Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.     

Induction and catalytic activities of cytochromes P-450b/e and P-450c in the liver microsomes of neonatal rats

The activities of cytochrome P-450-dependent monooxygenases has been investigated in the liver microsomes of newborn rats (3-16 days after birth) induced with PB or 3-MC. It has been shown that the induction by PB and 3-MC results in the increase of both the total amount of cytochrome P-450 as determined by the CO-reduced spectrum and the amount of induced forms P-450b/e and P-450c respectively. In the course of induction of the specific forms of cytochrome P-450 BP-hydroxylase and 7-ER-O-deethylase activities increased at 3-MC-induction, while BPh-N-demethylase and BP-hydroxylase increased at PB-induction. Analysis of inhibition of monooxygenase reactions with antibodies has showed that only P-450c was involved in metabolism of BP and 7-ER. Participation of P-450b/e in BPh N-demethylation was notably lower in the neonates in comparison to the adult rats. In the one-week-old rats induced with 3-MC a considerable rate of BP hydroxylation and 7-ER O-deethylation (2-4.5 nmol of product min-1 mg-1) has been observed despite a small amount of P-450 (0.02-0.1 nmol/mg of protein). This fact shows the higher catalytic activity of this cytochrome P-450 in the neonates compared to similar characteristics of P-450c in the 3-MC-induced microsomes. Metabolism of BP in the PB-microsomes of the neonatal rats was inhibited neither by anti-P-450b/e nor anti-P-450c in contrast to the adults, where this reaction was inhibited by antibodies against P-450b/e.     

Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception

We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.     

Purification of cytochrome P-450 catalyzing 25-hydroxylation of vitamin D3 from rat liver microsomes

Cytochrome P-450 catalyzing 25-hydroxylation of cholecalciferol (cytochrome P-450 cc25 ) was purified from rat liver microsomes based on its catalytic activity at each purification step. The specific cytochrome P-450 content of the final preparation was 15.1 nmol/mg of protein. Reconstituted activity of 25-hydroxylation of cholecalciferol with the purified enzyme was 2.3 nmol/min/mg of protein, which was 4,300 times as high as that in microsomes. The minimum molecular weight of the enzyme was 50,000 based on SDS-polyacrylamide gel electrophoretogram. Amino terminal sequence of the P-450 cc25 was H2N-Met-Asp-Pro-Val-Leu-Val-. Immunochemical study showed that the purified P-450 cc25 was homogeneous and the cytochrome was immunochemically different from either cytochrome P-450(PB-1) or cytochrome P-448(MC-1).     

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 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

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.     

Perfluorocarbons do not induce cytochrome P-448 in the rat liver

Cytochrome P-450 forms appearing in the liver after injection of methylcholanthrene, polychlorinated biphenyls and perfluorochemical emulsion to rats were studied. Activities of marker enzymes, benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, as well as the interaction of liver microsomal membranes with antibodies against different cytochrome forms were investigated. It was shown that fluorocarbon emulsion containing perfluorodecalin did not induce cytochrome P-448 in the rat liver.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.     

Comparison of cytochrome P-450 forms induced by phenobarbital and 1,4-bis[3,5-dichloropyridyloxy)]benzene in the mouse and rat liver

A form of cytochrome P-450 (P-450PB) with a molecular weight of 53.5-54.0 kD possessing a high benzphetamine-N-demethylase activity (100-120 nmol formaldehyde/min/nmol cytochrome) was isolated from liver microsomes of phenobarbital-induced C57Bl/6 mice. This cytochrome P-450 form is immunologically identical to its rat liver counterpart-P-450b (Mr = 52 kD) which is also characterized by a high rate of benzphetamine-N-demethylation. It was shown that 1.4-bis[2-(3.5-dichloropyridyloxy])benzene (TCPOBOP) induces in mouse liver the synthesis of the monoxygenase form whose substrate specificity and immunologic properties are identical to those of cytochromes P-450PB and P-450b. The immunochemically quantitated content of this form makes up to 20% of the total P-450 pool in liver microsomes of phenobarbital- or TCPOBOP-induced mice. Immunochemical analysis of microsomes with the use of antibodies to cytochromes P-450PB and P-450b revealed the presence on the electrophoregrams of phenobarbital-induced rat liver microsomes of two immunologically identical forms of cytochrome P-450, i.e., P-450b and P-450e (the latter had a low ability to benzphetamine N-demethylation). Liver microsomes of phenobarbital- or TCPOBP-induced mice gave only one precipitation band corresponding to cytochrome P-450PB.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Purification and characterization of two forms of chicken liver cytochrome P-450 induced by 3,4,5,3',4'-pentachlorobiphenyl

