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1.
Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.  相似文献   

2.
The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [14C]CCNU (1-(2-[14C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [14C]CCU (1-(2-[14C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [14C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [14C]CCNU was shown to undergo denitrosation to [14C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450.  相似文献   

3.
A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4-14C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.  相似文献   

4.
Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [14C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [14C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [14C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 in vivo, these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.  相似文献   

5.
The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-14C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (14CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-14C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of 14CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which 14CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-14C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of 14CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.  相似文献   

6.
Radiometric methods for the assay of deoxycorticosterone 11beta-hydroxylase and for the determination of NADP on a microscale were developed. The determination of NADP was based on the quantitative conversion of 6-phospho[1-14C]gluconate to 14CO2 by the action of 6-phosphogluconate dehydrogenase. Using these methods NADPH oxidase activity of the adrenodoxin reductase-adrenodoxin system as well as kinetic properties of deoxycorticosterone 11beta-hydroxylase (cytochrome P-450) were investigated. The NADPH oxidase activity observed in the presence of adrenodoxin reductase, adrenodoxin, and O2, but in the absence of cytochrome P-450 and deoxycorticosterone, were functions of O2 and adrenodoxin concentrations and represented the autooxidation of reduced adrenodoxin which resulted in the production of H2O2. Due to the rapid autooxidizability of reduced adrenodoxin, only a small fraction of electrons conveyed from NADPH to adrenodoxin by way of adrenodoxin reductase was utilized for the deoxycorticosterone 11beta-hydroxylase reaction under the conditions employed.  相似文献   

7.
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   

8.
The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.Abbreviations BAT Brown Adipose Tissue - MDA malondialdehyde  相似文献   

9.
K Zachariah  M R Juchau 《Life sciences》1975,16(11):1689-1692
Spectral analyses of the carbon monoxide (CO) complex of human placental microsomal cytochrome P-450 revealed absorption maxima at 426 and 450 nm when NADPH (2×10−4M) was utilized as a reducing agent. Additional NADPH or NADH did not produce any further increases in the absorption maximum at 450 nm. A period of 10–15 minutes was required for the complete reduction. Various steroids were added to both sample and reference cuvettes to examine their interactions with the CO-cytochrome P-450 complex. The resulting spectral changes indicated that low concentrations of steroids (≃10−7M) such as androstenedione, 19-hydroxyandrostenedione, 19-oxoandrostenedione and testosterone completely eliminated the absorbance maxima at 450 nm while 19-norandrostenedione, 19-nortestosterone, pregnenolone and benzo[a]-pyrene did not eliminate this peak. Since ample time was allowed to reduce the cytochrome P-450 with NADPH, the observed interaction of steroids with cytochrome P-450 in the presence of CO does not represent an effect on reductase activity, but on the formation of the CO-cytochrome P-450 complex.  相似文献   

10.
1. Ecdysone 20-monooxygenase activity has been found in hepatopancreas, gonads, epidermis and muscle of the crab Gecarcinus lateralis. Activity was assayed by measuring the in vitro conversion of [3H]-ecdysone to [3H]-20-hydroxyecdysone. Maximal activity is obtained at 30°C and pH 8.0 in sodium phosphate buffer.2. Activity from hepatopancreas is localized in a fraction which sediments at 10,000 g, probably mitochondria.3. NADPH stimulates activity and metyrapone or oxygen deprivation inhibits it, as has been observed for cytochrome P-450-dependent monooxygenases.4. Changes in ecdysone 20-monooxygenase activity at different stages of the molt cycle are not directly correlated to changes in ecdysteroid levels in the hemolymph.  相似文献   

11.
Using sonicated mitochondria fraction prepared from bovine adrenal glomerulosa cells, aldosterone biosynthesis from 18-hydroxycorticosterone was examined as its final step, as production of [3H]-aldosterone from [3H]-corticosterone was strongly reduced by addition of non-radioactive 18-hydroxycorticosterone during the incubation. Significant conversion of 18-hydroxycorticosterone to aldosterone by the mitochondria sonicate was observed in the presence of NADPH, but not NADP+. This reaction was almost completely inhibited in the atmosphere of 100% carbon monoxide in the presence of either NADP+, or NAD+, and significantly reduced in the mixture of carbon monoxide and oxygen (90:10) in the presence of NADPH. Several drugs, such as SU compounds, spironolactone, amphenone B and SKF 525A which affect cytochrome P-450 blocked production of aldosterone from 18-hydroxycorticosterone. From these results, we conclude that a mixed function oxidase involving a cytochrome P-450 is engaged in the final course of aldosterone biosynthesis.  相似文献   

