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R H Tukey

Publications and source records attributed to R H Tukey.

At least 73 records · Page 4Linked to original sources
PubMed1992

High-level expression of functional human cytochrome P450 1A2 in Escherichia coli.

Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA. The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase.

Amino Acid Sequence↗
PubMed1992

Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques.

The molecular basis for the use of caffeine (CA; 1,3,7-trimethylxanthine) as a probe for specific human cytochromes P450 has been investigated. The CA 1-, 3- and 7-demethylations (to form theobromine, paraxanthine and theophylline, respectively) all followed biphasic kinetics in human liver microsomes. Mean apparent Km values for the high- and low-affinity components of the demethylations ranged from 0.13-0.31 nM and 19.2-30.0 mM, respectively. cDNA-expressed CYP1A2 catalysed all three CA demethylations, and the apparent Km for CA 3-demethylation (the major metabolic pathway in humans) by the expressed enzyme was similar to the Km for the high-affinity liver microsomal CA 3-demethylase. IC50 values for inhibition of the CA demethylations by alpha-naphthoflavone were similar for both expressed CYP1A2 and the high-affinity microsomal demethylases. Moreover, CA was a competitive inhibitor of expressed CYP1A2 catalysed phenacetin O-deethylation, with the apparent Ki (0.080 mM) closely matching the apparent Km (0.082 mM) for CA 3-demethylation by the expressed enzyme. Expressed CYP1A1 was additionally shown to catalyse the 3-demethylation of CA, although activity was lower than that observed for CYP1A2. While these data indicate that CYP1A2 is responsible for the high-affinity component of human liver CA 3-demethylation, two limitations associated with the use of CA as an in vitro probe for CYP1A2 activity have been identified: (i) CA 3-demethylation reflects hepatic CYP1A2 activity only at appropriately low substrate concentrations; and (ii) CA is a non-specific CYP1A substrate and CYP1A1 may therefore contribute to CA 3-demethylase activity in tissues in which it is expressed. An anti-CYP3A antibody essentially abolished the 8-hydroxylation of CA to form trimethyluric acid, suggesting formation of this metabolite may potentially serve as a marker of CYP3A isozyme(s) activity.

Benzoflavones↗
PubMed1991

Localization of CYP1A1 and CYP1A2 messenger RNA in normal human liver and in hepatocellular carcinoma by in situ hybridization.

To better characterize the precise cellular distribution of CYP1A gene products in man, we have undertaken Northern-blot and in situ hybridization analyses of CYP1A expression in human liver. Using riboprobes transcribed from both CYP1A1 and CYP1A2 complementary DNAs to probe a series of Northern blots of 23 human liver messenger RNA samples, CYP1A1 expression was demonstrated in 11 samples and CYP1A2 expression was evident in 22 samples. The level of expression of both CYP1A enzymes in these livers demonstrated marked variability. The CYP1A1 and CYP1A2 riboprobes were then used for in situ hybridization localization of CYP1A1/1A2 messenger RNA sequences on paraffin-embedded, formalin-fixed human liver sections. These studies demonstrated that both CYP1A1 and CYP1A2 messenger RNAs are distributed nonuniformly across the human liver acinus, with levels highest in hepatocytes surrounding terminal hepatic venules and intercalated veins. Immunohistochemistry with an anti-rabbit CYP1A1 serum demonstrated a corresponding distribution for the translated CYP1A proteins. In situ hybridization analysis was also performed on sections of hepatocellular carcinoma, demonstrating a significant down-regulation in CYP1A expression. Functional studies using the activation of the food-derived heterocyclic amine MeIQ (2-amino-3,4-dimethylimadazo [4,5-f] quinoline) to a mutagen in the Ames test as an indicator of CYP1A expression confirmed this down-regulation. These results demonstrate heterogeneity of hepatic CYP1A expression both between individuals and in different acinar zones. This variation in expression may be of significance in assessing cell specific toxicities of various drugs and carcinogens.

Carcinoma, Hepatocellular↗
PubMed1991

Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor.

