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.     

Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

The roles of rabbit liver cytochrome b₅, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450_LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450_LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450_LM3. Cytochrome b₅ enhanced cytochrome P-450_LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450_LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b₅ was present in the membrane.     

Maintenance of microsomal cytochromes b5 and P-450 in primary cultures of parenchymal liver cells on collagen membranes

In previous reports from various laboratories, the levels of the microsomal cytochromes b₅ and P-450 in hepatocytes in primary culture have been found to be very low and difficult to measure. The studies reported in this paper demonstrate that cytochromes b₅ and P-450 in hepatocytes cultured on floating collagen membranes for periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of liver $\text{in}$$\text{vivo}$. Addition of high levels of hydrocortisone (10^?4M) to the culture medium for periods up to 10 days resulted in further increases in the levels of these cytochromes. Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained.     

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.     

Purification and properties of P-450LM3b, a constitutive form of cytochrome P-450, from rabbit liver microsomes

This laboratory has previously reported the occurrence in rabbit liver microsomes of a non-inducible form of cytochrome P-450, designated P-450lm_3b because of its electrophoretic mobility relative to that of phenobarbital-inducible P-450lm₂ and 5,6-benzoflavone-inducible P-450lm₄. In the present study, P-450lm_3b was purified to electrophoretic homogeneity and a specific content of over 19 nmol per mg of protein by chromatographic procedures carried out in the presence of detergents. The isolated cytochrome has a minimal molecular weight of 52,000 and exhibits absorption maxima at 418, 537, and 571 nm in the oxidized state, 412 and 547 nm in the reduced state, and 451 and 555 nm as the CO complex. In a reconstituted system containing NADPH-cytochrome P-450 reductase and phosphatidylcholine, P-450lm_3b has relatively high activity in the hydroxylation of testosterone in the 6β and 16α positions as well as significant activity toward a number of other substrates tested. The NADPH oxidase activity of P-450lm_3b is less than half that of P-450lm₂ and lm₄.     

Induction by Triacetyloleandomycin and partial purification of a LM3 form of cytochrome P-450 from rabbit liver microsomes

Treatment of rabbits with Triacetyloleandomycin (a currently used antibiotic in human therapy) at 1 mmol per kg of body weight daily for 5 days results in a significant induction of liver microsomal cytochrome P-450, (2.6 nmol/mg proteins). Electrophoresis in SDS polyacrylamide gels shows this increase in P-450 is associated to the appearance of a strong band in a zone located between the major bands of microsomes induced by phenobarbital and β-naphtoflavone (LM₃ forms in Coon's terminology). Partial purification of this P-450 LM₃ (TAO) was undertaken by chromatographic procedures (CM cellulose and hydroxylapatite). Its subunit molecular weight is 52 000; the absolute spectra in the oxidized, ferrous and CO-ferrous forms present maxima at 417, 536, and 570 nm; 415 and 548 nm; 450 and 555 nm respectively. Monooxygenase activity of LM₃ (TAO) was compared with that of LM₂ and LM₄ in a reconstituted system containing NADPH cytochrome P-450 reductase and phosphatidylcholine; the activity of P-450 LM₃ (TAO) was higher than that of LM₂ and LM₄ with chlorcyclizine as a substrate. According to these observations, LM₃ (TAO) resembles LM₃ (b), a constitutive form of untreated rabbit liver microsomes.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

Highly purified microsomal P-450: the oxyferro intermediate stabilized at low temperature.

The oxyferro intermediate of highly purified microsomal P-450 from rabbit liver was formed and stabilized at ?30°C in a mixture of aqueous buffer and glycerol ($\text{1}\text{1}$). Absolute and difference (Fe^2·+O₂-Fe³⁺) spectra of this intermediate appear to be very similar to those obtained under either steady state kinetics or stopped flow conditions on the same cytochrome as well as on bacterial P-450_cam. (Absolute and difference spectra present maxima at 420 and 557–558 nm and a broad maximum at 442 nm respectively). As temperature increases the oxyferro intermediate autoxidizes and ferric cytochrome P-450 is restored. This reaction appears to follow biphasic first order kinetics. The rate constant of both phases decreases with temperature and increases with protons concentrations.     

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.     

Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

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 $\text{c}$ 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.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

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.     

Structural resemblance of cytochrome P-450 isolated from Pseudomonas putida and from rabbit liver microsomes

The cytochrome P-450 of $\text{Pseudomonas putida}$ (P-450_cam) and that of phenobarbital-induced liver microsomes (P-450_LM) 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-450_cam and P-450_LM 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.     

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.     

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.     

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phenobarbital administration to rats on the level of the in vitro synthesis of cytochrome P-450 directed by total rat liver RNA.

Total liver RNA has been isolated from male rats at different time points subsequent to a single injection of phenobarbital, and the level of cytochrome P-450 synthesis directed by these RNA preparations in a cell-free translation system has been determined. It is observed that the maximum $\text{in vitro}$ synthesis of cytochrome P-450 occurs at 16 hours (3-fold above uninduced level) which is approximately 30 hours prior to the maximum induction of spectrophotometrically detectable cytochrome P-450 measured in liver homogenates. Thus, while cytochrome P-450 mRNA is involved in the induction process, its synthesis does not appear to be rate limiting. In addition, phenobarbital induced cytochrome P-450 is not synthesized $\text{in vitro}$ in a form larger than that isolated from endoplasmic reticulum, but rather is also found to have a molecular weight of 50,000.     

Purification and characterization of a new form of cytochrome P-450 (LM2b) from untreated male rabbit liver microsomes

A new form of cytochrome P-450 has been purified from untreated male rabbit liver microsomes. This form was designated P-450 LM2b on the basis of its electrophoretic mobility on SDS polyacrylamide gel, where it migrates as a polypeptide of apparent molecular weight of 50,250. This hemoprotein exhibits a maximum at 448.5 nm in the Soret band of the CO-Ferrous state spectrum. On the basis of its molecular, spectral, enzymologic and immunologic data, P-450 LM2b was shown to be distinct from the other P-450 forms, already characterized in rabbit liver microsomes. However P-450 LM2b and P-450 LM3b appear to be immunologically related proteins.     

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.     

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.