Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cDNA containing the full coding sequence of human NADPH-P450 oxidoreductase was isolated and completely sequenced. The cDNA contained 2398 base pairs, including 9 and 358 base pairs of 5' and 3' noncoding sequences, respectively. The human NADPH-P450 oxidoreductase protein deduced from the cDNA has 677 amino acids, with a calculated molecular weight of 76,656. The cDNA nucleotide and deduced amino acid sequences displayed 83 and 92% similarities, respectively, with those of the rat NADPH-P450 oxidoreductase. By use of somatic cell hybrids, the NADPH-P450 oxidoreductase gene was regionally localized to human chromosome 7 (7p15-q35). The levels of NADPH-P450 oxidoreductase protein and mRNA were analyzed in 13 human liver specimens and less than 3-fold variation was found among the different livers. The NADPH-P450 oxidoreductase cDNA was inserted into vaccinia virus and expressed in cell culture. The cDNA-expressed enzyme was active in reducing the electron acceptor cytochrome c. In addition, the NADPH-P450 oxidoreductase stimulated the enzymatic activity of vaccinia virus-expressed human P3(450) when both recombinant viruses were used to coinfect human cells in culture. An approximate equal mole level of NADPH-P450 oxidoreductase and P3(450) was required to achieve maximal activity for both ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase.
Mol Pharmacol 1989 Jul
PMID:Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7. 250 55

Irradiation of microsomes with visible light in the presence of externally-added acridine orange results in O2 uptake, malondialdehyde accumulation, and inactivation of the microsomal drug-metabolizing system. The latter effect is reflected by a decrease in NADPH-cytochrome P450- and NADH-cytochrome b5 reductase activities and cytochromes P450 and b5 content by 88-, 85-, 60-, and 34%, respectively, after 5-min irradiation. Anoxia prevented inactivation of both reductases by 70-90%, whereas it prevented completely cytochrome b5 destruction. The presence of reducing equivalents, at the expense of NADPH and NADH, exert a partial protection (40-54% residual activities) against photosensitization damage on both reductase activities, whereas it almost fully protected cytochrome b5. Photosensitization of lipid peroxidation, as well as inactivation of the microsomal drug-metabolizing system, appears to involve both a type I and type II process. Products of lipid peroxidation might also play a role in enzyme inactivation and cytochrome destruction, as suggested by kinetic and time course studies and the redox state of microsomes. The uptake of acridine orange by isolated lysosomes is linearly dependent on the concentration of added dye and the distribution between extra- and intralysosomal acridine orange is strongly dependent on the amount of lysosomes. Irradiation of acridine orange-loaded lysosomes (light intensity at the sample position approximately 320 mW/cm2) produces an impairment of the membrane which leads to a rapid release of enzyme (N-acetyl-beta-glucosaminidase activity) into the medium, accompanied by a loss of activity in the lysosome-containing pellet and a partial photodamage of the enzyme. Concomitantly, thiobarbituric acid-reactive material accumulation increases in the reaction mixture with increasing irradiation time. When light intensity at the position was reduced to approximately 3.6 mW/cm2, photodamage of lysosomes was of a lesser magnitude, allowing the demonstration of a lag phase, which decreased with irradiation time, probably reflecting the so-called first-stage activation of lysosomes, preceding the release of lysosomal enzymes.
Virchows Arch B Cell Pathol Incl Mol Pathol 1989
PMID:Acridine orange-mediated photodamage of microsomal- and lysosomal fractions. 256 19