Dioxin stimulates transcription from the cytochrome P-450IA1 promoter by interaction with the intracellular dioxin receptor. Upon binding of ligand, the receptor is converted to a form which specifically interacts in vitro with two dioxin-responsive positive control elements located in close proximity to each other about 1 kb upstream of the rat cytochrome P-450IA1 gene transcription start point. In rat liver, the cytochrome P-450IA1 gene is marked at the chromatin level by two DNase I-hypersensitive sites that map to the location of the response elements and exist prior to induction of transcription by the dioxin receptor ligand beta-naphthoflavone. In addition, a DNase I-hypersensitive site is detected near the transcription initiation site and is altered in nuclease sensitivity by induction. The presence of the constitutive DNase I-hypersensitive sites at the dioxin response elements correlates with the presence of a constitutive, labile factor which specifically recognizes these elements in vitro. This factor appears to be distinct from the dioxin receptor, which is observed only in nuclear extract from treated cells. In conclusion, these data suggest that a certain protein-DNA architecture may be maintained at the response elements at different stages of gene expression.

Animals↗
PubMed1990

Characterization of the CYP2C5 gene in 21L III/J rabbits. Allelic variation affects the expression of P450IIC5.

Rabbits exhibit two heritable phenotypes, 21L and 21H, with the latter displaying roughly 10-fold higher liver microsomal progesterone 21-hydroxylase activity. The higher activity of the 21H animals reflects the elevated expression of cytochrome P-450 1 (P450IIC5). Breeding studies indicate that this phenotypic difference is linked to allelic CYP2C5 genes (Johnson, E. F., Finlayson, M., Husjsak, C. M., Pendurthi, U. R., and Tukey, R. H. (1989) Arch. Biochem. Biophys. 273, 273-280). In this study, we report on the expression and structure of the CYP2C5 gene in the 21L inbred strain III/J. A cDNA encoding P450IIC5 was generated from III/J rabbit liver mRNA using polymerase chain reaction to amplify this sequence. DNA sequence analysis revealed that the cDNA encoded the same protein as the cDNA cloned previously from a 21H rabbit, demonstrating that structural differences in P450IIC5 do not underlie the phenotypic difference in the expression of 21-hydroxylase activity. DNA sequence analysis of the exons and surrounding intron regions in two III/J genomic clones that encode portions of the CYP2C5 gene showed that the exon sequences were identical to that of the P450IIC5 mRNA and that the codon positions of the exon/intron junctions are conserved with other class II genes. Southern blot analysis of DNA digested with BamHI and probed with a portion of DNA isolated from intron 5 revealed that the gene characterized in this study is an allele of the gene conferring the 21H phenotype. Primer extension analysis with a P450IIC5-specific oligonucleotide demonstrated that the CYP2C5 gene uses several transcriptional start sites in both 21H and 21L rabbits. Analysis of mRNA from livers of 21H and 21L rabbits with the P450IIC5-specific oligonucleotide showed a greater than 20-fold difference in the relative abundance of mRNA, which accounts quantitatively for the phenotypic difference in progesterone 21-hydroxylase activity.

Alleles↗
PubMed1990

Metabolism of 2-acetylaminofluorene and benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450.

The human P-450 CYP1A1 gene and a P450IA2 complementary DNA have been expressed in Cos-1 cells and the expressed proteins were assayed for their capacity to metabolize the carcinogens 2-acetylaminofluorene (AAF), benzo(a)pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined. The expressed human P450IA1 and P450IA2 proteins, when run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, migrated with different mobilities, with the former displaying the lower molecular weight. In human liver microsomes from 18 subjects, only a protein band corresponding to P450IA2 was detectable. Cos-1 cell-expressed P450IA1 and P450IA2 were capable of N-hydroxylating AAF and these activities were inhibited by alpha-naphthoflavone. In human liver microsomes, a correlation of r = 0.76 (P less than 0.05; n = 18) was obtained between AAF N-hydroxylase activity and P450IA2 content. AAF N-hydroxylase activity of human liver microsomes was also strongly inhibited by alpha-naphthoflavone. Except in the case of PhIP, where both proteins exhibited similar activities, P450IA2 was at least an order of magnitude more efficient than P450IA1 in activating IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline to mutagens as measured in the Ames test. Statistically significant correlations were obtained between IQ activation and P450IA2 content (r = 0.75, r2 = 0.56) and PhIP activation and P450IA2 content (r = 0.71, r2 = 0.5) in human liver microsomes. The activation of both IQ and PhIP by expressed proteins and human liver microsomes was strongly inhibited by alpha-naphthoflavone. The above data suggest a major role for P450IA2 in activation (N-hydroxylation) of aromatic amides and amines in human liver. When benzo(a)pyrene hydroxylase activity was determined, only Cos-1 cell-expressed P450IA1 exhibited appreciable activity. While alpha-naphthoflavone inhibited Cos-1 cell-expressed P450IA1 benzo(a)pyrene hydroxylase activity, it caused a marked stimulation of this activity in human liver microsomes, which lack P450IA1 protein. The lack of a role for P450IA proteins in benzo(a)pyrene metabolism is further supported by the poor correlation (r = 0.43, P greater than 0.05) between this activity and P450IA2 content of human liver microsomes. However, when P450IIIA3 content of the above human liver microsomes was determined by using the Western blot technique and correlated with benzo(a)pyrene metabolism, an r value of 0.70 (P less than 0.5) was obtained. These data suggest that human P450IIIA proteins are involved in benzo(a)pyrene metabolism.