3,4,5,3',4'-Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of liver microsomal monooxygenases, particularly 7-ethoxyresorufin (7-ER) O-deethylase, benzo(a)pyrene (BP) 3-hydroxylase, and testosterone 16 alpha-hydroxylase in chickens, but not NADPH-cytochrome c(P-450) reductase and cytochrome b5. Two forms of cytochrome P-450 (P-450) in liver microsomes of PenCB-treated chickens were purified and characterized. The absorption maxima of the CO-reduced difference spectra of both enzymes (chicken P-448 L and chicken P-448 H) were at 448 nm. From the oxidized form of their absolute spectra, chicken P-448 L was a low-spin form and chicken P-448 H was a high-spin form. They had molecular masses of 56 and 54 kDa, respectively. In a reconstituted system, 7-ER O-deethylation, BP 3-hydroxylation, and testosterone 16 alpha-hydroxylation were catalyzed at high rates by chicken P-448 L but not by chicken P-448 H. Chicken P-448 L also catalyzed N-demethylation of aminopyrine, benzphetamine, and ethylmorphine with relatively low activity. On the other hand, chicken P-448 H functioned only in catalyzing estradiol 2-hydroxylation. These results were supported by an inhibition study of microsomal monooxygenases using an antibody against each enzyme. Immunochemical studies revealed that the enzymes differ from each other but are both inducible by PenCB-treatment. Chicken P-448 L and chicken P-448 H respectively comprise about 82 and 7% of the total P-450 content in chicken liver microsomes.     

Functional characteristics of cytochromes P-4501A1 and P-4501A2 in rodent liver

Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) inhibit the 0-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benz(a)pyrene-induced (BP) mice but do not inhibit the 0-deethylase activity in liver microsomes of BP-induced rats. Anti-P3-450 and anti-P-450c inhibit BP-hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes. In a reconstituted monooxygenase system isolated cytochrome P3-450 metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, did not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min/nmol cytochrome. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes. The interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c was accompanied by the appearance of a single band (cytochrome P3-450).     

Isolation by ion-exchange high performance liquid chromatography of rabbit liver cytochrome P-450 with regioselectivity for omega-hydroxylation of prostaglandins

Previous studies suggested that rabbit liver microsomes contain cytochrome P-450 monooxygenase(s) with low affinity for (omega-1)-hydroxylation and high affinity for omega-hydroxylation of prostaglandins (Theoharides, A. D., and Kupfer, D. (1981) J. Biol. Chem. 256, 2168-2175). The current investigation describes the isolation from livers of untreated rabbits of a cytochrome P-450 catalyzing, with regioselectivity, the omega-hydroxylation of prostaglandins E1 and E2. The isolation of the enzyme involved enrichment of the omega-hydroxylase activity by polyethylene glycol 8000 fractionation, followed by ion-exchange high performance liquid chromatography. Based on Mr of 59,000-60,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated enzyme is referred to as P-450 form 7. This P-450 exhibits a low spin spectrum (lambda max = 417 nm) and a difference spectrum of the CO-reduced complex versus reduced (lambda max = 451 nm). For catalytic activity, the P-450 form 7 was reconstituted with NADPH-P-450 reductase, cytochrome b5, and lipid. There was no activity in the absence of the reductase, and deletion of cytochrome b5 yielded a minimal amount of product (heme could not substitute for cytochrome b5), demonstrating an absolute requirement for these components.     

Identification of the forms of cytochrome P-450 induced in rat liver by 2-acetylaminofluorene using immunoblotting and partial purification

The amounts of 5 different forms of cytochrome P-450 in liver microsomes from rats treated with 2-acetylaminofluorene were determined and compared with the corresponding patterns in microsomes from control, 3-methylcholanthrene- and phenobarbital-treated animals. 2-Acetylaminofluorene was found to increase the amount of cytochromes P-450b + e 10-fold and of cytochrome P-450d 3-fold, while there was a 54% increase in the level of cytochrome P-450 PB/PCN-E. Cytochrome P-450c was increased from a level too low to detect (less than 0.001 pmol/mg protein) to 0.019 pmol/mg protein. These findings were also confirmed by partial purification of cytochromes P-450b + e and c after 2-acetylaminofluorene treatment.     

Characteristics of 3,4-benzopyrene-induced cytochrome P-448 forms isolated from mouse liver microsomes

Two cytochrome P-448 fractions, B1 and B2, were isolated from liver microsomes of 3,4-benzpyrene-induced inbred C57Bl/6 mice, using chromatography on octyl-Sepharose CL-4B and on Whatman 52E. During subsequent chromatography on hydroxylapatite fraction B1 was separated into 2 subfractions, G1 and G2. Cytochrome fractions B1, G1 and G2 have similar "peptide maps" differing from that of fraction B2. Cytochrome fraction B1 is immunologically identical to G2, partly to fraction B2 but is distinct from fraction G1. Fraction G2 is identified as the form of cytochrome P-448 catalyzing the hydroxylation of 3,4-benzpyrene and 7-ethoxyresorufin and existing in a low spin form. Cytochrome fraction G1 is apparently identical to the form P3-450. Fraction B2 was not yet described in current literature, since cytochrome P-448 (Mr = 53,000 Da) was identified only after the induction of mice with 3,4-benzpyrene but not with other inducers, e.g., polycyclic aromatic hydrocarbons.     

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.