Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.     

Gene structure of cytochrome P-450(M-1) specifically expressed in male rat liver

Cytochrome P-450(M-1) [P-450(M-1)] is specifically expressed in adult male rat liver [Yoshioka, H., Morohashi, K., Sogawa, K., Miyata, T., Kawajiri, K., Hirose, T., Inayama, S., Fujii-Kuriyama, Y., & Omura, T. (1987) J. Biol. Chem. 262, 1706-1711]. Isolation and analysis of the gene for P-450(M-1) revealed that the coding region of the gene is interrupted by eight introns and is dispersed over a 35-kilobase pair region of chromosomal DNA. Intron insertion sites of the P-450(M-1) gene are located at equivalent positions to those of cytochrome P-450b and P-450e, which are phenobarbital-inducible. Sequence analysis of the 5'-upstream region of the P-450(M-1) gene shows that there is a homologous sequence to glucocorticoid regulatory elements (GRE) identified in glucocorticoid-responsive 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.     

Existence of multiple forms of cytochrome P-450scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH2-terminal amino-acid sequences of cytochrome P-450scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450scc mRNA (Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647-4651), whereas the sequence of fraction c showed a missing of isoleucine at the NH2-terminal. COOH-terminal ámino-acid sequences of fractions a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.     

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.     

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.     

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.     

Induction of mRNA coding for phenobarbital-inducible form of microsomal cytochrome P-450 in rat liver by administration of 1,1-Di(p-chlorophenyl)-2,2-dichloroethylene and phenobarbital

In the preceding paper (Yoshioka, H., et al. (1984) J. Biochem. 95, 937-947), we reported that 1,1-di(p-chlorophenyl)-2,2-dichloro-ethylene (DDE) induced the phenobarbital (PB)-inducible form of microsomal cytochrome P-450 (P-450(PB-1) in rat liver. In order to study more precisely the molecular events responsible for the induction of this particular form of cytochrome P-450 by the two chemical compounds, we determined the amounts of the mRNA coding for P-450(PB-1) in the liver of rats given a single dose of PB or DDE. RNA was extracted from the livers of the treated rats and the determination of the specific mRNA was carried out by using the rabbit reticulocyte lysate translation system and by a dot hybridization method using cloned P-450(PB-1) cDNA (Fujii-Kuriyama, Y., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) as the probe. The amounts of P-450(PB-1) mRNA determined by these two methods at various time points of the induction process showed good agreement. These observations further confirmed the induction of an identical form of cytochrome P-450 by DDE and PB. The maximum level of P-450(PB-1) mRNA, which was about 8-fold higher than the control level, was attained at 20-30 h and at 48-72 h after the administration of PB and DDE, respectively. The mRNA level showed a rapid decrease after the peak in the liver of PB-treated rats, but the decrease was much slower with DDE-treated rats. We conclude that DDE had a more persistent inducing effect on the mRNA level than PB, although these two compounds induced an identical form of cytochrome P-450 in the liver microsomes of the animals.     

Secondary structure prediction of liver microsomal cytochrome P-450; proposed model of spatial arrangement in a membrane

The secondary structure of rabbit liver microsomal cytochrome P-450 LM2, rat liver microsomal cytochromes P-450b and P-450e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450c, P-450d (3-methylcholanthrene-inducible) was predicted by a combination of methods (i) identifying the transmembrane parts of integral membrane proteins, and (ii) statistically predicting the secondary structure of globular proteins. The results are similar for all phenobarbital-inducible enzymes and make it possible to construct two structural models with seven or four transmembrane alpha-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning alpha-helices. In both cases, a large ectodomain with several consecutive alpha-helices, which may provide the heme-binding pocket, is exposed out of the membrane.     