2-Acetylaminofluorene↗
PubMed1990

Identification of cytochrome P450IA2 as a human autoantigen.

Autoantibodies occurring in a patient with idiopathic autoimmune type chronic active hepatitis (CAH) were found to react with purified rabbit cytochrome P450IA2 and to a much lesser extent with P450IA1. Both cytochrome P450s are known to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rabbit, and the expression of the microsomal protein recognized by the patient serum was induced in adult rabbit livers after treatment with TCDD. This protein is only weakly detected in liver microsomes from neonatal rabbits exposed to TCDD in utero, which is consistent with the age-dependent induction of P450IA2 by TCDD. The serum specifically reacted with a protein of similar size in microsomes prepared from COS-1 cells transfected with an expression vector containing the full length human P450IA2 cDNA. This reactivity was not detected in the cells transfected with the vector alone, indicating that the antibody recognizes human P450IA2. In addition, the serum extensively inhibited 7-ethoxyresorufin O-deethylation catalyzed by isolated human liver microsomes. This catalytic activity is associated with class IA P450s in other species. A screen of sera from patients with various hepatic and nonhepatic diseases indicates that the autoantibody to P450IA2 occurs rarely in CAH. Cytochrome P450IA2 becomes the third P450 identified as an autoantigen in inflammatory liver diseases.

Adult↗
PubMed1989

Regulation of the rabbit cytochrome P-450 3c gene. Age-dependent expression and transcriptional activation by rifampicin.

The macrolide antibiotic rifampicin is a potent inducer of cytochrome P-450-mediated drug metabolism in humans and rabbits. In this report, we demonstrate that in immature rabbits, rifampicin activates the transcription of the Cyp3A6 gene which encodes P450IIIA6 (cytochrome P-450 3c). The maximum increase in transcription was seen at 12 h following administration of rifampicin. Northern and slot blot analyses indicate that mRNAs corresponding to P450IIIA6 accumulates during the period of increased transcription and persist at 18 h when the rate of transcription has returned to basal levels. P450IIIA6 protein accumulates in liver microsomes over this period. At 24 h a greater than 10-fold increase in microsomal P450IIIA6 protein is detected by immunoblotting using a monoclonal antibody to P450IIIA6. The rate of microsomal progesterone 6 beta-hydroxylase activity is also elevated when compared to untreated rabbits, and this enzyme is activated in vitro by alpha-naphthoflavone. To determine whether this enzyme is stimulated by alpha-naphthoflavone in intact cells, COS-1 cells were transfected with an expression vector harboring the coding region of a P450IIIA6 cDNA. Our results demonstrate that the transfected COS-1 cells exhibit progesterone 6 beta-hydroxylase activity that is stimulated by alpha-naphthoflavone added to the culture medium.

Aging↗
PubMed1989

Genetic contributions to the variation among rabbits of liver microsomal deoxycorticosterone synthesis.

