Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.     

cDNA cloning and expression of the mRNA for cytochrome P-450kd which shows a fatty acid omega-hydroxylating activity

We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.     

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.     

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Microheterogeneity in the major phenobarbital-inducible forms of rabbit liver microsomal cytochrome P-450 as revealed by nucleotide sequencing of cloned cDNAs

We have isolated one full-length cDNA clone, termed pHP1, and a number of clones of shorter insert lengths, tentatively called b14, b46, etc., all encoding phenobarbital- (PB-) inducible forms of rabbit liver microsomal cytochrome P-450, and determined their nucleotide sequences. The polypeptides encoded by these cDNAs can be classified into five types, represented by HP1, b14, b46, b52, and b54, the deduced amino acid sequences of which are more than 95% similar to one another. Amino acid differences among them total 24 positions, which are distributed over the entire sequence, in contrast to the microheterogeneity observed in two PB-inducible rat liver microsomal cytochromes P-450 (P-450b and P-450e). The primary structure deduced for the HP1 protein is 97% similar to that determined for rabbit P-450 LM2 (form 2), which has been purified by Coon and co-workers [van der Hoeven, T. A., Haugen, D. A., & Coon, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569-675; Haugen, D. A., & Coon, M. J. (1976) J. Biol. Chem. 251, 7929-7939] as the major PB-inducible form of rabbit liver microsomal cytochrome P-450. The amino acid sequence of P-450(1), which we have purified as the major PB-inducible rabbit liver cytochrome P-450, was partially determined with the sequence reported for P-450 LM2 as a reference. The two sequences are closely similar to each other, but at least two amino acid differences can be detected between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.     

Nucleotide sequence of a human liver cytochrome P-450 related to the rat male specific form

P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.     

Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme

Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064-8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203-217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.     

Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P-450 (form 2), the major phenobarbital-inducible form

We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins.     

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.     

Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase

Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH2-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulatory characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.     

Cloned cytochrome P-450 cDNA. Nucleotide sequence and homology to multiple phenobarbital-induced mRNA species

Phenobarbital (PB) treatment of rats of various strains leads to the accumulation of liver mRNAs which encode two or three immunochemically related but electrophoretically separable cytochrome P-450 polypeptides. These mRNAs hybridize efficiently to a single cloned cDNA derived from mRNA of PB-treated rats and, therefore, must have extensive sequence homology. The nucleotide sequence of this cloned cDNA was determined and shown to encode the COOH-terminal 211 amino acids of one of the major cytochrome P-450 isozymes induced in rat liver by PB. Together with the recently reported sequence data of Fujii-Kuriyama et al. (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sagawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797) for cloned rat cytochrome P-450 cDNA, our data suggest that differences between two closely related P-450 isozymes are restricted to the COOH-terminal half of the polypeptides, with highly divergent regions flanking a tridecapeptide which has been previously shown to be highly conserved in two dissimilar forms of rabbit liver cytochrome P-450. The significance of other interesting features of the cDNA sequence such as a second long (409 residues) open frame, an unusual poly(A) addition signal, and the absence of long hydrophobic stretches in the encoded polypeptide is discussed.     

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2)

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.     

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

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

Comparison of primary structures deduced from cDNA nucleotide sequences for various forms of liver microsomal cytochrome P-450 from phenobarbital-treated rabbits

cDNA clones, termed pHP2, b32-3, b43, and b43-1, encoding cytochromes P-450 that are expressed in the liver of phenobarbital- (PB-) treated rabbits were isolated, and their nucleotide sequences were determined. pHP2 cDNA contains an open reading frame for a 490-residue protein and is a full-length counterpart of pP-450PBc2 [Leighton, J. K., Debrunner-Vossbrinck, B. A., & Kemper, B. (1984) Biochemistry 23, 204-210]. The b32-3 insert has a sequence for a protein whose primary structure is 91% similar to that of progesterone 21-hydroxylase P-450 1, though this cDNA lacks the sequence encoding the amino-terminal 110 residues. The overlapping clones b43 and b43-1 together encode an ethanol-inducible form of cytochrome P-450, though the amino-terminal five or more residues are missing in the composite b43/b43-1 sequence. Northern blot analysis showed that the b43/b43-1 protein is more strongly inducible by polycyclic aromatic hydrocarbons and isosafrole than by PB, in contrast to the case of the HP2 and b32-3 proteins. A comparison of the primary structures of eight forms of cytochrome P-450, including the HP2, b32-3, and b43/b43-1 proteins, that are expressed in the liver of PB-treated rabbits showed that 149 out of 487-492 amino acid residues are conserved in these cytochromes P-450. The eight forms can be assigned to three rabbit cytochrome P-450 gene subfamilies, P450IIB, P450IIC, and P450IIE. It was also shown that the members of the rabbit P450IIC subfamily can be further classified into three subgroups on the basis of their sequence similarity.     

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.     

cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6

A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.     

Sex differences in cytochrome P-450-dependent 25-hydroxylation of C27-steroids and vitamin D3 in rat liver microsomes

Monoclonal antibodies directed against the cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 (Andersson, S., Holmberg, I., and Wikvall, K. (1983) J. Biol. Chem. 258, 6777-6781) have been prepared by immunization of mice in hind footpads. Lymph node cells from the mice were fused with the Sp 2/0-Ag 14 line of mouse myeloma cells. A monoclonal antibody, designated MAb-25-6, monospecific for cytochrome P-450(25) was, after coupling to Sepharose, able to bind to cytochrome P-450(25) and to immunoprecipitate the activity for 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol and vitamin D3 as well as that for 16 alpha-hydroxylation of testosterone when assayed in a reconstituted system. Two-dimensional gel electrophoresis of adult male rat liver microsomes and immunoblotting with MAb-25-6 showed a single spot with an apparent isoelectric point of 7.4 and Mr approximately 51,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with MAb-25-6, cytochrome P-450(25) was shown to be male-specific and not detectable in adult female rat liver microsomes. N-terminal sequence analysis of cytochrome P-450(25) showed a structure identical with that of the male-specific steroid 16 alpha-hydroxylase isolated in several laboratories.