共查询到20条相似文献,搜索用时 15 毫秒
1.
R H Stanton F W Plapp R A White M Agosin 《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,61(2):297-305
1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains. 相似文献
2.
Ken Higashi Kie Ikeuchi Masanobu Obara Yuji Karasaki Hideyasu Hirano Sadao Gotoh 《Bioscience, biotechnology, and biochemistry》2013,77(8):2399-2405
Microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L., Balalaika) was purified to an almost electrophoretically homogeneous preparation. The specific content of cytochrome P-450 in the final preparation was 6.68 nmol/mg protein, which was 30-fold enriched from that of the solubilized fractions of microsomes. The molecular weight of purified cytochrome P-450 by SDS-gel electrophoresis is 52,500. The Oxidized form of the purified cytochrome P-450 had absorption peaks at 392, 552, and 645 nm and the absolute reduced CO spectrum peaked at 448 nm. Judged spectrally, the purified cytochrome P-450 is in high spin in the oxidized state. Antiserum against this cytochrome P-450 previously has shown to be highly specific for its antigen but showed a single precipitin line with solubilized microsomal proteins from tulip bulbs of several other cultivars. The physiological role of this cytochrome P-450, however, is unknown in these dormant tulip bulbs. 相似文献
3.
Evidence for cytochrome P-450 and P-450-mediated benzo(a) pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium 总被引:2,自引:0,他引:2
Segula Masaphy Dan Levanon Yigal Henis K. Venkateswarlu Steven L. Kelly 《FEMS microbiology letters》1996,135(1):51-55
Abstract The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. K m values for activity showed a difference between the cellular fractions with a K m of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The V max values observed were 0.83 nmol min− (nmol microsomal P-450) −1 and 0.4 nmol min−1 (nmol cytosolic P-450)−1 . The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes. 相似文献
4.
Variations in hepatic progesterone 21-hydroxylase activity reflect differences in the microsomal concentration of rabbit cytochrome P-450 1 总被引:1,自引:0,他引:1
A monoclonal antibody specific for cytochrome P-450 1 that extensively (greater than 95%) inhibits the hepatic 21-hydroxylation of progesterone was used in a two-site immunoradiometric assay to estimate the concentration of cytochrome P-450 1 in microsomes prepared from 24 individual, untreated New Zealand White rabbits. The progesterone 21-hydroxylase activities of these microsomes ranged from 0.2 to 5.8 nmol min-1 mg microsomal protein-1. Scatchard analysis revealed similar slopes and thus apparent affinities between the antibody and microsome samples that varied greater than 10-fold in 21-hydroxylase activity. The maximal extent of binding of the antibody to different microsomal preparations was greater for microsomes exhibiting high as compared to low 21-hydroxylase activity, suggesting that the level of binding reflects the microsomal content of P-450 1. Quantitation was based on the extent of binding of the 125I-labeled monoclonal antibody to P-450 1 sequestered from a sample by a heterologous monoclonal antibody adsorbed to the wells of a microtiter plate. These results indicate that the microsomal content of P-450 1 varies from less than 0.05 to 0.5 nmol/mg microsomal protein. The microsomal content of this antigen as determined in the two-site immunoradiometric assay was highly correlated (r = 0.97) with progesterone 21-hydroxylase activity. Linear regression analysis was used to estimate the turnover number for progesterone in situ, yielding a value of 11 nmol deoxycorticosterone formed min-1 nmol microsomal P-450 1(-1). This is similar to the value of 14 nmol deoxycorticosterone formed min-1 nmol-1 obtained for the reconstituted, purified P-450 1 used as a standard in the immunoquantitation assay. 相似文献
5.
Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system. 总被引:5,自引:5,他引:0
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The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions. 相似文献
6.
A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450
A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs. 相似文献
7.
Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney. 总被引:4,自引:0,他引:4
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M L Schwartzman N G Abraham M A Carroll R D Levere J C McGiff 《The Biochemical journal》1986,238(1):283-290
Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla. 相似文献
8.
Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min. 相似文献
9.
Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450 enzyme system. 总被引:3,自引:2,他引:1
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Oxidative demethylation of dimethylnitosamine was studied with both reconstituted and unresolved liver microsomal cytochrome P-450 enzyme systems from rats and hamsters. Proteinase treatment of liver microsomal preparations yielded cytochrome P-450 particulate fractions. Both cytochrome P-450 and NADPH- cytochrome c reductase fractions were required for optimum demethylation activity. Particulate cytochrome P-450 fractions were more effecient than either Triton X-100- or cholatesolubilized preparations of these particles in demethylation activity with rat and hamster liver preparations appear to be due to differences in specificity in their cytochrome P-450 fractions. 相似文献
10.