Outbred New Zealand white rabbits exhibit two phenotypes, 21H and 21L, corresponding to rates greater than or less than 1 nmol/min/mg, respectively, for liver microsomal progesterone 21-hydroxylase activity. In contrast, the inbred strain III/J exhibits only the 21L phenotype. Two 21H male New Zealand white rabbits were mated with several female III/J rabbits to produce a total 46 progeny. Both the 21H and 21L phenotypes were evident among male and female offspring in roughly equal numbers. Backcrosses between 21L progeny and III/J rabbits exhibit only the 21L phenotype, whereas 21H offspring yield both 21H and 21L progeny when backcrossed to the 21L inbred strain III/J. These results are consistent with autosomal dominant inheritance of the 21H phenotype. Analysis of Southern blots of genomic DNA digested with the restriction endonuclease KpnI reveals 20-, 13-, and 9-kb fragments that hybridize with a probe derived from the 3'-untranslated region of the 21-hydroxylase cDNA. The 13-kb band is not observed for strain III/J or 21L progeny of strain III/J crossed with 21H rabbits, but it is detected for both 21H fathers and 21H progeny indicating that the genetically determined difference of 21-hydroxylase expression is inherited cis to the gene for P450IIC5, the hepatic progesterone 21-hydroxylase. Electrophoretic analysis of P450IIC5 synthesized in vitro from mRNA isolated from 21L and 21H rabbits reveals that little or no P450IIC5 is synthesized from 21L mRNAs. A second immunoreactive, electrophoretically distinct protein is synthesized from both 21L and 21H mRNAs to a similar extent but in lesser amounts than P450IIC5. The second protein could represent either an allozymic form of the enzyme or the product of a distinct locus. Thus, it is likely that distinct structural genes for P450IIC5 contribute to the differences in P450-mediated metabolism in 21L as compared to 21H rabbits.

Animals↗
PubMed1989

The human cytochrome Cyp1A2 gene contains regulatory elements responsive to 3-methylcholanthrene.

The regulation of the human cytochrome Cyp1A2 gene by 3-methylcholanthrene was studied through the transfection of 5'-flanking sequences into human cells. The Cyp1A2 promoter sequence and 3700 bases 5' to the cap site were linked to the procaryotic chloramphenicol acetyltransferase gene. Transfection of this construct into HepG2 cells generated a 2-3-fold increase in Cyp1A2-directed chloramphenicol acetyltransferase activity when the cells were treated with 3-methylcholanthrene. Deletion of flanking sequence to -1079 resulted in a loss of 3-methylcholanthrene-induced chloramphenicol acetyltransferase activity. When 5'-flanking sequences of the Cyp1A2 gene were inserted into a plasmid containing the chloramphenicol acetyltransferase gene under control of the simian virus 40 promoter, 3-methylcholanthrene-enhanced chloramphenicol acetyltransferase activity was observed. The strongest 3-methylcholanthrene-induced chloramphenicol acetyltransferase activity, a 4-fold increase, was observed for a DNA fragment located at -3202 to -1595. When this Cyp1A2 responsive element was transfected into human breast carcinoma MCF-7 cells, 3-methylcholanthrene did not stimulate chloramphenicol acetyltransferase activity. In comparison, when a DNA fragment that contained a copy of the human Cyp1A1 xenobiotic-responsive element was analyzed for enhancer activity, 3-methylcholanthrene initiated chloramphenicol acetyltransferase activity in both HepG2 cells and MCF-7 cells. These results suggest that the 3-methylcholanthrene-responsive Cyp1A2 element may be regulated in a tissue-specific manner.

Base Sequence↗
PubMed1987

Characterization of multiple human cytochrome P-450 1 cDNAs. The chromosomal localization of the gene and evidence for alternate RNA splicing.

Employing the rabbit liver progesterone-21-hydroxylase P-450 1 cDNA as a probe (Tukey, R.H., Okino, S., Barnes, H., Griffin, K.J., and Johnson, E.F. (1985) J. Biol. Chem. 260, 13347-13354), we have identified a highly homologous (81% within the coding region) human liver cDNA, termed Hp1-1, that encodes a 490-amino acid protein. Comparison of the predicted translation products between the human and rabbit homologues demonstrates that the two proteins are 73% homologous, while increasing to 82% similarity when allowing for conserved amino changes. The human P-450 1 is 82% homologous to the s-mephenytoin 4-hydroxylase (Umbenhauer, D. R., Martin, M. V., Lloyd, R. S., and Guengerich, F. P. (1987) Biochemistry 26, 1094-1099). Southern blot analysis using various portions of the human P-450 1 cDNA as probes indicates that the human P-450 1 gene is part of a larger gene family but can be selectively identified by using a 3'-noncoding portion of the cDNA. Identification of the gene from a panel of human-rodent somatic cell hybrids using the conserved 3' portion of the human P-450 1 cDNA as a probe places the location of the gene on human chromosome 10. Results are also presented which demonstrate that the human P-450 1 gene transcript is processed by an alternate RNA-splicing mechanism that generates two mRNA products, one which represents the functional transcript, and the other a form of mRNA that is not capable of encoding a functional P-450.

