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1.
Hydroxylation of dibenzo- p-dioxin (DD), dibenzofuran (DF), biphenyl (BP) and diphenyl ether (DPE) by the white-rot fungus Phlebia lindtneri GB-1027 was studied. DD and DF were rapidly degraded in a culture of P. lindtneri. The initial oxidation products were identified by gas chromatography-mass spectrometry. P. lindtneri oxidized DD to 2-hydroxy-DD, and DF to 2- and 3-hydroxy-DF. BP and DPE were also oxidized to p-hydroxy-BP and p-hydroxy-DPE, respectively. The oxidation catalyzed by P. lindtneri with each substrate was position-specific, because the hydroxyl group was introduced to the molecular edge of every substrate. Significant inhibition of the degradation of DD and DF was observed in incubation with the cytochrome P-450 monooxygenase inhibitors 1-aminobenzotriazole and piperonyl butoxide. These experiments with cytochrome P-450 inhibitors, and formation of the mono-hydroxyl metabolites suggest that P. lindtneri initially oxidizes DD, DF, BP, and DPE by a cytochrome P-450 monooxygenase and that it directly introduces a hydroxyl group to each of these substrates. 相似文献
2.
Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus 总被引:4,自引:0,他引:4
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The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene. 相似文献
3.
The white rot fungus Pleurotus ostreatus, grown for 11 days in basidiomycetes rich medium containing [14C] phenanthrene, metabolized 94% of the phenanthrene added. Of the total radioactivity, 3% was oxidized to CO2. Approximately 52% of phenanthrene was metabolized to trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) (28%), 2,2'-diphenic acid (17%), and unidentified metabolites (7%). Nonextractable metabolites accounted for 35% of the total radioactivity. The metabolites were extracted with ethyl acetate, separated by reversed-phase high-performance liquid chromatography, and characterized by 1H nuclear magnetic resonance, mass spectrometry, and UV spectroscopy analyses. 18O2-labeling experiments indicated that one atom of oxygen was incorporated into the phenanthrene trans-9,10-dihydrodiol. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was 9R,10R, which is different from that of the principal enantiomer produced by Phanerochaete chrysosporium. Significantly less phenanthrene trans-9,10-dihydrodiol was observed in incubations with the cytochrome P-450 inhibitor SKF 525-A (77% decrease), 1-aminobenzotriazole (83% decrease), or fluoxetine (63% decrease). These experiments with cytochrome P-450 inhibitors and 18O2 labeling and the formation of phenanthrene trans-9R,10R-dihydrodiol as the predominant metabolite suggest that P. ostreatus initially oxidizes phenanthrene stereoselectively by a cytochrome P-450 monoxygenase and that this is followed by epoxide hydrolase-catalyzed hydration reactions. 相似文献
4.
V E Kozlovskaia A Iu Grishanova V M Mishin V V Liakhovich 《Biokhimii?a (Moscow, Russia)》1992,57(10):1592-1596
Effects of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes have been studied. Pyridine (200 mg/kg) increased total cytochrome P-450 content and activated metabolism of some specific substrates 24 hours after injection. There was an increase in the degree of p-nitrophenol and chlorzoxazone hydroxylation due to increasing ethanol-induced cytochrome P-450IIE1 content. Pyridine was also able to induce cytochrome P-450IIB1 in rat microsomes; this reaction was accompanied by acceleration of 7-pentoxyresorufin 0-dealkylation. Cytochrome P-450IA1 appearance in liver microsomes was associated with increasing content of cytochrome P-450IA2. Dealkylation rates for specific substrates (7-ethoxyresorufin and 7-methoxyresorufin) were also increased. Similar to pyridine, pyridine-7-oxide induced cytochromes P-450IIE1, P-450IIB1/B2, and P-450IA1/A2, resulting in activation of specific substrate metabolism. Hence, pyridine and its derivative pyridine-N-oxide can be regarded as effective inducers of cytochrome P-450. 相似文献
5.
Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes 总被引:3,自引:0,他引:3
W Pyerin H Taniguchi F Horn F Oesch Z Amelizad T Friedberg C R Wolf 《Biochemical and biophysical research communications》1987,142(3):885-892
A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase. 相似文献
6.
