Characterization of complementary DNA clones coding for two forms of 3-methylcholanthrene-inducible rat liver cytochrome P-450

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.     

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Structural analysis and specific expression of microsomal cytochrome P-450(M-1) mRNA in male rat livers

cDNA clones for the P-450(M-1) mRNA, which exhibits a male-specific expression in rat livers, were isolated by using synthetic oligonucleotides as the probes. Sequence analysis of the cDNAs showed that P-450(M-1) mRNA contains 1,853 nucleotides in addition to a poly(A) chain, and a single open reading frame of 1,500 nucleotides encodes a polypeptide of 500 amino acids with a Mr = 57,187. The predicted NH2-terminal sequence of 30 amino acids agrees well with that of the purified protein determined by Edman degradation, and the predicted primary structure included all the partial sequences of six internal peptides of P-450(M-1) obtained by the proteolytic digestion and a conserved amino acid sequence containing a putative heme-binding cysteine, proximate to the COOH terminus of the molecules. P-450(M-1) showed relatively high sequence similarity with P-450b (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797) (52% similarity), P-450-3b (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1985) J. Biol. Chem. 260, 5427-5434) (64%), P-450-1 (Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., and Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354) (74%), P-450PBc1 (Leighton, J. K., DeBrunner-Vossbrinck, B. A., and Kemper, B. (1984) Biochemistry 23, 4598-4603) (71%), while its sequence similarity with 3-methylcholanthrene-inducible P-450c and P-450d is rather low. Consequently, P-450(M-1) could be structurally classified into the phenobarbital-inducible type of P-450 gene family. RNA blot analysis using a synthetic oligonucleotide specific for P-450(M-1) revealed that P-450(M-1) mRNA was expressed exclusively in the livers of mature male rats in a sex-specific manner, but not in other tissues so far examined.     

Interaction of the 4 S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene

The induction of cytochrome P-450c, the isozyme most closely associated with aryl hydrocarbon hydroxylase activity in the rat, is mediated through a cytosolic polycyclic aromatic hydrocarbon (PAH)-binding protein(s). We have reported on the purification and characterization of a 4 S protein that interacts in a specific and saturable manner with [3H]benzo[a]pyrene and other PAHs. (W. H. Houser et al. (1985) Biochemistry 24, 7839-7845). We have also reported on the specific and saturable interaction of the 4 S protein with a plasmid containing 1.9 kbp of cloned rat P-450c sequence including exon 1, the 5' half of intron 1, and approximately 882 bp upstream information. Our investigations now show that incubation of this protein with a portion of the rat P-450c gene, followed by digestion with either lambda exonuclease or exonuclease III, tentatively identified two protected regions at -225 and -455 bp 5' from exon 1. To further study the significance of these protected regions, a 3.4-kbp fragment containing cytochrome P-450c promoter and 5'-upstream sequences (-882 to +2545) was fused to the chloramphenicol acetyl transferase (CAT) reporter gene and transfected into either rat epithelial RL-PR-C cells or rat hepatoma H-4-II-E cells. Both cell lines expressed CAT activity in response to induction by 3-methylcholanthrene (3MC), indicating that important regulatory regions responsive to 3MC are present in these constructs. However, neither cell line expressed CAT activity in response to 3MC when transfected with plasmids containing deletions (-95 to -724 or -240 to -720) in the regions shown to be protected by our footprinting studies. These results corroborate previous studies which indicated that the 4 S PAH-binding protein interacts in a specific manner with regions of the rat cytochrome P-450c gene. We conclude that the 4 S protein may play an important role in the regulation of expression of cytochrome P-450c in the rat.     

The cDNA and protein sequence of a phenobarbital-induced chicken cytochrome P-450