The effect of DNA damage induced by the carcinogen chromium(VI) on the function of DNA as a template for transcription of constitutive and inducible genes was examined in chick embryo liver in vivo. Changes in gene expression, determined using solution hybridization and northern blot analyses to measure steady-state mRNA levels and a nuclear run-off assay to measure gene transcription rates, were compared to chromium-DNA binding and to chromium(VI)-induced DNA damage as previously measured by DNA alkaline elution. Chromium(VI) treatment had little or no effect on either the steady-state mRNA levels or the transcription rates of the constitutively expressed genes for albumin, conalbumin (avian transferrin), or beta-actin. In contrast, chromium(VI) treatment had significant but opposite effects on the basal and drug-inducible expression of 5-aminolevulinate synthase and cytochrome PB1 P450. The changes in steady-state expression of these two inducible genes were similar to the changes in transcription rate, indicating that the effects of chromium were principally transcriptional. Chromium(VI) treatment increased the basal expression of both inducible genes four- to fivefold at maximum, and the time course of this effect was similar to the time course for chromium(VI)-induced DNA damage and repair. In contrast, chromium(VI) pretreatment suppressed by 60-70% at maximum the subsequent induction of these genes by glutethimide, a phenobarbital analog, and the time course of this effect also corresponded to that of chromium(VI)-induced DNA damage and repair. The time courses of the changes in expression of these genes were bimodal, with the second peak corresponding closely to that of chromium(VI)-induced DNA cross-links. However, the first peak occurred during a period when no DNA cross-links or strand breaks were detectable by alkaline elution, although significant levels of chromium were bound to DNA. This suggests that chromium(VI), like cisplatin, may initially produce a DNA monoadduct that subsequently leads to DNA cross-link formation and that both types of chromium(VI)-induced lesions have a significant effect on the expression of targeted genes.
Mol Carcinog 1989
PMID:Differential effects of chromium(VI) on constitutive and inducible gene expression in chick embryo liver in vivo and correlation with chromium(VI)-induced DNA damage. 260 65

An immunohistochemical technique was used to localize the major constitutive cytochrome P450 isozyme, P450 LM2, and the major beta-naphthoflavone-inducible isozyme, P450 LM4b, in the livers of untreated and aflatoxin B1 (AFB1)-initiated, tumor-bearing rainbow trout. In hepatic tissue sections from untreated trout, no regular anatomical pattern within the hepatic parenchymal cells could be discerned for either isozyme. Immunostaining was observed for P450 LM2 along the sinusoidal border of some of the parenchymal cells, there was moderate staining within the cytoplasm of most cells, and there were focal areas of increased staining. There was intense, uniform immunostaining for P450 LM2 within the cytoplasm of the bile duct cells, in the endothelial lining of arterioles, and along the epithelial surface of the gall bladder. Staining for P450 LM4b in livers from untreated trout was barely detectable. In liver tissue sections from AFB1-treated tumor-bearing fish, P450 LM2 appeared to be reduced and P450 LM4b was absent in the hepatocellular carcinoma nodules. An apparent increase in immunostaining for P450 LM4b was observed in nonneoplastic cells juxtaposed next to neoplastic cells as well as in areas distant to the tumors. These results may indicate that the pattern of P450 isozymes is altered in nonneoplastic cells of tumor-bearing trout livers.
Exp Mol Pathol 1989 Jun
PMID:Cytochrome P450 isozyme distribution in normal and tumor-bearing hepatic tissue from rainbow trout (Salmo gairdneri). 265 90