12.
Incubation of R(+)-[14C]pulegone with rat liver microsomes in the presence of NADPH resulted in covalent binding of radioactive material to macromolecules. Covalent binding was much higher in phenobarbital-treated microsomes as compared to 3-methylcholanthrene treated or control microsomes. The Km and Vmax of covalent binding was 0.4 mM and 1.7 nmol min-1 mg-1, respectively. Covalent binding was drastically inhibited (93%) in the presence of piperonyl butoxide. Antibodies to phenobarbital-induced cytochrome P-450 and NADPH-cytochrome P-450 reductase inhibited covalent binding to an extent of 72% and 47%, respectively. Cysteine and semicarbazide also inhibited NADPH dependent binding of radiolabel from R(+)-[14C]pulegone to microsomal proteins. The results suggest the involvement of liver microsomal cytochrome P-450 in the bioactivation of R(+)-pulegone to reactive metabolite(s) which might be responsible for covalent binding to macromolecules resulting in toxicity.  相似文献   

13.
1-Ethynylpyrene, trans-, & cis-1-(2-bromovinyl)pyrene, methyl 1-pyrenyl acetylene, and phenyl 1-pyrenyl acetylene are substrates for cytochrome P-450 dependent monooxygenases and also inhibitors of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activities in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, trans-1-(2-bromovinyl)pyrene, and methyl 1-pyrenyl acetylene cause a mechanism based inhibition (suicide inhibition) of the benzo[a]pyrene hydroxylase activities in microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, while cis-1-(2-bromovinyl)pyrene only causes suicide inhibition of the hydroxylse activities in the 5,6-benzoflavone induced microsomes and phenyl 1-pyrenyl acetylene does not cause a detectable suicide inhibition of these activities in either type of microsome. Incubation with NADPH and 1-ethynylpyrene, trans-, or cis-1-(2-bromovinyl)pyrene causes a loss of the P-450 content in the microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, but incubations with methyl 1-pyrenyl acetylene or phenyl 1-pyrenyl acetylene did not cause a loss of the P-450 content of either microsomal preparation.  相似文献   

14.
V.L. Kubic  M.W. Anders 《Life sciences》1980,26(25):2151-2155
Aerobic incubation of hepatic microsomal fractions in the presence of carbon tetrachloride, NADPH and cysteine resulted in the formation of phosgene which was identified by gas chromatography/mass spectrometry as the adduct, 2-oxothiazolidine-4-carboxylic acid, formed by its reaction with cysteine. [13C]-Carbon tetrachloride was metabolized to 2-[13C]-oxothiazolidine-4-carboxylic acid the , when carbon tetrachloride was incubated in the presence of [18O]-O2, 2- [18O]-oxothiazolidine-4-carboxylic acid was formed. The reaction was inhibited by carbon monoxide showing the involvement of the cytochrome P-450-dependent mixed function oxidase system. The metabolism of carbon tetrachloride to phosgene may play a role in the production of hepatotoxicity by this compound.  相似文献   

15.
In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.  相似文献   

16.
Cytochrome P-450scc (P-450 XIA1) from bovine adrenocortical mitochondria was investigated using a suicide substrate: [14C]methoxychlor. [14C]Methoxychlor irreversibly abolished the activity of the side-chain cleavage enzyme for cholesterol (P-450scc) and the inactivation was prevented in the presence of cholesterol. The binding of [14C]methoxychlor and cytochrome P-450scc occurred in a molar ratio of 1:1 and the cholesterol-induced difference spectrum of cytochrome P-450scc was similar with the methoxychlor-induced difference spectrum. [14C]Methoxychlor-binding peptides were purified from tryptic-digested cytochrome P-450scc modified with [14C]methoxychlor. Determination of the sequence of the amino-acid residues of a [14C]methoxychlor-binding peptide allowed identification of the peptide comprising the amino-terminal amino-acid residues 8 to 28.  相似文献   

17.
Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from 18O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H218O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from 18O2 into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O2 plus NADPH.  相似文献   

18.
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   

19.
Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-3H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O2 for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.  相似文献   

20.
The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe3+, ethylenediaminetetra-acetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe3+. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.  相似文献   

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