Several cDNA clones complementary to a chicken phenobarbital-inducible cytochrome P-450 have been isolated and sequenced, representing the first non-mammalian eukaryotic cytochrome P-450 sequence to be analyzed. The cDNA clones hybridized to two mRNAs of 3.5 and 2.5 kilobases in length, but further analysis indicated that the clones were derived from the larger mRNA. The sequence contains a 5'-noncoding region of 39 nucleotides and an open reading frame of 1473 nucleotides. The remainder of the sequence is due to the 3'-noncoding region and poly(A) tail. The open reading frame encodes a protein of 491 amino acids with a molecular weight of 56,196. The chicken cytochrome P-450 shows an overall homology of 45-54% compared with the mammalian phenobarbital-induced cytochrome P-450s. The degree of homology is not uniform, with some short regions showing much greater levels of sequence conservation. In particular, the chicken cytochrome P-450 contains the conserved cysteinyl domain near the carboxyl terminus, found in all cytochrome P-450s and which is thought to be involved in heme binding. Using the chicken sequence, a more accurate estimate of the evolutionary rates of cytochrome P-450s has been made. It is suggested that the phenobarbital-, 3-methylcholanthrene, and pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 gene families diverged from a common ancestral gene 600 million years ago. Furthermore the phenobarbital-inducible gene apparently underwent gene duplication events at about the time of the divergence of the chicken and mammalian lineages. The results imply that most mammals should have at least four rather distantly related phenobarbital-inducible gene subfamilies.     

Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam. Indications for a similar overall topology

An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam). All sequences available on-line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography. While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms. Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms. Other helices which seem to exist in all P-450 forms include helices C, D, G and J. K. In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences.     

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.     

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.     

Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver

The gene structure of cytochrome P-450b, a major form of phenobarbital-inducible cytochrome P-450 in rat livers was elucidated by sequence analysis of the cloned genomic DNAs and was compared with the previously determined gene structures of cytochrome P-450e, a minor form of phenobarbital-inducible cytochrome P-450 and two forms of 3-methylcholanthrene-inducible cytochrome P-450 (P-450c and -d). The gene for cytochrome P-450b is 23 kilobase pairs (kb) long and is separated into 9 exons by 8 intervening sequences. This gene structure is very similar to that of cytochrome P-450e except for the first intron, the first intron being much longer in cytochrome P-450b gene (approximately 12 kb) than in cytochrome P-450e gene (3.2 kb), but differs greatly from the gene structures of two 3-methylcholanthrene-inducible cytochrome P-450s as pointed out previously (Sogawa, K., Gotoh, O., Kawajiri, K. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5066-5070). The nucleotide sequences in all 9 exons and their flanking regions in introns show very close homology between the two phenobarbital-inducible cytochrome P-450 genes. Forty base substitutions are found in approximately 1900 nucleotides of all exonic sequences, and 15 of them result in 14 amino acid replacements. These base substitutions occur in relatively limited regions of the gene sequences. Most of them are found in exons 6, 7, 8, and 9, most frequently in exon 7 as described previously (Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958-3962). The close sequence homology between the two phenobarbital-inducible cytochrome P-450 genes is also found to extend to the promoter region with one notable exception. The simple repeated sequences of (CA)n which is present at -254 position in cytochrome P-450e gene is also observed at the equivalent position in cytochrome P-450b gene, but the repetitiveness is greatly reduced in cytochrome P-450b gene ((CA)5 for P-450b versus (CA)19 for P-450e), and this may somehow be related to the difference in the level of cytochrome P-450b and P-450e in the inductive phase of phenobarbital administration.     

Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium

Cytochrome P-450BM-3 (P-450BM-3) from Bacillus megaterium incorporates both a P-450 and an NADPH:P-450 reductase in proteolytically separable domains of a single, 119-kDa polypeptide and functions as a fatty acid monooxygenase independently of any other protein. A 5-kilobase DNA fragment which contains the gene encoding P-450BM-3 was sequenced. A single continuous open reading frame starting at nucleotide 1541 of the 5-kilobase fragment correctly predicted the previously determined NH2-terminal protein sequences of the trypsin-generated P-450 and reductase domains and, in toto, predicted a mature polypeptide of 1,048-amino acid residues with Mr = 117,641. The trypsin site was found at arginine residue 471. The P-450 domain is most similar (about 25%) to the fatty acid omega-hydroxylases of P-450 family IV, while the reductase domain exhibits some 33% sequence similarity with the NADPH:P-450 reductases of mammalian liver. Both the P-450 and reductase domains of P-450BM-3 define new gene families but contain highly conserved segments which display as much as 50% sequence similarity with P-450s and reductases of eukaryotic origin. The mRNA for P-450BM-3 was found by S1 mapping to be 3,339 +/- 10 nucleotides in length. In the accompanying paper, two regions in the 1.5 kilobases 5' to the P-450BM-3 coding region have been implicated in the regulation of P-450BM-3 gene expression.     