A filamentous fungus Cunninghamella elegans IM 1785/21Gp which displays ability of 17alpha,21-dihydroxy-4-pregnene-3,20-dione (cortexolone) 11-hydroxylation (yielding epihydrocortisone (eF) and hydrocortisone (F)) and polycyclic aromatic hydrocarbons (PAHs) degradation, was used as a microbial eucaryotic model to study the relationships between mammalian steroid hydroxylation and PAHs metabolization. The obtained results showed faster transformation of phenanthrene in Sabouraud medium supplemented with steroid substrate (cortexolone). Simultaneously phenanthrene stimulated epihydrocortisone production from cortexolone. In phenanthrene presence the ratio between cortexolone hydroxylation products (hydrocortisone and epihydrocortisone) was changed from 1:5.1-6.2 to 1:7.6-8.4 in the culture without phenanthrene. Cytochrome P-450 content significantly increased after the culture supplementation by the second substrate, phenanthrene or cortexolone, adequately. To confirm the involvement of cytochrome P-450 in phenanthrene metabolism, the inhibition studies were performed. The cytochrome P-450 inhibitors SKF 525-A (1.5mM) and 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) (2mM) inhibited phenanthrene transformation by 80 and 62%, respectively. 1-aminobenzotriazole (1mM) completely blocked phenanthrene metabolism. The obtained results suggest a presence of connections between steroid hydroxylases and enzymes involved in PAH degradation in C. elegans. 相似文献
7.
The activity of three enzymatic systems of xenobiotic metabolism (cytochrome P-450-dependent monooxygenases, non-specific esterases and glutathione S-transferases) was studied at different stages of development of the Colorado potato beetle (Leptinotarsa decemlineata). Significant sex and ontogenetic differences in the content of cytochrome P-450, position of maxima of CO-difference spectra of the cytochrome P-450 reduced form and substrate specificity of cytochrome P-450 were revealed. The activity of non-specific esterases was shown to increase depending on the age of larvae. The insecticide 1-naphtholenol methylcarbamate which is metabolized by the system of cytochrome P-450-dependent monooxygenases is more toxic in the larvae than at the imago stage, which is correlated with the activity of this system at different stages of ontogenesis. The microsomal monooxygenase inhibitor, piperonylbutoxide, increases the insecticide toxicity in the Colorado beetle imago more than 2-fold. The experimental results testify to different contribution of the detoxication systems to the sensitivity of the Colorado beetle to insecticides at various metamorphosis stages. 相似文献
8.
Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift. 相似文献
9.
V V Obratsov A Iu Grishanova O V Gudkova D G Shekhtman 《Biokhimii?a (Moscow, Russia)》1992,57(7):1011-1020
The inducer of the liver monooxygenase system perfluorodecalin added to microsomes as a submicron emulsion forms an enzyme-substrate complex with cytochrome P-450. The K(app) values for the perfluorodecalin binding to cytochrome P-450 in microsomes isolated from the livers of control and phenobarbital-treated rats are 5 x 10(-5) M and 2.3 x 10(-6) M, respectively. Perfluorodecalin competitively inhibits the binding of substrates to cytochrome P-450 and decreases the rates of monooxygenase reactions. Perfluorodecalin extrusion from the active center of cytochrome P-450 occurs when an excess of perfluorocarbons non-interacting with cytochrome P-450 is added to microsomes. There is a significant vagueness in the rates of various monooxygenase reactions because of simultaneous induction and inhibition of monooxygenase enzymes after perfluorodecalin administration to rats. The data obtained are consistent with the hypothesis that constitutive forms of cytochrome P-450 are primary receptors for xenobiotic-inducers of phenobarbital-type cytochrome P-450 isoforms. 相似文献
10.
M T Huang E F Johnson U Muller-Eberhard D R Koop M J Coon A H Conney 《The Journal of biological chemistry》1981,256(21):10897-10901
The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system. 相似文献
11.
Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications 总被引:2,自引:0,他引:2
Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high specific activity from the type II methanotroph Methylosinus trichosporium OB3b were compared to the same reactions catalyzed by methane monooxygenase from the type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome P-450. The two methane monooxygenases produced nearly identical product distributions, in accord with physical studies of the enzymes which have shown them to be very similar. The products obtained from the oxidation of a series of deuterated substrates by the M. trichosporium methane monooxygenase were very similar to those reported for the same reaction catalyzed by liver microsomal cytochrome P-450, suggesting that the enzymes use similar mechanisms. However, differences in the product distributions and other aspects of the reactions indicated the mechanisms are not identical. Methane monooxygenase epoxidized propene in D2O and d6-propene in H2O without exchange of substrate protons or deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T., Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J. Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of olefins by methane monooxygenase differs at least in part from that of cytochrome P-450. Hydroxylation of alkanes by methane monooxygenase revealed close similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of 3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in the case of methane monooxygenase, whereas 33% was reported for this reaction catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J. Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo greater than or equal to 5.5, was calculated for this reaction. However, the intermolecular kinetic isotope effect on Vm for methane oxidation was small, suggesting that steps other than C-H bond breakage were rate limiting in the overall enzymatic reaction. Similar isotope effects have been observed for cytochrome P-450. These observations indicate a stepwise mechanism of hydroxylation for methane monooxygenase analogous to that proposed for cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
I B Tsyrlov K E Gerasimov N I Gutkina V V Liakhovich 《Biokhimii?a (Moscow, Russia)》1987,52(8):1307-1314
Using the previously obtained data on the substrate-type induction of monooxygenase by xenobiotics of phenobarbital type, the method of conversion of typical substrates for cytochrome P-450 into inducers of biosynthesis of this enzymatic system by blocking in the substrate molecule of the position subjected to oxidative conversion in the enzyme active center was tested. The introduction of the methyl group in the omega-1 position of amobarbital, of Cl- into positions 2 and 4 of biphenyl and the substitution of methyl groups for the isopropyl groups in the 4-N(CH3)2 position of aminopyrine provides for marked induction of these derivatives of cytochrome P-450 and some monooxygenase activities. 相似文献
13.
Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate. 总被引:7,自引:1,他引:6
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U Karlson D F Dwyer S W Hooper E R Moore K N Timmis L D Eltis 《Journal of bacteriology》1993,175(5):1467-1474
A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G + C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450RR1 was induced by 2-ethoxyphenol, and cytochrome P-450RR2 was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (KS = 4.2 +/- 0.3 microM) and 2-methoxyphenol (KS = 2.0 +/- 0.1 microM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450RR2 by both 4-methoxybenzoate (KS = 2.1 +/- 0.1 microM) and 4-ethoxybenzoate (KS = 1.6 +/- 0.1 microM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-450RR2, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers. 相似文献
14.
Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 总被引:1,自引:0,他引:1
When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (Mr 120000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its Mr of 38000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH2-terminal amino acid is proline; the penultimate NH2-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase. 相似文献
15.
Chinese hamster V79 fibroblasts, frequently used as target cells in short-term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA-encoding rat liver cytochrome P-450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so-called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16 alpha- and 16 beta-hydroxy-T as well as 4-androsten-3,17-dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio- and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P-450IIB1 in a reconstituted system. 相似文献
16.
B G Niranjan N M Wilson C R Jefcoate N G Avadhani 《The Journal of biological chemistry》1984,259(20):12495-12501
Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination. 相似文献
17.
M C Romano R D Eckardt P E Bender T B Leonard K M Straub J F Newton 《The Journal of biological chemistry》1987,262(4):1590-1595
omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins. 相似文献
18.
J T Borlakoglu J P Wilkins M P Quick C H Walker R R Dils 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1991,99(3):287-291
1. Analysis of individual PCB-isomers and congeners in extracts of adipose tissue from N = 12 razorbills suggested that 4-chlorobiphenyl was subjected to metabolism. 2. In vitro metabolism studies using [14C]-4-chlorobiphenyl as substrate showed that razorbills metabolise this substrate to [14C]4-chloro-4'-hydroxybiphenyl at an average rate of 20 pmol/mg microsomal protein/min. For comparison, the metabolism of [14C]-4-chlorobiphenyl by pigeons and rats was also studied, and average rates in the formation of [14C]-4-chloro-4'-hydroxybiphenyl of 12 pmol/mg microsomal protein min and 342 pmol/mg microsomal protein min were estimated. 3. A comparison of the hepatic drug metabolising enzyme system of razorbills and pigeons showed similar concentrations of cytochrome P-450, cytochrome b5 and comparable catalytic activities of cytochrome P-450-dependent monooxygenase, when assessed for HHDN epoxidase, PROD, EROD and the Phase II enzymes glutathiones-S-transferase, but were significantly lower, when compared with rats. The results obtained suggest fundamental differences in the catalytic activities of cytochrome P-450-dependent monooxygenase between avian and mammalian species. 4. The present study, however, provides evidence that fish-eating seabirds have the ability to metabolically dispose of certain PCB isomers and congeners, which are amongst the most ubiquitously distributed pollutants in the ecosystem. 相似文献
19.
Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver 总被引:11,自引:0,他引:11
The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories. 相似文献
20.
L Richert C Bergmann J P Beck S Rong B Luu G Ourisson 《Biochemical and biophysical research communications》1983,117(3):851-858
A soluble cytochrome P-450-dependent fatty acid monooxygenase activity obtained from Bacillus megaterium ATCC 14581 can be induced by at least 13 different barbiturates. In general, the potency of these compounds as inducers increases with their increasing lipophilicity. We have now shown that at least 4 of these barbiturates (phenobarbital, secobarbital, pentobarbital and methohexital) seem to induce the same active cytochrome P-450-containing enzyme by a non-substrate type mechanism. The partially purified enzymes obtained from cultures induced with each of the 4 barbiturates tested were all of similar molecular size (Mr = 130,000 +/- 10,000) and had similar turnover numbers (1400-1800 +/- 300) with either palmitoleate or myristate as substrates. None of the tested barbiturates served as substrates, activators or inhibitors of any of the monooxygenase preparations, nor did they appear to interact in any way with the monooxygenase enzyme or the P-450 component. 相似文献