Isolation of immunochemically distinct form of cytochrome P-450 from microsomes of tulip bulbs

A highly purified cytochrome P-450 was obtained from the microsomes of tulip bulbs (Tulipa gesneriana L.). The molecular weight (Mr = 52,500) and amino acid composition of this plant cytochrome P-450 are similar to those reported for rat livers. On the contrary, Ouchterlony double diffusion analyses indicated that cytochrome P-450 isolated from tulip bulbs shares no common antigenic determinants with those of 9 other plants, in spite of the presence of comparable contents of cytochrome P-450 and/or trans-cinnamate 4-monooxygenase with tulip bulbs.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.

A solubilized preparation of cytochrome P-450, obtained by treatment of mitochondria from bovine corpora lutea with phospholipase A, contained all of the necessary components for the cholesterol side chain cleavage activity. The solubilized cytochrome -450 preparation could be isolated essentially free of endogenous cholesterol side chain cleavage activity by various fractionation techniques. A cholesterol side chain cleavage enzyme system was reconstituted using the isolated cytochrome P-450 preparation and purified adrenodoxin and adrenodoxin reductase (components of the enzyme system purified from the adrenal cortex). Protein kinase was partially purified from the cytosol fraction of bovine corpora lutea. It was purified 43-fold and the activity was highly dependent on cyclic adenosine 3:5-monophosphate (cyclic AMP). When ATP and this partially purified cyclic AMP-dependent protein kinase were added to the reconstituted cholesterol side chain cleavage enzyme assay in which cytochrome P-450 was limiting, a stimulation (20 to 74%) of the conversion of cholesterol into pregnenolone was observed. This stimulation was statistically significant with p value less than 0.001. The stimulatory effect of the protein kinase appeared to be dependent on ATP and was not mimicked by bovine serum albumin, indicating that the effect was specific for protein kinase. Protein kinase caused a phosphorylation of the cytochrome P-450 preparation when large amounts of this preparation were used in the assay. It is concluded from these results that the direct activation of the cytochrome P-450 component of the cholesterol side chain cleavage by protein kinase may be one of the ways by which cyclic AMP mediates the effect of luteinizine.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl-3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and alpha and beta bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. 1 alpha-hydroxycholecalciferol (at 25), testosterone (at 2 alpha and 16 alpha) and dehydroepiandrosterone (at 16 alpha). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglobulin per nmol of cytochrome P-450.     

Interaction of cytochrome P-450 with hydrocarbons

Hydrocarbons of different structures interact with microsomal and solubilized cytochrome P-450 from liver of phenobarbital-pretreated rats forming a high spin enzyme-substrate type complex. The affinity of cytochrome P-450 for hydrocarbons increases with increasing lipophilicity independently of the chemical structure. The binding capacity of microsomal P-450 for aliphatic hydrocarbons is generally higher than for aromates. Mutual influence in binding of two different hydrocarbons by microsomal P-450 is stronger among aromatic than among aliphatic hydrocarbons; in both cases it appears to be effected rather by specific interaction of both substances with the binding site than by a nonspecific influence on the microsomal membrane. Only one fraction of low spin form of solubilized cytochrome P-450 from rat liver interacts with hydrocarbons. The binding capacity for aromatic and aliphatic substances corresponds to that found in microsomes. The affinity for the most lipiphilic substrate, perhydrophenanthrene, is equal in microsomal and solubilized preparation; with decreasing lipophilicity the affinity of solubilized P-450 decreases faster than in microsomes. The LM2 fraction of cytochrome P-450 from phenobarbital-pretreated rabbits interacts only with aliphatic hydrocarbons with wide variation of the binding capacity. The affinity is generally one order of magnitude lower than in microsomes. Active fractions of solubilized P-450 from both species are rapidly converted to P-420 by dithionite. The extent of this conversion is strongly reduced by saturation with substrate.     