Amino Acid Sequence↗
PubMed1987

Characterization of a second gene product related to rabbit cytochrome P-450 1.

A cDNA, p1-88, was cloned from a library constructed using rabbit liver mRNA. Sequence analysis indicates that p1-88 is highly similar (congruent to 95%) to the cDNA, p1-8, that encodes rabbit liver cytochrome P-450 1 and that had been isolated from the same library. The predicted amino acid sequence of the protein encoded by p1-88, P-450 IIC4, differs at 25 of 487 amino acids from that encoded by p1-8. P-450 IIC4 was synthesized in vitro using rabbit reticulocyte lysate primed with RNA transcribed from the coding sequence of p1-88 using a bacteriophage T7 RNA polymerase/promoter system. P-450 IIC4 reacts with two monoclonal antibodies that recognize P-450 1 and exhibits the same relative electrophoretic mobility as P-450 1. In contrast, the reactivity of a third monoclonal antibody recognizing P-450 1, 1F11, toward P-450 IIC4 synthesized in vitro is greatly diminished. The latter antibody extensively inhibits hepatic progesterone 21-hydroxylase activity and recognizes phenotypic differences among rabbits in the microsomal concentration of P-450 1. This difference in the immunoreactivity of P-450 IIC4 and P-450 1 with the 1F11 antibody suggests that P-450 IIC4 does not contribute significantly to hepatic progesterone 21-hydroxylase activity. S1 nuclease mapping demonstrates that the expression of mRNAs corresponding to p1-88 are expressed to equivalent extents in rabbits exhibiting high and low expression of mRNAs corresponding to p1-8. Thus, P-450 1 differs from the protein encoded by p1-88, in its regulation, immunoreactivity, and by inference its catalytic properties although the amino acid sequences of P-450 1 and P-450 IIC4 are highly similar (congruent to 95%).

Amino Acid Sequence↗
PubMed1987

Intraspecies individuality for the metabolism of steroids.

A variety of regulatory factors contribute to differences in the rates of 6 beta-hydroxylation, 16 alpha-hydroxylation and 21-hydroxylation of progesterone as catalysed by liver microsomes prepared from individual rabbits. It is likely that the 6 beta-hydroxylation of progesterone is catalysed primarily by cytochrome P-450 3c, an enzyme that exhibits allosteric activation by alpha-napthoflavone, and by a form of P-450 3b, 6 beta+, that is expressed in some rabbits in an autosomal dominant manner. The mechanism of activation for P-450 3c appears to reflect an effector mediated increase of the affinity of the enzyme for substrate as judged by substrate binding studies. A second form of P-450 3b, 6 beta-, catalyses a major portion of hepatic progesterone 16 alpha-hydroxylation and exhibits activation by a variety of C21 steroids of which 5 beta-pregnane-3 beta,20 alpha-diol is the most efficacious. P-450 1, which catalyses the 21-hydroxylation of progesterone, is expressed at 10-fold higher levels in the 21H phenotype than the 21L phenotype, and the former is inherited as an autosomal dominant characteristic. A cDNA encoding a P-450 1-related gene product exhibits a predicted amino acid sequence that is 95% homologous to that of P-450 1. The P-450 1-related gene product is expressed in liver to a similar degree in both 21H and 21L rabbits.

Allosteric Regulation↗
PubMed1986

Human cytochrome P-450 4 mRNA and gene: part of a multigene family that contains Alu sequences in its mRNA.

