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1.
In order to define the site of bioactivation of CCl4, CHCl3 and CBrCl3 in the NADPH cytochrome reductase-cytochrome P-450 coupled systems of liver microsomes, the 14C-labeled hepatotoxins were incubated with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O2 (8:2) and addition of a cytochrome reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome reductase activity was unchanged, the covalent binding of CCl4, CHCl3, and CBrCl3 was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N2 enhanced the binding of CCl4, inhibited the binding of CHCl3 and did not influence the binding of CBrCl3. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome reductase and that CCl4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl3 activation proceeds by cytochrome P-450 dependent oxidative pathways. 相似文献
2.
S.D. Nelson J.R. Mitchell E. Dybing H.A. Sasame 《Biochemical and biophysical research communications》1976,70(4):1157-1165
2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome reductase and 70% in an atmosphere of CO:O2 (9:1) when compared to an atmosphere of N2:O2 (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion. 相似文献
3.
Robert L. Barbieri Rapin Osathanondh Jacob A. Canick Robert J. Stillman Kenneth J. Ryan 《Steroids》1980,35(3):251-263
The effects of danazol on steroidogenesis in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant KI = 0.8 μM) and the mitochondrial 11β-hydroxylase (KI = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal . 相似文献
4.
John W. Greiner Robert E. Kramer Howard D. Colby 《Prostaglandins & other lipid mediators》1979,17(4):587-597
Studies were carried out to investigate the effects of prostaglandins (PG) on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1α or PGF2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism. 相似文献
5.
Addition of -nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b5. Addition of partially purified cytochrome b5 to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of -nitroanisole. -Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of -nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b5 and that cytochrome P-450 is the enzyme which catalyzes -nitroanisole O-demethylation. 相似文献
6.
Bengt Jernström Jorge Capdevila Sten Jakobsson Sten Orrenius 《Biochemical and biophysical research communications》1975,64(3):814-822
Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino--octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome 5 and NADPH-cytochrome reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found. 相似文献
7.
The effect of pretreatment with phenobarbitone, rifampicin, β-naphthoflavone, antipyrine and spironolactone on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes has been examined and the corresponding changes in microsomal P-450 content and cytochrome reductase activity measured. Rifampicin produced the greatest increase (220%) in irreversible binding while phenobarbitone produced the greatest increase in both microsomal P-450 content (172%) and cytochrome reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome reductase activity. 相似文献
8.
H A Sasame M M Ames S D Nelson 《Biochemical and biophysical research communications》1977,78(3):919-926
Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O2 (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein. 相似文献
9.
Kathryn M. Ivanetich Anita K. Costa Tom Brittain 《Biochemical and biophysical research communications》1982,105(4):1322-1326
Limitations on the determination of the concentration of the major phenobarbital inducible form of cytochrome P-450 (P-450b) in hepatic microsomes by the metyrapone assay of Luu-The (1) are reported. Compounds which bind to the Type I, II and IR binding sites, or convert cytochrome P-450 to P-420, decrease the apparent concentration of cytochrome P-450b by 20 to 100% in hepatic microsomes from untreated and pregnenolone-16α-carbonitrile or phenobarbital treated rats. It is calculated that errors of greater ca. 40% in the concentration of cytochrome P-450b can arise in the presence of appreciable quantities of the major pregnenolone-16α-carbonitrile or polycyclic hydrocarbon inducible forms of cytochrome P-450. 相似文献
10.
The role of cytochrome 5 in the -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 5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome 5 reductase, and Triton X-100 in addition to cytochrome 5. The omission of cytochrome 5 from the complete system entirely abolished the activity. These results clearly show that cytochrome 5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome 5 in such a way that the second electron is transferred from cytochrome 5 and thus exhibits the demethylase activity. 相似文献
11.
Karl Dus William J. Litchfield Anne G. Miguel Theodore A. van der Hoeven David A. Haugen William L. Dean Minor J. Coon 《Biochemical and biophysical research communications》1974,60(1):15-21
The cytochrome P-450 of (P-450cam) and that of phenobarbital-induced liver microsomes (P-450LM) differ markedly in substrate specificity, solubility, and the requirement of the former for an iron-sulfur protein and the latter for a phospholipid for hydroxylation activity. Despite these differences, highly purified P-450cam and P-450LM show immunological cross reaction by competitive binding and inhibition of catalytic activity and are of similar subunit molecular weight and amino acid composition. Upon treatment with cyanogen bromide they yield small heme-containing peptides of highly similar amino acid composition. 相似文献
12.