Human placenta estrogen synthetase (aromatase) purified by affinity chromatography

Microsomal estrogen synthetase (cytochrome P-450ES), also known as aromatase, was purified from fresh human placenta microsomes by DEAE-Trisacryl and testosterone-agarose chromatography. Estrogen synthetase assays were done with androstenedione as substrate, NADPH as electron donor, and a partially purified P-450 reductase from human placenta as the electron carrier. The specific cytochrome P-450 content of the purified P-450 was 0.67 nmol mg-1 of protein, and the preparation contained no cytochrome P-420. The absorbance maximum was 448.5 nm. The specific estrogen synthetase activity of the purified P-450ES fraction was 35 nmol min-1 nmol-1 of cytochrome P-450 or 23.3 nmol min-1 mg-1 of protein. The latter value shows a 179-fold purification with a yield greater than 1% in the two-step procedure. Kinetic constants for the reaction were measured with androstenedione as the aromatizable substrate. The Km was 1.4 nM and the Vmax was 37 nmol min-1 nmol-1 of P-450. The purified enzyme aromatized androstenedione and testosterone at identical rates; androstenedione gave only estrone, and testosterone gave only estradiol-17 beta. Dehydroepiandrosterone was not detectably aromatized or otherwise metabolized. Neither 16 alpha-hydroxytestosterone nor 16 alpha-hydroxyandrostenedione was aromatized. No hydroxysteroid dehydrogenase or reductase was detected in direct assays. No free reaction intermediates were detected in aromatization assay incubation mixtures. The purity of the product and the simplicity of the preparation recommend it for use in further studies of the enzyme.     

Characterization and identification of a pyrazole-inducible form of cytochrome P-450

In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from rat livers to electrophoretic homogeneity and its biochemical, spectral, and immunological properties characterized. The final preparation had a specific content of 11 nmol of cytochrome P-450/mg of protein. A single band with an apparent molecular weight of 52,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the isolated pyrazole cytochrome P-450 displayed peaks at 648 and 396 nm, suggestive of a high spin cytochrome. The ethylisocyanide difference spectrum exhibited two maxima, one at 457 nm, the other at 428 nm. Pyrazole and dimethyl sulfoxide produced binding spectra with the purified P-450, with peaks at 425 or 419 nm and troughs at 390 or 386 nm, respectively. K8 values for dimethyl sulfoxide and pyrazole were 21 and 0.04 mM, respectively. The catalytic activity of the pyrazole cytochrome P-450 was elevated with aniline and dimethylnitrosamine (low Km) but not with aminopyrine, benzphetamine, ethoxycoumarin, or ethoxyresorufin as substrates. An antibody against pyrazole cytochrome P-450 recognized a 52,000 molecular weight protein upon reaction with saline microsomes. The intensity of the immunoblot was increased when microsomes isolated from pyrazole, 4-methylpyrazole-, acetone-, or chronic ethanol-treated rats were utilized, but not after phenobarbital or 3-methylcholanthrene treatment. Homology at the amino terminus of 19 amino acids was observed between pyrazole P-450 and the isoniazid-inducible P-450j. Based upon the above catalytic, spectral, and immunological properties, it appears that pyrazole induces a form of cytochrome P-450 which is identical to that induced by ethanol and isoniazid.     

A highly purified preparation of cytochrome P-450 from microsomes of anaerobically grown yeast.

Cytochrome P-450 was purified from microsomes of anaerobically grown yeast to a specific content of 12–15 nmoles per mg of protein with a yield of 10–30%. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis, the purified preparation yielded a major protein band having a molecular weight of about 51,000 together with a few faint bands. It was free from cytochrome b₅, NADH-cytochrome b₅ reductase, and NADPH-cytochrome c (P-450) reductase. In the oxidized state it exhibited a low-spin type absorption spectrum, and its reduced CO complex showed a Soret peak at 447–448 nm. It was reducible by NADPH in the presence of an NADPH-cytochrome c reductase preparation purified from yeast microsomes. Its conversion to the cytochrome P-420 form was much slower than that of hepatic cytochrome P-450.     

The active form of cytochrome P-45011beta from adrenal cortex mitochondria.

