Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver.

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 $\text{c}$ 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.     

Purification of cytochrome P-450 from liver microsomes of phenobarbital-treated guinea pigs

Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b₅ and NADPH-cytochrome $\text{c}$ reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome $\text{c}$ reductase and phosphatidylcholine.     

Purification of the major cytochrome P-450 of liver microsomes from rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Cytochrome P-450 was isolated in highly purified form from liver microsomes of adult male rabbits treated with 2,3,7,8-tetrachlorodibenzo-$\text{p}$-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.     

Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: catalytic activity and other properties

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 $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.     

Purification of a substrate complex of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-treated rabbits.

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 $\text{c}$ reductase was active in NADPH-dependent hydroxylation of benzo[α]pyrene.     

A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits

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 $\text{b}{}_5$, NADH-cytochrome $\text{b}{}_5$ reductase, and NADPH-cytochrome $\text{c}$ reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome $\text{c}$ reductase preparation (purified by a detergent method) and phosphatidyl choline.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ 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.     

Cytochrome P-450 and NADPH cytochrome c reductase in rat brain: formation of catechols and reactive catechol metabolites.

Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome $\text{c}$ 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 $\text{c}$ 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.     

Demonstration of a cytochrome P-450-dependent steroid 15β-hydroxylase in Bacillus megaterium

The steroid 15β-hydroxylase system of $\text{Bacillus megaterium}$ was obtained in a cell-free preparation through sonication. The strictly NADPH-dependent 15β-hydroxylase activity, measured using progesterone as substrate, was inhibited by carbon monoxide, SKF 525-A, imidazole and metyrapone, indicating that the reaction is cytochrome P-450-dependent. A 40-fold purification of cytochrome P-450 in cell-free extracts was obtained by chromatography on DEAE-cellulose yielding a concentration of 0.32 nmoles of cytochrome P-450 per mg of protein. This partially purified cytochrome P-450 preparation catalyzed 15β- and 15α-hydroxylation of progesterone in the presence of NaIO₄ or NaClO₂ but not in the presence of NADPH or NADH.     

Multiple forms of cytochrome P-450: resolution and purification of rabbit liver aryl hydrocarbon hydroxylase.

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-$\text{p}$-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($\text{a}$)pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase.     

Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene

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 $\text{N}$-and ring-hydroxylation require both cytochrome P-450 fraction and the reductase fraction. $\text{N}$-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.     

The effect of enzyme induction on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes

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 $\text{c}$ 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 $\text{c}$ reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome $\text{c}$ reductase activity.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-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 $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ 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 $\text{c}$ 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 $\text{c}$ 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 $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

The involvement of cytochrome P-450 in the NADH-dependent O-demethylation of p-nitroanisole in phenobarbital-treated rabbit liver microsomes.

Addition of $\text{p}$-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 $\text{p}$-nitroanisole. $\text{p}$-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 $\text{p}$-nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b₅ and that cytochrome P-450 is the enzyme which catalyzes $\text{p}$-nitroanisole O-demethylation.     

Induction of polysubstrate monooxygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus NRRL 3240

The presence of the components of polysubstrate monooxygenase (PSMO) activity, viz., cytochrome P-450 and NADPH cytochrome P-450 reductase has been established for the first time in the microsomes of $\text{Aspergillus parasiticus}$. The microsomes were able to metabolize benzphetamine. NADPH cytochrome P-450 reductase, benzphetamine metabolism and aflatoxin production was increased by the presence of phenobarbitone (PB, 2mg/ml) in the medium. These results demonstrate that induction of PSMO activity could be a prerequisite for increased production of aflatoxins, since hydroxylation of intermediates is an obligatory step in aflatoxin biosynthesis.     

The effect of extra bound cytochrome b-5 on cytochrome P-450-dependent enzyme activities in liver microsomes

Binding of increasing amounts of detergent-purified cytochrome $\text{b}{}_5$ 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 $\text{b}{}_5$ in these two reactions functions as an intermediate electron carrier to cytochrome P-450.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ 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 $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. 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 $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ 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 ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). 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.     

Esherichia coli pyruvate dehydrogenase complex: improved purification and the flavin content.

The $\text{E. coli}$ pyruvate dehydrogenase complex, when purified by published procedures, contains phosphotransacetylase and coenzyme A as trace contaminants as well as one or more spectral contaminants which interfere with spectral and radiochemical experiments. They can be removed by further chromatographic purification on columns of calcium phosphate gel-cellulose. The resulting complexes from $\text{E. coli}$ K12 or Crookes strain are indistinguishable with respect to visible spectrum, catalytic activity, and flavin content. The activity is the highest so far reported, 40–42 μmoles DPNH per min per mg of protein, and the flavin content is 1.8–2.4 nanomoles per mg of protein.