Previous studies have shown that high levels of cytochrome P450 can occur in cardiac microsomes of vertebrates [Mol. Pharmacol. 21:517-526, (1982)]. Here we identify the dominant cardiac P450 in the marine fish scup as P450E, a teleost representative of P450IA1, and we describe its restricted cellular localization in the heart. Treatment of scup with beta-naphthoflavone produced an unusually strong (10-fold) induction of spectrally measured P450 in cardiac microsomes, with specific content reaching levels (0.5 nmol/mg) similar to those induced in scup liver. Microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities, catalytic functions of scup P450E, were induced in parallel with P450 content. Similar induction was seen in both atrium and ventricle. Immunoblot analysis with monoclonal antibody 1-12-3, specific to scup P450E and other vertebrate P450IA1 proteins, showed that this hydrocarbon-inducible P450 is the dominant and possibly sole P450 form in heart microsomes of experimentally induced animals. Immunohistochemical analysis of scup heart sections (2-4-microns) with monoclonal antibody 1-12-3 revealed that P450E was detectable only in endothelial cells of the endocardium and of the coronary vasculature. A similar endothelial cell localization of the monoclonal antibody 1-12-3 epitope was observed in heart of rainbow trout, induced with beta-naphthoflavone, indicating a general nature for the endothelial localization of induced cardiac P450. Morphometric analysis showed that endothelium could constitute 8-9% of the volume of teleost heart, from which we calculate that P450IA1 could account for as much as 25% of the endothelial cell microsomal protein. Heart microsomes of untreated animals from contaminated environments also contained high levels of P450E, indicating that induction like that caused by beta-naphthoflavone could occur with chemicals in the environment. Strongly induced P450E (P450IA1) in endothelium could play a critical role in chemical-biological interactions involving xenobiotics affecting the vasculature of the heart or other organs.
Mol Pharmacol 1989 Nov
PMID:Cytochrome P450IA1 induction and localization in endothelium of vertebrate (teleost) heart. 268 43

Of four monoclonal antibodies to purified rat liver cytochrome P450s, including those from 3-methylcholanthrene-, phenobarbital-, ethanol-, and pregnenolone-16-alpha-carbonitrile-treated rats, only the monoclonal antibody against pregnenolone-16-alpha-carbonitrile-inducible P450 immunodetected proteins in chicken liver microsomes after blotting from sodium dodecyl sulfate-polyacrylamide gels. This protein migrated identically with the pregnenolone-16-alpha-carbonitrile-inducible P450 detected in microsomes from dexamethasone-treated rats. It was most predominant in liver microsomes from chickens at 1 day posthatching, whereas much lower levels were observed in the embryo and at 36 days posthatch. Phenobarbital and dexamethasone were both effective inducers of this protein. The developmental profile and induction by phenobarbital and dexamethasone of several cytochrome P450-associated catalytic activities were compared with those of the immunodetected protein. Chicken liver microsomal erythromycin demethylase, a characteristic activity of rat pregnenolone-16-alpha-carbonitrile-inducible P450, was similar in developmental profile and induction to the immunodetected protein, with a high degree of augmentation at 1 day posthatch compared with that in the embryo and at 36 days posthatch; aldrin epoxidase, benzphetamine demethylase, ethylmorphine demethylase, and aminopyrine demethylase were more similar to each other in development and induction and were less well correlated with the immunodetected protein. This evidence suggests the presence in chicken liver of at least two types of P450, one a form related to the pregnenolone-16-alpha-carbonitrile-inducible P450 family. All of the catalytic activities were induced after pretreatment of chickens with phenobarbital but aldrin epoxidase was most effectively induced. Aldrin epoxidase was also detected in microsomes from untreated embryos as early as 7 days of incubation. Erythromycin demethylase was the only catalytic activity induced by dexamethasone. There was a trend of increased specific activity toward all the substances after hatching, indicating a more efficient P450 system, possibly due to a sharp increase in some isozymes, including the form from the pregnenolone-16-alpha-carbonitrile-inducible P450 family. This evidence for a pregnenolone-16-alpha-carbonitrile-inducible P450 in chickens agrees with sequence information that suggests the early evolution of this form and demonstrates the suitability of the chicken for studies of P450 evolution.
Mol Pharmacol 1989 May
PMID:Evidence for a PCN-P450 enzyme in chickens and comparison of its development with that of other phenobarbital-inducible forms. 272 70

A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P450(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P450(17 alpha), 64% similarity with bovine P450(17 alpha), and 47% similarity with chicken P450(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P450(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P450(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta 4-C21 steroids to delta 4-C19 steroids at significant rates.
Mol Endocrinol 1989 Jun
PMID:Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17,20-lyase reactions. 278 90