Cytochrome P-45011beta has been solubilized and partially purified from bovine adrenal cortex mitochondria by means of chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but had a low specific content of P-450 (between 1 and 2 nmol of P-450 per mg of protein). In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11beta-hydroxylation of unconjugated and sulfoconjugated deoxycorticosterone. In the reconstituted system the hydroxylation of deoxycorticosterone sulfate proceeded at a much higher rate than in intact mitochondria, indicating that in the former case interactions between the hydrophilic substrate and P-450 were facilitated. In the presence of Triton X-100 the partially purified cytochrome P-45011beta had a Stokes radius of 4.5 nm, a sedimentation coefficient of 3.1 S, and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011beta . Triton X-100 complex had a molecular weight of about 100,000 and that P-45011beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011beta . Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011beta is the active form of the protein.     

Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung

1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.     

Molecular properties of cytochrome P-45011 beta from adrenal cortex mitochondria.

Cytochrome P-45011 beta has been solubilized and partially purified from bovine adrenal cortex mitochondria using chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by SDS-polyacrylamide gel electrophoresis. In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11 beta-hydroxylation of unconjugated and sulphoconjugated deoxycorticosterone. In presence of Triton X-100 the partially purified cytochrome P-45011 beta had a Stoke's radius of 4.5 nm, a sedimentation coefficient of 3.1 S and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011 beta-Triton X-100 complex has a molecular weight of about 100 000 and that P-45011 beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011 beta-Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011 beta is an active form of the protein.     

Purification and characterization of a form of cytochrome P-450 with high specificity for aflatoxin B1 from 3-methylcholanthrene-treated hamster liver

A form of cytochrome P-450 highly active in inducing mutagenicity of aflatoxin B1 was purified to a specific content of 15.1 nmol/mg of protein from 3-methylcholanthrene-treated hamster liver. This species of cytochrome P-450, having its absorption maximum at 448.5 nm in carbon monoxide-complex of reduced form and low spin ferric ion, is of molecular weight of 56,000 and distinctly different in physicochemical and catalytic properties from major forms of cytochrome P-450 purified from phenobarbital- or 3-methylcholanthrene-treated rat liver. In the induction of aflatoxin B1 mutagenicity, this hamster cytochrome P-450 is 50 times more potent than those from rat liver.     

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (Mr 120000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its Mr of 38000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH2-terminal amino acid is proline; the penultimate NH2-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Mouse liver testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha). Purification, regioselectivity, stereospecificity, and immunochemical characterization

Microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) was purified from the livers of male 129/J mice based on enzyme activity in the eluates from columns of DEAE Bio-Gel A, hydroxylapatite, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified P-450(16) alpha fraction was 9.5 nmol/mg of protein. The specific testosterone 16 alpha-hydroxylation activity of the purified P-450(16) alpha fraction was 80 nmol/min/nmol of cytochrome P-450 or 764 nmol/min/mg of protein, and these values were about 40- and 400-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(16) alpha showed extremely high regioselectivity and stereospecificity for testosterone hydroxylation; more than 90% of the testosterone metabolites formed by the purified P-450(16) alpha fraction was 16 alpha-hydroxytestosterone. The purified anti-P-450(16) alpha antibody exhibited absolute specificity for inhibition of testosterone 16 alpha-hydroxytestosterone was inhibited by the anti-P-450(16) alpha. Anti-P-450(16) alpha inhibited the 16 alpha-hydroxylation activity of intact microsomes prepared from livers of male or female 129/J mice more than 90%, indicating that P-450(16) alpha is the major cytochrome P-450 isozyme catalyzing 16 alpha-hydroxylation activity of testosterone in these microsomal preparations. The purified P-450(16) alpha fraction also possessed high benzphetamine N-demethylation activity relative to the rates found with other xenobiotic substrates tested in this report.     

Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes

Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14 alpha-demethylase and cholesterol 7 alpha-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel and adsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mg of protein and a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 51,000. A Soret maximum for the reduced/CO binding complex at 448 nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochrome-c reductase, dilaurylphosphatidylcholine, NADPH, and O2 supports the demethylation process which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediates with single catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P-450 is responsible for all three oxidations and the lyase activity involved in the lanosterol C-32 demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterol substrates is observed for the purified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutive P-450 involved in anabolic biochemical processes.     

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.