Response of the mixed function oxidase system of rat hepatic microsomes to parathion and malathion and their oxygenated analogs 总被引:1,自引:0,他引:1
inhibition of ethylmorphine metabolism in rat hepatic microsomes by parathion, malathion, malaoxon and paraoxon was not well correlated with their effects on NADPH oxidation, cytochrome C reduction or the reduction of cytochrome P-450. A parallel relationship was observed between inhibition of ethylmorphine metabolism by parathion, malathion and malaoxon and the binding affinity of these agents to microsomal cytochrome P-450 obtained from rats pretreated with an anticholinesterase agent, Bis-[?-nitrophenol] phosphate. 相似文献
13.
Both the cytochrome b5 level and NADH cytochrome b5 reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. , drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b5 and NADH cytochrome b5 reductase in rat liver microsomes. 相似文献
14.
Dipak K. Ghosh Debjani Dutta Timir B. Samanta Ajit K. Mishra 《Biochemical and biophysical research communications》1983,113(2):497-505
Microsomal preparations of TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and O2 and exhibits a pH optimum of 8.0–8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistant with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus. 相似文献
15.
Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene 总被引:6,自引:0,他引:6
P D Lotlikar L Luha K Zaleski 《Biochemical and biophysical research communications》1974,59(4):1349-1355
A procedure is described for the isolation of cytochrome P-450 fraction from hamster liver microsomes. It involves removal of NADPH-cytochrome c reductase activity by treatment with bacterial protease before solubilization with Triton X-100 and precipitation with ammonium sulfate. Reconstitution studies indicate that 2-acetylaminofluorene -and ring-hydroxylation require both cytochrome P-450 fraction and the reductase fraction. -hydroxylation activity of cytochrome P-450 fraction from 3-methylcholanthrene pretreated hamsters is different and severalfold greater than that of cytochrome P-450 fraction from controls. These results demonstrate for the first time an activation of a chemical carcinogen by a reconstituted cytochrome P-450 enzyme system. 相似文献
16.
The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome 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 content, of the presence of type I and type II substrates for cytochrome . 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 . The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome 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 (). 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
17.
The effect of extra bound cytochrome b-5 on cytochrome P-450-dependent enzyme activities in liver microsomes 总被引:1,自引:0,他引:1
Binding of increasing amounts of detergent-purified cytochrome to rabbit liver microsomes produces a progressive inhibition of NADPH-cytochrome P-450 reductase activity which is accompanied by a similar inhibition of NADPH-supported benzphetamine demethylation. In contrast, NADH-cytochrome P-450 reductase activity in the enriched microsomes is markedly enhanced and this stimulation is accompanied by a similar increase in NADH-peroxidase activity, suggesting that cytochrome in these two reactions functions as an intermediate electron carrier to cytochrome P-450. 相似文献
18.
J J Bradshaw M R Ziman K M Ivanetich 《Biochemical and biophysical research communications》1978,85(3):859-866
Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl--propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and -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--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--propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital. 相似文献
19.
Evelyne Rouer Patrick Dansette Philippe Beaune Jean-Paul Leroux 《Biochemical and biophysical research communications》1980,95(1):41-46
The thermally-induced changes of a cytochrome P-450 dependent activity (ethoxycoumarin-O-deethylase) and of the fluorescence polarization of diphenylhexatriene were compared in microsomes from lean, and mice. In lean mice, biphasic plots were obtained with break points in the same range of temperature by both methods, whereas, in and mice, no discontinuities were observed. These results may be related to a modified fatty acid composition of microsomal membranes in mutant mice. They exemplify the influence of the lipid environment on the monooxygenase system as also shown by the modified binding constants of cytochrome P-450 towards type II substrates in mice. 相似文献
20.
NADPH reduces both liver microsomal cytochrome P-450 and cytochrome . In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome directly reduces cytochrome P-450 in rat liver microsomes. 相似文献