Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450

A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.     

Isolation and characterization of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.     

Differential induction and tissue-specific expression of closely related members of the phenobarbital-inducible rabbit cytochrome P-450 gene family

We have examined the tissue-specific expression of three rabbit genes that are closely related members of a subfamily of the phenobarbital-inducible cytochrome P-450 gene family. Analysis of the levels of mRNA in liver revealed that (a) cytochrome P-450PBc1 mRNA was not detectable in livers from control animals but was present in livers from animals treated with phenobarbital, (b) cytochrome P-450PBc2 was present in control tissue and was increased by about 3-fold 24 h after phenobarbital treatment, and (c) the levels of cytochrome P-450PBc3 mRNA was the same in livers from control and treated animals. In the kidney, only P-450PBc2 mRNA was detected at a level 15% of that in the liver, and the levels increased about 3-fold after phenobarbital treatment. None of the mRNAs was detected in lung tissue. Multiple species of RNA were observed that hybridized to probes for cytochrome P-450PBc1 and P-450PBc2 cDNAs by Northern blot analysis ranging in size from 2300 to 4000 nucleotides. Differential sites for polyadenylation probably cause the heterogeneity in size. A single species of RNA of 2200 nucleotides that hybridized to cytochrome P-450PBc3 cDNA probes was observed. These data demonstrate that three closely related cytochrome P-450 genes are differentially responsive to phenobarbital treatment and that they exhibit different tissue-specific patterns of expression.     

Isolation and partial purification of cytochrome P-450 from uninduced rat liver

Carbon-13 Fourier transform nmr has been used to make the first observation of a carbon-13-iron-57 spin-spin coupling constant in a protein, sperm whale carbonyl myoglobin enriched to 90% in both iron-57 and carbon-13. The coupling constant, 27.1±0.2 Hz, is found to be essentially identical to that of a model compound, supporting the view that the carbonyl is not tilted with respect to the heme plane in solution. Such carbon-13-iron-57 couplings, and the resultant iron-57 chemical shifts obtained from decoupling experiments, should provide valuable new tools for studying the different affinity states of tetrameric hemoglobins.     

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.     

Isolation of a cytochrome P-450e gene variant and characterization of its 5' flanking sequences

A cytochrome P-450e gene variant has been isolated from the rat liver genomic library. It is a typical e gene clone but unique in having b-like single base substitutions at specific sites in the 5' flanking region. It also appears to have certain additional restriction sites in the introns. When compared with the cytochrome P-450b gene, the e gene has some of the repetitive motifs interrupted in the 5' flanking region. In addition, this region is characterized by the presence of alternating pyrimidine-purine stretch, steroid hormone regulatory elements, consensus eukaryotic enhancer sequence and sequences involved in general amino acid regulation.     

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.     

Hepatic mitochondrial cytochrome P-450 system. Identification and characterization of a precursor form of mitochondrial cytochrome P-450 induced by 3-methylcholanthrene

Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.     

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1

Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of thromboxane synthase from human platelets as a cytochrome P-450 enzyme

Thromboxane synthase from human platelets was purified to apparent homogeneity by conventional chromatographic techniques. A 423-fold enrichment over the specific content in the 100,000 X g sediment from platelet homogenates was obtained. The enzyme gave a single band on sodium dodecyl sulfate-gel electrophoresis corresponding to a monomeric molecular weight of 58,800. One heme per polypeptide chain was present, and by optical and EPR spectroscopy a close analogy to the group of cytochrome P-450 proteins was established. From its substrate prostaglandin H2, the stable end product thromboxane B2 is formed with a specific activity of 24.1 mumol min-1 mg of protein-1 which corresponds to a molecular activity of 1628 min-1. The enzyme formed 12L-hydroxy-5,8,10-heptadecatrienoic acid together with thromboxane B2 in a 1:1 ratio. Both products were identified by gas chromatography-mass spectrometry analysis. As reported previously for platelet microsomes (Ullrich, V., and Haurand, M. (1983) Adv. Prostaglandin Thromboxane Leukotriene Res. 11, 105-110), the pure hemoprotein spectrally interacts with pyridine- or imidazole-based inhibitors and for the potent inhibitor imidazo-(1,5-a)pyridine-5-hexanoic acid a stoichiometric binding to the heme was shown. Substrate analogs with a methylene group replacing the oxygen in either the 9- or 11-position caused difference spectra showing spectral shifts towards 387 and 407 nm, respectively. The identification of thromboxane synthase as a P-450 protein suggests that the heme-thiolate group of the enzyme is required to split and activate the endoperoxide bond of prostaglandin H2.     

Cloning and characterization of bovine cytochrome P-450(11 beta) genes

We isolated 4 different clones of the P-450(11 beta) gene from a bovine genomic library. These genomic clones were highly homologous with each other. Two of the isolated clones were pseudogenes. Determination of its nucleotide sequences indicated that the bovine P-450(11 beta) gene is divided into 9 exons by 8 introns and that it is about 8.5 kb in total length. The number of exons and the locations of intron insertion into the P-450(11 beta) gene are identical with those in the case of P-450(SCC), but different from those of other microsomal P-450s.     

Purification and partial characterization of hepatic microsomal cytochrome P-450s from phenobarbital- and 3-methylcholanthrene-treated rats.

Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's procedures )1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16 nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities. In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous conbinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common. Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.     

Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.     

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.     

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.