V I Popova L M Va?ner I I Gorshkova O A Gromova V V Liakhovich 《Biokhimii?a (Moscow, Russia)》1985,50(1):53-62
The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type. 相似文献
11.
Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats. 总被引:2,自引:0,他引:2
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In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO. 相似文献
12.
以谷胱甘肽为电子供体的细胞膜氧化还原系统 总被引:1,自引:0,他引:1
内载谷胱甘肽(GSH)的大豆(Glycine max L.)下胚轴正向型质膜囊泡具有以GSH为电子供体的跨膜电子传递活性,能还原膜外电子受体FeCN和细胞色素(Cyt)C,其还原速率分别为(21.6±0.6)nmolFeCN·min~(-1)·mg~(-1)蛋白和(6.6±1.0)nmol Cyt C·min~(-1)·mg~(-1)蛋白。这种跨膜电子传递能引起膜上Cyt P-450吸收光谱标志带(Soret带)的变化,表明Cyt P-450参与了这一氧化还原过程。在跨质膜电子传递的同时伴随着H~ 运输和膜电位的改变。 相似文献
13.
Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver. 相似文献
14.
Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo
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Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring. 相似文献
15.
A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase). 相似文献
16.
A comparison of methods used in isolating microsomes and in measuring microsomal cytochrome P-450 demonstrated that separation following protoplast lysis gave the best results. By this latter technique a high amount of cytochrome P-450 (0.2–0.3 nmol/mg) was recovered but cytochrome P-420, considered as the denatured form, was absent.The alkanes specifically induce cytochromes P-450 and b5 localized on the microsomes. The denaturation in vivo of cytochrome P-450 into cytochrome P-420 even occurs during storage at 1 °C. This degradation is increased during preparation of subcellular fractions if no preventive measures are taken. 相似文献
17.
A A Akhrem M A Kisel' S A Usanov D I Metelitsa 《Prikladnaia biokhimiia i mikrobiologiia》1980,16(1):100-105
The effect of different phospholipids on the functional activity of highly purified cytochrome P-450 used as a co-substrate of cumene hydroperoxide was examined. At the molar ratio of phospholipids to cytochrome P-450 that was equal to 30, phosphatidyl serine, phosphatidyl inositol, the total fraction of microsomal phospholipids, and lysophosphatidyl choline increased the hydroxylation rate of aniline and naphthalene. The effect of the above phospholipids on the rate of naphthalene oxidation was much more pronounced. Phosphatidyl choline and sphingomyelin in similar large quantities did not exert a stimulating effect on the reactions studied. The kinetic parameters of aniline oxidation in the systems containing phospholipids that produced an activating effect were investigated. 相似文献
18.
A V Klotz J J Stegeman B R Woodin E A Snowberger P E Thomas C Walsh 《Archives of biochemistry and biophysics》1986,249(2):326-338
Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist. 相似文献
19.
Expression of rabbit cytochrome P-450IIE2 in yeast and stabilization of the enzyme by 4-methylpyrazole 总被引:1,自引:0,他引:1
S J Pernecky T D Porter M J Coon 《Biochemical and biophysical research communications》1990,172(3):1331-1337
A rabbit cytochrome P-450IIE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP1) and the iso-1-cytochrome c gene terminator (CYC1), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450IIE2 per mg of protein. The P-450IIE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450IIE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized. 相似文献
20.
J L Rivière 《Biochemical and biophysical research communications》1980,97(2):546-552
Hepatic and duodenal microsomes were prepared from partridge by conventional procedures. The duodenal homogenates were stable, avoiding the use of protease inhibitors in the preparation of microsomes. Both microsomal fractions were able to dealkylate 7-ethoxycoumarin, showing the characteristics of a cytochrome P-450 dependent reaction. Parameters of the reaction (cofactor requirements, optimal pH, Km) were established. Typical type I difference spectrum was obtained upon addition of 7-ethoxycoumarin to hepatic and duodenal microsomes; with liver, Km and Ks values were similar. The concentration of cytochrome P-450 was very high and similar in both organs, but the specific activity of duodenal 7-ethoxycoumarin dealkylase was about 10% and NADPH-cytochrome c reductase 50% that of liver. 相似文献