Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

mRNA quantitation by a simple and sensitive RNAse protection assay.

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.     

Limitations on the metyrapone assay for the major phenobarbital inducible form of cytochrome P-450

Limitations on the determination of the concentration of the major phenobarbital inducible form of cytochrome P-450 (P-450b) in hepatic microsomes by the metyrapone assay of Luu-The $\text{et al.}$ (1) are reported. Compounds which bind to the Type I, II and IR binding sites, or convert cytochrome P-450 to P-420, decrease the apparent concentration of cytochrome P-450b by 20 to 100% in hepatic microsomes from untreated and pregnenolone-16α-carbonitrile or phenobarbital treated rats. It is calculated that errors of greater ca. 40% in the concentration of cytochrome P-450b can arise in the presence of appreciable quantities of the major pregnenolone-16α-carbonitrile or polycyclic hydrocarbon inducible forms of cytochrome P-450.     

A Link between cholesterol levels and phenobarbital induction of cytochromes P450

Squalestatin1 (SQ1), a potent inhibitor of squalene synthase produced a dose-dependent induction of cytochromes P450 CYP2H1 and CYP3A37 mRNAs in chicken hepatoma cells. The effect of SQ1 was completely reversed by 25-hydroxycholesterol. Bile acids elicited an induction of CYP3A37 and CYP2H1 mRNA. Bile acids also reduced the phenobarbital induction of CYP2H1 but not of CYP3A37 mRNA. The effects of SQ1 and its reversal by 25-hydroxycholesterol and the effects of bile acids were reproduced in reporter gene assays with a phenobarbital-responsive enhancer unit of CYP2H1. These data suggest that an endogenous molecule related to cholesterol homeostasis regulates induction of drug-inducible CYPs.     

Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

Identification of human cytochromes P-450 analogous to forms induced by phenobarbital and 3-methylcholanthrene in the rat.

Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have been used to make a structural and functional comparison of rat and human cytochromes P-450. Proteins from both species were identified on Western blots by their reaction with these antibodies. In the human liver preparations, structurally related proteins to PB1 and to PB2 were identified in all the samples tested with apparent Mr values of 51 800 and 54 800 for PB1 and 53 600 and 57 200 for PB2. Considerable variation in the content of the lower-Mr proteins was measured between samples and, as with the rat enzymes, samples which reacted well with anti-PB1 also reacted with anti-PB2, indicating that these proteins are regulated at least to some degree, co-ordinately. The apparent Mr values of the major human proteins identified with anti-MC1 and anti-MC2 were 54 400 and 57 000 respectively. Only six (of 31) human samples contained significant amounts of these proteins. The same six samples which reacted with anti-MC1 also reacted with anti-MC2, again indicating co-ordinate regulation of these two proteins. Antibody inhibition of microsomal 7-ethoxycoumarin and 7-ethoxyresorufin metabolism demonstrated a degree of conservation of substrate specificity related to specific P-450 isoenzymes between the species. However, the contributions of the different P-450 isoenzymes to the human microsomal activity were not always related to the rat enzyme with the highest activity towards these substrates.     

A constitutive form of guinea pig liver cytochrome P450 closely related to phenobarbital inducible P450b(e)

In order to provide evidence that a cytochrome P450 belonging to the IIB subfamily is expressed as a constitutive form in the guinea pig, we tried to purify an isozyme from liver microsomes of untreated guinea pigs by assessing its reactivity with anti-P450b antibody in the present study. One form of cytochrome P450, named P450GP-1, was obtained. The minimum molecular weight of this isozyme was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino terminal sequence up to the 33rd amino acid of P450GP-1 was determined. As expected, comparison of the amino acid sequence with those of cytochrome P450 isozymes from other species reported so far indicated that P450GP-1 was highly homologous to P450s categorized in the IIB subfamily; that is, 67% similarity to rat P450b, 82% to rabbit LM2, 76% to dog PBD-2, 70% to mouse pf 3/46, and 73% to human IIB1. On the other hand, P450GP-1 showed only low similarity, less than 41%, to other cytochrome P450s of the II subfamily and those of the I, III, and IV families. Affinity of P450GP-1 to anti-P450b immunoglobulin G was confirmed to be comparable with that of a principal antigen, P450b. Immunoblot analysis revealed that P450GP-1 in the guinea pig liver microsomes was induced by phenobarbital treatment, but the increase was not as large as in the rat. P450GP-1 efficiently catalyzed benzphetamine N-demethylation, strychnine 2-hydroxylation, and testosterone 16 beta-hydroxylation, all of which are also catalyzed by P450b. Based on these results, it was strongly suggested that the IIB-type of cytochrome P450 in guinea pigs, at least one of them, is a constitutive form which is moderately induced by phenobarbital.     

Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice

Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host’s health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum ^NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum ^NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum ^NIZO2877; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.     

Prediction of binding modes for ligands in the cytochromes P450 and other heme-containing proteins

The cytochromes P450 (P450s) are a family of heme-containing monooxygenase enzymes involved in a variety of functions, including the metabolism of endogenous and exogenous substances in the human body. During lead optimization, and in drug development, many potential drug candidates are rejected because of the affinity they display for drug-metabolising P450s. Recently, crystal structures of human enzymes involved in drug metabolism have been determined, significantly augmenting the prospect of using structure-based design to modulate the binding and metabolizing properties of compounds against P450 proteins. An important step in the application of structure-based metabolic optimization is the accurate prediction of docking modes in heme binding proteins. In this paper we assess the performance of the docking program GOLD at predicting the binding mode of 45 heme-containing complexes. We achieved success rates of 64% and 57% for Chemscore and Goldscore respectively; these success rates are significantly lower than the value of 79% observed with both scoring functions for the full GOLD validation set. Re-parameterization of metal-acceptor interactions and lipophilicity of planar nitrogen atoms in the scoring functions resulted in a significant increase in the percentage of successful dockings against the heme binding proteins (Chemscore 73%, Goldscore 65%). The modified scoring functions will be useful in docking applications on P450 enzymes and other heme binding proteins.     

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min⁻¹) and 2-MCDD (13 min⁻¹) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichloro-dibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzo-p-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

The active sites of cytochromes P450 IA1, IIB1, IIB2, and IIE1. Topological analysis by in situ rearrangement of phenyl-iron complexes

The reactions of cytochromes P450 IA1, IIB1, IIB2, and IIE1 with phenyldiazene yield complexes with absorption maxima at either 474 or 480 nm. Anaerobic extraction of the prosthetic group from the phenyldiazene-treated proteins followed by its exposure to oxygen and strong acid produces an equal mixture of the four possible N-phenylprotoporphyrin IX regioisomers. Exposure of the anaerobically extracted heme complexes to oxygen in the absence of strong acid results in their decomposition to heme and products other than N-phenylprotoporphyrin IX. These results show that the 474/480 nm absorption maxima are due to sigma phenyl-iron complexes. Treatment of the intact hepatic cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 474/480 nm band. Extraction of the prosthetic group then yields only the two N-phenylprotoporphyrin IX regioisomers with the N-phenyl group on pyrrole rings A and D. The same regioisomer pattern is obtained if the cytochrome P450IA1 phenyl-iron complex is simply warmed to 37 degrees C, but this thermal rearrangement occurs much less readily, if at all, with the complexes of the other isozymes. The regioisomers with the N-phenyl on pyrrole rings A and D are obtained in a 2:1 ratio with isozyme IA1, 1:1 with IIB2, 1:1.7 with IIB1, and 1:2 with IIE1. These results establish that the active sites of these cytochrome P450 isozymes have a common architecture despite their gross differences in substrate specificity. In this architecture, the region directly above pyrrole rings A and D is relatively open whereas that above pyrrole rings B and C is occluded by protein residues.     

Characterization of a cDNA encoding a new member of the glucocorticoid-responsive cytochromes P450 in human liver

