Biomedical subjects

C B Kasper

Publications and source records attributed to C B Kasper.

At least 37 records · Page 2Linked to original sources

PubMed1993

Expression of rat microsomal epoxide hydrolase in Escherichia coli. Identification of a histidyl residue essential for catalysis.

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.

Amino Acid Sequence↗

PubMed1992

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.

Animals↗

PubMed1992

Geometric relationship between the nicotinamide and isoalloxazine rings in NADPH-cytochrome P-450 oxidoreductase: implications for the classification of evolutionarily and functionally related flavoproteins.

The stereospecificity of hydride abstraction from NADPH and the conformation of the nicotinamide ring around the glycosidic bond have been determined for the flavoprotein NADPH-cytochrome P-450 oxidoreductase (P-450R). The A-side (pro-R) hydrogen is abstracted from NADPH, and the nicotinamide ring is in the anti conformation. These results are consistent with the apparently strong correlation between A-side stereospecificity and anti conformation and between B-side stereospecificity and syn conformation [You, K. (1985) CRC Crit. Rev. Biochem. 17, 313]. This correlation reveals how the flavin and nicotinamide rings are oriented relative to each other. In P-450R, the flavin is then "on top of" (on the exo side of) the nicotinamide ring. In another flavoprotein dehydrogenase, glutathione reductase, which is a B-side/anti enzyme [Pai, E. F., & Schulz, G. E. (1983) J. Biol. Chem. 258, 1752], the flavin is "underneath" (on the endo side of) the nicotinamide ring. We argue that all enzymes that are evolutionarily related to these two flavoproteins should have their respective overall configurations. The overall configuration is defined by the following five properties: (1) relative orientation of the isoalloxazine and nicotinamide rings, (2) stereospecificity of hydride transfer to/from the nicotinamide ring, (3) conformation of the nicotinamide ring around the glycosidic bond, (4) stereospecificity of hydride transfer to/from the flavin, and (5) conformation of the flavin around its N5-N10 axis. There are only eight possible overall configurations, and a knowledge of only three of the five properties is needed to determine which one is present (as long as the combination of properties is not 1, 2, 3 or 1, 4, 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding.

Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected. The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions. These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.

Amino Acid Sequence↗

PubMed1991

Transcriptional regulation and localization of the tissue-specific induction of epoxide hydrolase by lead acetate in rat kidney.

Intraperitoneal administration of lead acetate to male Sprague-Dawley rats resulted in the tissue-specific transcriptional activation of the microsomal epoxide hydrolase gene in kidney. This response was followed by a 15-fold increase in the level of kidney epoxide hydrolase mRNA, while no change in mRNA level was noted in liver. Treated animals also showed no increase in mRNA levels for NADPH-cytochrome P-450 oxidoreductase, cytochrome P-450b/e, cytochrome P-450PCN (where PCN is pregnenolone 16 alpha-carbonitrile), or serum albumin in either kidney or liver. Immunoquantitation of the enzyme revealed a 16-fold increase in kidney upon treatment with lead acetate, but significant changes in epoxide hydrolase levels were not noted in liver, heart, spleen, lung, small intestine, or testis. The enzymatic activity for liver and kidney paralleled the immunochemical results; however, the activity increase in kidney was only one-third of the increase noted for total enzyme protein. Immunohistochemical analysis of epoxide hydrolase protein in sections of rat kidney demonstrated that in lead acetate-treated animals there was a marked increase in staining of the cytoplasm of the proximal tubular cells in the outer cortex as compared with kidneys from control animals. In contrast, considerable protein was also localized to collecting ducts, but no change was evident in the content of the epoxide hydrolase gene product in these structures in control and lead acetate-treated animals. Immunohistochemical differences were not noted between livers from control and lead-treated animals. Furthermore, the staining patterns for NADPH-cytochrome P-450 oxidoreductase were the same for control and treated animals in both kidney and liver. Quantitative measurements of lead uptake by various rat tissues showed liver, spleen, and small intestine reaching a maximum of approximately 12,000 ng of lead/g of dry tissue at 8, 8, and 16 h, respectively, while kidney, lung, and testis peaked (approximately 3,000 ng of lead/g of dry tissue) at 16, 16, and 12 h, respectively.

Animals↗

PubMed1990

Activation of mitomycin C by NADPH:cytochrome P-450 reductase.

Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase. In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions. However, under hypoxic conditions increased sensitivity was not observed. This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells. These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance. P-450 reductase expressed in S. typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway.

Animals↗

PubMed1990

NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein.

cDNA clones to rat liver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 lambda-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.

Amino Acid Sequence↗

PubMed1990

Glucocorticoid repression and basal regulation of the epoxide hydrolase promoter.

