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1.
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
2.
Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: catalytic activity and other properties 总被引:3,自引:0,他引:3
T A van der Hoeven D A Haugen M J Coon 《Biochemical and biophysical research communications》1974,60(2):569-575
Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and 5 and NADPH-cytochrome reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate. 相似文献
3.
A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits 总被引:9,自引:0,他引:9
Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome , NADH-cytochrome reductase, and NADPH-cytochrome reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome reductase preparation (purified by a detergent method) and phosphatidyl choline. 相似文献
4.
Cytochrome P-450 was purified as a 3-methylcholanthrene complex from liver microsomes of 3-methylcholanthrene-treated rabbits to a specific content of 17 to 18 nmoles per mg of protein with a yield of about 10 %. The purified protein gave only a single protein band on sodium dodecylsulfate-urea-poly-acrylamide gel electrophoresis, and its apparent molecular weight was estimated to be about 54,000, a value which is higher than that for cytochrome P-450 from phenobarbital-treated rabbits by about 4,000. The reconstituted system containing the purified cytochrome and NADPH-cytochrome reductase was active in NADPH-dependent hydroxylation of benzo[α]pyrene. 相似文献
5.
NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-polyacrylamide gel electrophoresis, exhibits a minimum molecular weight of 80,000 daltons and contains 1 molecule each of FAD and FMN per 80,000 molecular weight. The specific activity for cytochrome as electron acceptor is 48.8 μmoles per min and for substrate hydroxylation of benzphetamine measured as NADPH oxidation in the presence of cytochrome P-450 and phosphatidylcholine is 2.5 μmoles per min. 相似文献
6.
Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes 总被引:6,自引:0,他引:6
NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 μmoles of cytochrome per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 μmoles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate. 相似文献
7.
E F Johnson U Muller-Eberhard 《Biochemical and biophysical research communications》1977,76(3):652-659
Cytochrome P-450 was isolated in highly purified form from liver microsomes of adult male rabbits treated with 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD). Preparations average 17.8 ± 0.8 nmoles cytochrome P-450 per mg protein and have an estimated molecular weight of 54,500. The visible absorption spectrum of the purified cytochrome displays absorption spectral maxima characteristic of high spin forms of cytochrome P-450. When reconstituted with highly purified NADPH-cytochrome P-450 reductase, this cytochrome catalyzes the hydroxylation of acetanilide and the O-deethylation of 7-ethoxyresorufin, two activities induced by TCDD. 相似文献
8.
K M Ivanetich I Aronson I D Katz 《Biochemical and biophysical research communications》1977,74(4):1411-1418
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 5 or NADPH-cytochrome reductase . 相似文献
9.
H A Sasame J R Gillette M R Boyd 《Biochemical and biophysical research communications》1978,84(2):389-395
An antibody prepared against purified rat liver NADPH-cytochrome reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b5, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome reductases, and was inactive against the respective NADPH-dependent cytochrome reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b5 is rate-limiting in lung microsomes. 相似文献
10.
Y Yoshida Y Aoyama H Kumaoka S Kubota 《Biochemical and biophysical research communications》1977,78(3):1005-1010
Cytochrome P-450 was purified from microsomes of anaerobically grown yeast to a specific content of 12–15 nmoles per mg of protein with a yield of 10–30%. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis, the purified preparation yielded a major protein band having a molecular weight of about 51,000 together with a few faint bands. It was free from cytochrome b5, NADH-cytochrome b5 reductase, and NADPH-cytochrome c (P-450) reductase. In the oxidized state it exhibited a low-spin type absorption spectrum, and its reduced CO complex showed a Soret peak at 447–448 nm. It was reducible by NADPH in the presence of an NADPH-cytochrome c reductase preparation purified from yeast microsomes. Its conversion to the cytochrome P-420 form was much slower than that of hepatic cytochrome P-450. 相似文献
11.
E F Johnson U Muller-Eberhard 《Biochemical and biophysical research communications》1977,76(3):644-651
We describe the resolution and partial purification of two minor forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo--dioxin. Both forms have different electrophoretic mobilities when compared to the major form of cytochrome P-450 isolated from this source. The two cytochromes show different activities with several substrates. One form is very active in the hydroxylation of benzo()pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase. 相似文献
12.
Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles 总被引:3,自引:0,他引:3
M Ingelman-Sundberg I Johansson 《Biochemical and biophysical research communications》1980,97(2):582-586
When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450LM2, cytochrome b5 enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or -nitroanisole about 5-fold. Cytochrome b5 did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b5-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H2O2 or in the system, thus strongly indicating cytochrome b5 being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase. 相似文献
13.
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
14.
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
15.
NADPH-cytochrome reductase (NADPH-cytochrome reductase, EC 1.6.2.4), the flavoprotein which is responsible for the NADPH-dependent reduction of cytochromes P-450 in hepatic microsomes, has been localized immunohistochemically at the light microscopic level in rat liver. Localization was achieved through the use of sheep antiserum to rat hepatic microsomal NADPH-cytochrome reductase in an unlabeled antibody peroxidase-antiperoxidase technique. Parenchymal cells throughout the liver lobule were found to be stained positively for NADPH-cytochrome reductase, although the intensity of immunostaining was slightly greater in the centrilobular regions. Immunostaining for NADPH-cytochrome reductase was not detected in Kupffer cells, connective tissue cells, or in cells of the hepatic vasculature. 相似文献
16.
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
17.
Jan I. Pedersen Helge Oftebro Tore Vänngård 《Biochemical and biophysical research communications》1977,76(3):666-673
An iron-sulfur protein has been isolated from bovine liver mitochondria and purified 140-fold on DEAE-cellulose and Sephadex G-100. During the isolation the protein was detected by its NADPH-cytochrome reductase activity in the presence of adrenal NADPH-ferredoxin reductase. The molecular weight of the protein (12,400), the optical spectrum (peaks at 414 nm and 455 nm which disappear upon reduction), and the EPR spectrum (gx = gy = 1.935 and gz = 2.02) were typical for a ferredoxin. In the presence of soluble adrenal cytochrome P450, ferredoxin reductase and NADPH, this protein could support the formation of pregnenolone from cholesterol. Under similar conditions, but in the presence of a cytochrome P450 solubilized from rat liver mitochondria, cholesterol was transformed into a more polar compound tentatively identified as 26-hydroxycholesterol. 相似文献
18.
P Beaune P Dansette J P Flinois S Columelli D Mansuy J P Leroux 《Biochemical and biophysical research communications》1979,88(3):826-832
Human liver cytochrome P 450 was partially purified by hydrophobic chromatography on Octyl-Sepharose, followed by ion-exchange chromatography on DEAE-cellulose. Two fractions (A and B) were obtained; cytochrome P 450 of fraction A was purified sixfold, with an overall yield of about 6 %. Its spectral properties were similar to those previously described in animal cytochromes P 450. Moreover, p-nitroanisole-O-demethylase activity could be obtained in a reconstituted system involving cytochrome P 450 of fraction A, human NADPH-cytochrome reductase and phospholipids. 相似文献
19.
Microsomal 11α-hydroxylation of progesterone in : Part I: Characterization of the hydroxylase system
C.R. Jayanthi Prema Madyastha K.Madhava Madyastha 《Biochemical and biophysical research communications》1982,106(4):1262-1268
Microsomes (105,000xg sediment) prepared from induced cells of was found to hydroxylate progesterone to 11α-hydroxyprogesterone (11α-OHP) in high yields (85–90% in 30 min.) in the presence of NADPH and O2. The pH optimum for the hydroxylase was found to be 7.7. However, for the isolation of active microsomes grinding of the mycelium should be carried out at pH 8.3. Metyrapone, carbon monoxide, SKF-525A, p-CMB and N-methyl maleimide inhibited the hydroxylase activity indicating the involvement of cytochrome P-450 system. The inhibition of the hydroxylase by cytochrome and the presence of high levels of NADPH-cytochrome reductase in induced microsomes suggest that the reductase could be one of the components in the hydroxylase system. 相似文献
20.
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