Several overlapping lambda gt11 cDNA clones have been sequenced and shown to encode for the full-length human cytochrome P-450 4. The structure and location of the exons and flanking intron regions were also identified from a lambda EMBL-3 human genomic clone that encodes the full-length human P-450 4 gene. The human P-450 4 mRNA is flanked by 62 base pairs of 5'- and 1508 base pairs of 3'-noncoding sequence, with 1548 bases that encode a protein of 516 amino acids (Mr, 58,376). The predicted amino acid sequence of human P-450 4 is 69% and 70% homologous to its equivalent in mouse and rat, respectively, 75% homologous to rabbit P-450 4, and 68% homologous to human P1-450. The 7.6-kilobase gene encodes 3118 nucleotides of exon sequence that is separated by six introns into seven exons. Exon 7, which is 1802 nucleotides, contains three inverse/complement Alu sequences that are organized in tandem. Comparison of the genomic DNA sequence of the human P-450 4 gene with the human P1-450 and related genes in rat and mouse and the identification of the amino acid residues and triplet codon at each exon-intron junction show that the location of each intron in the human P-450 4 gene is conserved within this gene family. Although the length and homology of the introns within a related gene family may not be conserved, the location of intronic sequences may be an important determinant in the identification of related P-450 genes.

Amino Acid Sequence↗
PubMed1985

Multiple gene-like sequences related to the rabbit hepatic progesterone 21-hydroxylase cytochrome P-450 1.

Rabbits exhibit phenotypic differences, 21H and 21L, in the rate of hepatic progesterone 21-hydroxylation that reflect 10-fold higher microsomal concentrations of cytochrome P-450 1 in 21H rabbits. A cDNA library in pBR322 was prepared from liver mRNA isolated from a 21H rabbit. A clone, p1-8, producing a hybrid protein resulting from the insertion of the cDNA into the beta-lactamase gene of the plasmid expressed 5 distinct epitopes that were recognized by a panel of monoclonal antibodies developed toward P-450 1. RNAs selected from total hepatic mRNA by filter hybridization with p1-8 yield at least two electrophoretically distinct proteins when translated in vitro and immunoprecipitated with the 3C3 monoclonal antibody. Only one of the two proteins is recognized by the 1F11 monoclonal antibody, which is highly specific for P-450 1, and the immunoprecipitated protein exhibits the electrophoretic mobility of P-450 1. The other protein remains unidentified. Northern blot analysis indicates that the 3' noncoding portion of p1-8 hybridizes to higher steady state concentrations of polyadenylated RNA in the 21H as compared to 21L rabbits. This correspondence in expression with that of P-450 1 in the 21H and 21L phenotypes further suggests that p1-8 encodes P-450 1 or a closely related protein. The cDNA is 1871 base pairs in length and encodes a protein of 487 amino acids. Southern blot analysis indicates that several independent, gene-like sequences hybridize with the 3' noncoding region of p1-8 under conditions of high stringency. These results indicate that P-450 1 is a member of an extensive multigene family.

Amino Acid Sequence↗
PubMed1985

Cloning and characterization of cDNAs encoding 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible rabbit mRNAs for cytochrome P-450 isozymes 4 and 6.

Monoclonal antibodies toward rabbit liver cytochrome P-450 isozyme 6 (P-450 6) were used to identify several recombinant clones from a pBR322 cDNA library that express beta-lactamase-P-450 6 hybrid proteins. The nucleic acid sequence and predicted amino acid sequence of a rabbit P-450 6 cDNA shows a high degree of homology with rat P-450c and mouse P1-450. When used as a probe to rescreen the library, the P-450 6 cDNA hybridized to several heterologous classes of cDNAs. One such class was shown to encode P-450 4 by comparison of its predicted amino acid sequence to amino acid sequences of cysteine-containing tryptic peptides derived from P-450 4. DNA sequence analysis of a cDNA clone belonging to a third class demonstrated that it contained a 131-base-pair intervening sequencing when compared to the cDNA coding for P-450 6. This sequence corresponds in location to intron E of the rat P-450c gene. Blot-hybridization analysis demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramatically induced P-450 4 and 6 mRNAs, which differ in size. The sizes of these mRNAs differ from their analogs in the mouse as a result of divergence in the 3' untranslated portions of the mRNAs.

Amino Acid Sequence↗