Molecular modeling of the 3-D structure of cytochrome P-450scc.

Sequence-alignment studies of the bovine mitochondrial cholesterol side-chain cleavage enzyme cytochrome P-450scc with the bacterial cytochrome P-450cam (camphor hydroxylating enzyme) have been undertaken. Our novel alignment of the sequences revealed 69 identical residues and many highly conserved regions. The results of the sequence alignment studies were used to model the 3-D structure of P-450scc based on the available crystal structure of P-450cam. The major insertions in the sequence are found mainly on four external-loop regions of the molecule, while the core structure of P-450cam is retained with subtle internal modifications. The most hydrophobic of these four external loops is proposed as a candidate for membrane attachment.     

The accumulation of distinct mRNAs for the immunochemically related cytochromes P-450c and P-450d in rat liver following 3-methylcholanthrene treatment

Treatment of rats with 3-methylcholanthrene leads not only to a marked accumulation in the liver of translatable mRNA coding for a 56-kilodalton polypeptide representing cytochrome P-450c, the major 3-methylcholanthrene-induced cytochrome P-450 of rat liver, but also to the accumulation of comparable amounts of mRNA encoding a 52-kilodalton polypeptide which is immunoprecipitated with antibodies prepared against rat liver cytochrome P-450c. Further electrophoretic and immunochemical characterization of the latter translation product demonstrates that it corresponds to cytochrome P-450d, the major isosafrole-induced form of rat liver cytochrome P-450. The mRNAs for cytochromes P-450c and P-450d can be completely separated by electrophoresis in denaturing agarose gels and have chain lengths of approximately 4000 and 2000 nucleotides, respectively. These two mRNAs do not show detectable sequence homology to the mRNAs coding for the major phenobarbital-induced forms of cytochrome P-450 (P-450b and P-450e) since in Northern blotting experiments they fail to hybridize under conditions of low to moderate stringency to cloned probes for the latter mRNAs.     

Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G

The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromes P-450 from rat, P-450BNF/MC-B and P-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) only P-450BNF/MC-B mRNA, (b) only P-450ISF/BNF-G mRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these two P-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primary in vitro translation products of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, the P-450BNF/MC-B mRNA was found to be 2900 bases long, while the P-450ISF/BNF-G mRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected for P-450BNF/MC-B mRNA, while a 3100-base precursor was detected for P-450ISF/BNF-G mRNA. These two mRNAs were induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, the P-450BNF/MC-B mRNA was consistently present at very low levels while the P-450ISF/BNF-G mRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs were measured by dot blot hybridization. P-450BNF/MC-B mRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholanthrene, while the P-450ISF/BNF-G mRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates; P-450BNF/MC-B mRNA dropped by about 20% during the next 24 h, while P-450ISF/BNF-G mRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete cDNA and protein sequence of a pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450. A representative of a new gene family

A full-length cDNA complementary to rat liver mRNA coding for pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 (P-450PCN) was isolated and completely sequenced. P-450PCN mRNA is 2038 nucleotides in length and has a continuous reading frame (82-1596) that encodes a protein of 504 amino acids (Mr = 57,917). The amino-terminal sequence of 18 residues of the purified P-450PCN protein agrees with the open reading frame of the cDNA sequence. The P-450PCN mRNA nucleotide and amino acid sequences clearly establish that this cytochrome is a member of a separate P-450 family different from the phenobarbital-induced (e.g. P-450e) and 3-methyl-cholanthrene-induced (e.g. P-450c) P-450 gene families. P-450PCN shares 38 and 37% nucleotide similarity and 33 and 33% amino acid similarity with P-450e and P-450c, respectively. P-450PCN, P-450e, and P-450c exhibit greater homology in the C-terminal half than in the N-terminal half of the proteins. Included in this region is the cysteinyl fragment (surrounding residue 443 in P-450PCN), which appears to be the most conserved among all fragments of other P-450 proteins. Of interest, the N-terminal region of P-450PCN does not contain the cysteine residue previously thought to contribute the thiolate ligand to the heme iron in P-450 proteins; these data establish more firmly the cysteine residue located in the carboxylterminal region as serving this function. These sequence studies further support the conclusion derived from chromosomal localization studies and Southern blot analyses that P-450PCN represents a member of a distinct third family of P-450 genes, which diverged from a common ancestor more than 200 million years ago.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.