Through a series of promoter deletions and gene transfer experiments we have examined the basal regulation and glucocorticoid-mediated repression of the rat epoxide hydrolase gene. Three regions of the 5' flanking sequence were found to influence the basal level of promoter function in H4IIE hepatoma cells. Region A (-891 to -355 bp) contains an apparent repressor of epoxide hydrolase expression, while regions B (-271 to -171 bp) and C (-141 to -85) were found to contain important sequences required for optimal promoter activity. Previous work has demonstrated that dexamethasone represses epoxide hydrolase transcription by approximately 50% in isolated rat liver nuclei, and, in this study, we have demonstrated that the ability of the epoxide hydrolase promoter to drive CAT expression is similarly repressed in H4IIE cells treated with 1 microM dexamethasone. Furthermore, the level of endogenous epoxide hydrolase mRNA is decreased by 70-88% in nontransfected H4IIE cells treated with dexamethasone. Interestingly, promoter activity was not decreased by dexamethasone in COS cells, which lack glucocorticoid receptors. The current data show that sequences from -42 to +110 bp are sufficient to support the dexamethasone response, and, furthermore, they suggest that repression may not require direct interaction of the ligand-receptor complex with the promoter region.

Animals↗

PubMed1989

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development.

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.

Adrenal Glands↗

PubMed1989

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis.

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.

Amino Acid Sequence↗

PubMed1987

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene.

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.

Amino Acid Sequence↗

PubMed1987

Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids. Transcriptional and post-transcriptional regulation.

Quantitative evaluation of transcriptional rates in isolated nuclei and intranuclear and cytoplasmic mRNA levels demonstrates that the catatoxic steroids pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone modulate microsomal drug metabolizing enzymes both positively and negatively at the transcriptional level. Additionally, a strong post-transcriptional influence, believed to be message stabilization, was found to be exerted by dexamethasone. Both NADPH-cytochrome P-450b-homologous mRNA are induced 7- and 12-fold, respectively, presumably through message stabilization. Under these conditions, however, the oxidoreductase content of the cell is only marginally increased while the cytochrome P-450b level is actually decreased by 40% within three days after a single injection of dexamethasone. In contrast, genes coding for cytochromes P-450PCN show a definite increase in transcription following administration of either pre gnenolone-16 alpha-carbonitrile or dexamethasone, whereas transcription of the epoxide hydrolase gene is markedly reduced by administration of these compounds.

Animals↗

PubMed1987

Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli.

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2'5'-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5-10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 mumol of cytochrome c reduced/mg reductase/min at 22 degrees C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.

Animals↗

PubMed1986

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins.

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.

Amino Acid Sequence↗

PubMed1986

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Amino Acid Sequence↗

PubMed1985

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

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

Amino Acid Sequence↗

PubMed1985

Chromosomal assignments of genes coding for components of the mixed-function oxidase system in mice. Genetic localization of the cytochrome P-450PCN and P-450PB gene families and the nadph-cytochrome P-450 oxidoreductase and epoxide hydratase genes.

Filter-hybridization studies show that major phenobarbital and pregnenolone-16alpha-carbonitrile-inducible cytochrome P-450 mRNAs in rats were encoded by members of separate, distinct gene families. These gene families are genetically divergent from each and show no cross-hybridization, even under low-stringency conditions. Furthermore, sequences contained in the P-450PB and P-450PCN gene families map to separate chromosomes of the mouse genome. Using mouse X Chinese hamster somatic cell hybrids (EBS cell lines), all distinguishable P-450PCN sequences were found to map to chromosome 6, whereas all P-450PB sequences were located on chromosome 7. Our data support the proposition that the region of the Coh locus on chromosome 7 is the site of the cytochrome P-450PB gene family. The presence of gene families for the cytochromes P-450 occurs in many mammalian species and is likely an important part of the mechanism by which the mixed-function oxidase system is capable of recognizing and metabolizing such a wide array of endogenous and foreign compounds. Conversely, NADPH-cytochrome P-450 oxidoreductase appears to be encoded in many vertebrate species by a single gene and is located on chromosome 6 of the mouse. Corroboratory data are presented to show that the Eph-1 locus on chromosome 1 is the site of at least one microsomal epoxide hydratase gene.

Animals↗

PubMed1985

Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains.

The coding nucleotide sequence of the mRNA for NADPH-cytochrome P-450 oxidoreductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) from rat liver was determined from two overlapping cDNA clones, pOR-7 and pOR-8, which together contain 2401 nucleotides complementary to rat liver oxidoreductase mRNA. The single open reading frame of 2034 nucleotides spanning these cDNAs codes for a 678 amino acid polypeptide with a molecular weight of 76,962. The deduced amino acid composition is in excellent agreement with that determined by direct amino acid analysis of purified rat liver P-450 oxidoreductase, and the amino-terminal region (residues 1-80) largely coincides with the amino-terminal sequence of the oxidoreductase isolated from rabbit liver. Comparison of the amino acid sequence to those of other flavoproteins revealed two separate domains that are likely to be involved in flavin binding: a long segment (residues 77-228) homologous with Desulfovibrio vulgaris flavodoxin, an FMN-containing protein, and a shorter segment (residues 452-477) homologous with the FAD-binding segment of fumarate reductase from Escherichia coli.

Amino Acid Sequence↗