Structural details of the human cytochrome P-450c gene

Aryl hydrocarbon hydroxylase activity is most closely associated with cytochrome P-450c in the rat and cytochrome P1-450 in the mouse. The sequence for the orthologous human gene coding for this enzymatic activity has been determined from several sources: cytochrome P-450c isolated from human embryonic DNA [K. Kawajiri, J. Watanabe, O. Gotoh, Y. Tagashiri, K. Sogawa, and Y. Fujii-Kuriyama (1986) Eur. J. Biochem. 139, 219-225], human lymphocytes in our own laboratory, and cytochrome P1-450 isolated from the established human breast carcinoma cell line, MCF-7 [A.K. Jaiswal, F. J. Gonzalez, and D. W. Nebert (1985) Nucleic Acids Res. 13, 4503-4520]. The data from our laboratory agree well with the sequence derived from human embryonic DNA, but differs significantly from that reported for the gene isolated from MCF-7 cells. Among these differences are a 320-bp insert and a 650-bp deletion in intron 1 relative to the sequence derived from the established cell line. We observe two mRNA species that hybridize to cytochrome P-450c probes, one expected at 2.7 kb and an additional 2.0-kb species. Finally, we note additional hybridization bands in 11% of the population examined by Southern blot analysis, representing either a second rare allele or, more likely, a duplication of at least a portion of the cytochrome P-450c gene.     

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.     

Molecular basis for a functionally unique cytochrome P450IIB1 variant.

Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.     

Evidence that the catalytic differences of two structurally homologous forms of cytochrome P-450 relate to their heme environment

Cytochromes P-450 PB3a and PB3b, which appear to be equivalent to forms b and e described by Ryan et al. [Ryan, D.E., Thomas, P.E., & Levin, W. (1982) Arch. Biochem. Biophys. 216, 272-288], have been shown to share 97% sequence homology [Suwa, Y., Mizukami, Y., Sogawa, K., & Fujii-Kuriyama, Y. (1985) J. Biol. Chem. 260, 7980-7984] yet exhibit an intriguing difference in enzymatic activity. Studies to establish the basis for this difference, including a development of the technique of surface-enhanced resonance Raman spectroscopy (SERRS), are reported. Studies on substrate binding, metabolism, and redox properties, as well as SERRS, indicate a significant difference in the heme environment of these two proteins. No significant difference in the interaction of the two proteins with P-450 reductase could be established. However, this interaction appeared sensitive to changes in ionic strength, suggesting ionic interactions are important in the functional coupling of these electron-transport components. A marked variation in the ratio of PB3a to PB3b activity in the metabolism of different substrates, which included a series of structurally similar resorufin analogues, provided further evidence that reductase coupling was not a critical factor. Therefore, the few amino acid differences observed between these proteins indicate sites that may be important in influencing the heme environment of these cytochrome P-450's.     

P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16 alpha-hydroxylase of dehydroepiandrosterone 3-sulfate

In a reconstituted system containing NADPH, dilauroyl-L-3-phosphatidylcholine, and NADPH-cytochrome P-450 reductase purified from rat liver microsomes, cytochrome P-450 (P-450 HFLa) purified from human fetal livers catalyzed the 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate). Addition of cytochrome b5 purified from rat liver microsomes to the reconstituted system resulted in a remarkable increase in the hydroxylase activity. The level of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Antibodies to P-450 HFLa inhibited the 16 alpha-hydroxylation of DHEA-sulfate in a dose-dependent manner. The NH2-terminal amino acid sequence of P-450 HFLa was similar to that of P-450NF (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068). We conclude that P-450 HFLa is a form of cytochrome P-450 involved in the 16 alpha-hydroxylation of DHEA-sulfate.     

Genetic polymorphisms for a phenobarbital-inducible cytochrome P-450 map to the Coh locus in mice

Southern blot analysis suggests that multiple sequences homologous to a phenobarbital-inducible cytochrome P-450 cDNA are present in the rat and mouse genomes. A cDNA (pP-450b-5) to a major phenobarbital-inducible cytochrome P-450 mRNA species in the rat detected 6 polymorphic DNA fragments when hybridized to DNA from C57BL/6J and DBA/2J mice restricted with endonucleases EcoRI, BamHI, and PvuII. Using the BXD recombinant inbred strains, five of these polymorphisms were mapped to the Coh (coumarin hydroxylase) locus on chromosome 7 of the mouse. The Coh locus has previously been shown to code for a phenobarbital-inducible enzyme, believed to be a cytochrome P-450, which catalyzes the conversion of coumarin to 7-hydroxycoumarin (umbelliferone). The DNA polymorphisms appear to reflect changes in either cytochrome P-450 genes or pseudogenes that are very closely linked to the gene responsible for differential coumarin hydroxylase in mice or it may represent a change(s) in the Coh gene itself. The region of the Coh locus on chromosome 7 may be the site of a cluster of cytochrome P-450 genes.     

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.     

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.