Adult human liver contains a form of cytochrome P450, termed HLp, that resembles the glucocorticoid-inducible cytochrome P450p in rat liver in its structure, function, and regulation and catalyzes the oxidation of such clinically important substrates as cyclosporin, nifedipine, erythromycin, and midazolam. Recent evidence, however, suggests that HLp may represent two or more closely related forms of cytochromes P450, one of which is termed P450nf. To search for additional members of the Class III human subfamily of HLp related genes, we screened a human liver cDNA library cloned in phage vector lambda gt11 with oligonucleotides and with a cDNA fragment related to HLp. We isolated a full-length cDNA (1709 nucleotides) encoding a new form of human cytochrome P450 termed HLp2. Analysis of HLp2 cDNA predicted a protein of 502 amino acids, weighing 57,294 Da 83% similar to HLp. HLp2 appears to represent a distinct gene as judged by partial sequence analysis of a cloned human gene and by hybridizations of Southern blots, under conditions of varying stringency, with a 3'-portion of HLp cDNA and with an oligonucleotide specific for HLp2. Northern blot analysis revealed that HLp/P450nf was present in all samples of liver mRNA from adult patients not treated with inducers of HLp, whereas HLp2 mRNA was undetectable in more than two-thirds. Human fetal liver RNA contained mRNA species 2.1 and 1.9 kb which hybridized with an HLp2 oligonucleotide. We conclude that HLp2 represents a third member of the Class III glucocorticoid-responsive gene family that is expressed in both fetal and adult human liver and may account for polymorphism in metabolism of clinically important drugs.     

Differential effects of phenobarbital on the constitutive and inducible expression of P450 2B and 3A subfamilies in sheep tissues.

The activity and expression of cytochromes P450 were determined in liver, kidneys, lungs, duodenum, jejunum, ileum, and caecum of adult Lacaune sheep. High expression of total P450, benzphetamine and erythromycin demethylase activities, and P450 2B isoforms, as two distinct proteins that were detected and called P4502 Bm and P4502 Bx, was found in the lungs (in addition to liver). By contrast, the P450 3A subfamily was only expressed in liver and duodenal mucosa of untreated sheep. Phenobarbital (PB) treatment led to significant increases in all measured hepatic parameters and in total P450 of each investigated organ with the exception of ileum and caecum. Benzphetamine demethylase activity increased in liver and kidneys, correlating with the expression of the two P450 2B proteins, which were also induced in duodenum and ileum. By contrast, benzphetamine demethylase activity and expression of the P450 2B isoforms in lungs were unchanged by PB treatment. Erythromycin demethylation activity and P450 3A subfamily expression was increased only in liver of PB-treated sheep.     

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ω3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10 μM of these two ω3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of ω3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.     

Electrochemistry of cytochromes P450: Analysis of current-voltage characteristics of electrodes with immobilized cytochromes P450 for the screening of substrates and inhibitors

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14α-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 4, pp. 542–549.     

Cloning and characterization of two major 3-methylcholanthrene inducible hamster liver cytochrome P450s

We have studied the immunochemical properties of two major 3-methylcholanthrene inducible hamster liver cytochrome P450 isozymes, P450 MC1 and P450 MC4. Immunoblots using specific antibodies against P450 MC1 and P450 MC4 demonstrated that these two P450s were present in very low levels in control hamster livers and were greatly induced by 3-methylcholanthrene treatment. P450 MC1 was immunochemically different from P450 MC4, rat P450c and P450d, and rabbit LM4. The immunorelated polypeptide to P450 MC1 was not present in the control or the 3-methylcholanthrene-treated rat liver microsomes, whereas it was present in two human liver microsomal preparations. On the other hand, P450 MC4 was immunochemically related to rat P450d and rabbit LM4. The immunorelated polypeptide to P450 MC4 was present in the human and 3-methylcholanthrene-treated rat liver microsomes. We also isolated full-length cDNA clones encoding P450 MC1 and P450 MC4 mRNAs from a 3-methylcholanthrene-induced hamster liver cDNA library. The full-length cDNA clones of P450 MC1 and P450 MC4 contained 1771 and 1868 base pairs, which encoded polypeptides of 494 and 513 amino acids, respectively. RNA blot analysis revealed that the mRNAs for P450 MC1 and P450 MC4 were 2100 and 2600 bases in length, respectively. 3-Methylcholanthrene pretreatment increased the P450 MC1 mRNA level by 16-fold and the P450 MC4 mRNA level by 11-fold in the hamster livers. A comparison of the deduced amino acid sequences with other cytochrome P450s revealed that P450 MC1 was most similar to the mouse P450(15) alpha with 75% sequence identity, whereas P450 MC4 shared 87% identity with the rat P450d or mouse P3(450). These results indicated that P450 MC1 was a unique member (CYP2A8) in the P450IIA subfamily, whereas P450 MC4 was the hamster P450IA2.