Of all of the tissues studied, in which immunoreactive cytochrome P-450 could be detected, only the olfactory epithelium failed to undergo enzyme induction following treatment with beta-naphthoflavone. 相似文献
17.
Immunoblot analysis of liver microsomes from nine patients demonstrated that each contained a cytochrome P-450 that reacted with an antibody directed against the ethanol-inducible rabbit liver cytochrome, P-450 3a. Two of the liver specimens exhibited high concentrations of the immunoreactive protein, high rates of aniline hydroxylation and N-nitrosodimethylamine demethylation, and extensive inhibition of activity in the presence of antibody to P-450 3a. One other liver specimen exhibited a very low rate of aniline hydroxylation with significantly less antibody inhibition. The variability witnessed was independent of the alcohol history of the individual patients, suggesting that the human cytochrome may be under some other environmental, dietary or genetic regulation. Its inducibility by ethanol was not directly studied in this investigation. However, we conclude that there is a cytochrome P-450 present in human liver which is immunochemically and catalytically similar to the ethanol-inducible P-450 of rabbit liver. 相似文献
18.
Species-dependent expression and induction of homologues of rabbit cytochrome P-450 isozyme 5 in liver and lung 总被引:2,自引:0,他引:2
The presence of homologues of rabbit cytochrome P-450 isozyme 5 in pulmonary and hepatic microsomal preparations from guinea pig, mouse, monkey, hamster, and rat was examined by immunoblotting and inhibition of metabolism of 2-aminofluorene with antibodies to isozyme 5. Homologues to isozyme 5 were detected in pulmonary preparations from all five species. However, only hepatic preparations from hamster, in addition to those from rabbit, contained detectable levels of this isozyme. With the exception of induction by phenobarbital in rabbit liver, treatment of animals with phenobarbital or tetrachlorodibenzo-p-dioxin did not increase hepatic or pulmonary content of isozyme 5 homologues or the amount of 2-aminofluorene metabolism inhibited by antibodies to isozyme 5. Metabolism of 2-aminofluorene was measured both colorimetrically (formation of a reduced iron chelate from the N-hydroxyfluorene metabolite) and radiochemically (separation of 3H-metabolites by high performance liquid chromatography and quantitation by scintillation counting). A turnover number of 48 nmol of product X min-1 X nmol of enzyme-1 for isozyme 5-catalyzed metabolism of 2-aminofluorene was determined with incubations containing isozyme 5 purified from rabbit lung. A similar turnover number was calculated from the rabbit hepatic microsomal activity inhibited by antibodies to isozyme 5 and the microsomal isozyme 5 content measured by immunoquantitation. In other species, amounts of metabolism inhibited by antibodies to isozyme 5 agreed qualitatively with relative staining intensities on immunoblots. In all species except the hamster, rates of total and isozyme 5-catalyzed metabolism of 2-aminofluorene were greater with pulmonary than with hepatic microsomal preparations from untreated animals. 相似文献
19.
To produce potent, isozyme-selective suicide inhibitors of cytochrome P-450 (P-450), a series of N-alkylated 1-aminobenzotriazole (ABT) derivatives was synthesized; these included the N-methyl, N-butyl (BuBT), N-benzyl (BBT), and N-alpha-methylbenzyl (alpha MB) analogues of ABT. The suicide inhibitors showing the greatest potency and isozyme selectivity were BBT and alpha MB, compounds which included molecular features for P-450 inactivation (the ABT moiety) and similarity to benzphetamine. ABT and its N-alkylated derivatives were tested as suicide inhibitors in rabbit lung microsomes, whose P-450 monooxygenase system has been well characterized in both untreated and beta-naphthoflavone- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated animals. ABT (10 mM) destroyed up to 99% of the total P-450 content of lung microsomes of untreated rabbits. At equimolar concentrations (10 microM), ABT was less effective than the N-alkylated compounds for the inhibition of P-450 isozyme 2-catalyzed benzphetamine N-demethylation (BND); in fact, BuBT, BBT, and alpha MB completely inhibited BND activity at this concentration and destroyed less than 40% of total pulmonary P-450. However, these compounds also inactivated 69-85% of isozyme 6-catalyzed 7-ethoxyresorufin O-deethylation. The most potent and isozyme-selective suicide inhibitor prepared was alpha MB: at 1 microM this compound inhibited approximately 80% of isozyme 2-catalyzed and 20% of isozyme 6-catalyzed monooxygenase activity but spared P-450 isozyme 5; at 2.5 microM it caused a near-complete loss (96 +/- 2%) of BND activity. The partition ratio of alpha MB, i.e., the molar ratio of inhibitor present to that of the P-450 destroyed, was 11 +/- 2, further demonstrating the potency of this compound. Experiments with BBT- and sodium phenobarbital-treated rats showed that the mechanism for suicidal inactivation of P-450 by this N-alkylated compound was by benzyne release, the same mechanism demonstrated earlier for the parent compound ABT. 相似文献