The metabolism of midazolam and triazolam to their 1'-hydroxy and 4-hydroxy metabolites was studied in microsomes of 15 human livers. The formation of both metabolites was inhibited by more than 90% by an antiserum directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P450 (P450PCN1) of rat liver. Moreover, midazolam hydroxylase activity was immunoprecipitated from solubilized human microsomes with polyclonal antibodies against rat P450PCN1 and the closely related human isozyme P450NF. A close correlation was observed between the amount of protein detected in immunoblots with these antibodies and the midazolam or triazolam hydrxylase activity. The formation of both metabolites of midazolam was inhibited by triacetyloleandomycin, a known inhibitor of cytochromes P450 of the IIIA family. Direct evidence that P450IIIA4 catalyzes the metabolism of midazolam was provided through the use of cDNA-directed expression. Monkey COS cells transfected with human P450PCN1 cDNA were able to catalyze both the 1'- and the 4-hydroxylation of midazolam. We conclude that the metabolism of midazolam and triazolam in human liver is predominantly mediated by cytochrome P450IIIA4. Two of 15 human livers expressed a second immunoreactive microsomal protein of higher apparent Mr and were more active in midazolam 1'-hydroxylation. Our data also provide evidence that the marked interindividual variation in the response to these widely used benzodiazepine drugs is due to variable hepatic metabolism.
Mol Pharmacol 1989 Jul
PMID:Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. 278 73

cDNAs for rodent P(1)450, P(3)450, and P450a were expressed in the modified vaccinia virus-T7 RNA polymerase system. Each P450 exhibited its appropriate molecular weight and characteristic enzyme activity. Aryl hydrocarbon hydroxylase activity was catalyzed by P(1)450, acetanilide hydroxylase by P(3)450, and testosterone 7 alpha-hydroxylase by P450a. Ethoxycoumarin deethylase was exhibited by both P(1)450 and P(3)450. Each expressed P450 was also analyzed for its ability to activate 19 carcinogens of diverse classes to their mutagenic forms. Most notable was the activation of several polycyclic aromatic hydrocarbons by P1 and the activation of acetylaminofluorene, 4-aminobiphenyl, and several heterocyclic amine food pyrolysate products by P(3)450. P450a, in contrast, showed slight mutagen activation only toward N-hydroxy-2-acetyl aminofluorene. The vaccinia virus-T7 RNA polymerase system described here can express cDNAs for diverse forms of P450, each of which can then be characterized for substrate and product specificity and for mutagen activation.
Mol Carcinog 1989
PMID:Mutagen activation by cDNA-expressed P(1)450, P(3)450, and P450a. 278 89

The vaccinia virus cDNA expression system was used to produce human cytochrome P450 IA2 in a hepatoma cell line that is devoid of significant basal levels of P450. The expressed enzyme yielded a reduced carbon monoxide-bound difference spectrum with a lambda max of 449 nm. Catalytic activities and mutagen activation ability of the human enzyme were assessed and directly compared with results obtained with the orthologous mouse IA2, which was also expressed using vaccinia virus. Both the human and mouse enzymes were able to catalyze efficiently the p-hydroxylation of aniline. Mouse IA2 also catalyzed ethoxyresorufin O-deethylation, and its activity was sevenfold greater than expressed human IA2. The mouse and human enzymes also activated several promutagens and procarcinogens. Mouse IA2 was five- to sevenfold more active than the human enzyme for activation of the procarcinogens 2-acetylaminofluorene and benzo[a]pyrene-trans-7,8-dihydrodiol and the promutagens Glu-P-2 and Trp-P-1. Comparable activities were observed with 2-aminoanthracene, 2-aminofluorene, and Glu-P-1. These data demonstrate the utility of cDNA expression for examining the activities of human P450s and further suggest potentially important differences in catalytic activities of orthologous P450s found in different species.
Mol Carcinog 1989
PMID:Human cDNA-expressed cytochrome P450 IA2: mutagen activation and substrate specificity. 280 20


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