Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “P450”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1993

Differential role in lipid peroxidation between rat P450 1A1 and P450 1A2.

The role of cytochrome P450 (P450) in lipid peroxidation induced by NADPH or peroxide was investigated in a reconstituted system. When cumene hydroperoxide, t-butyl hydroperoxide and hydrogen peroxide were used as initiators, the rates of malondialdehyde (MDA) formation were much higher in a reconstituted system containing P450 1A1 than those observed in a reconstituted system containing P450 1A2. In contrast to peroxide-induced lipid peroxidation, P450 1A2 catalysed NADPH-induced lipid peroxidation more effectively than did P450 1A1 regardless of the presence of ADP-Fe(NO3)3. Carbon monoxide inhibited NADPH-induced formation of MDA in a reconstituted system containing P450 1A2, but not P450 1A1. In addition, superoxide dismutase (SOD) was an effective inhibitor in a NADPH-induced lipid peroxidation system catalysed by P450 1A2 but not by P450 1A1. These results suggest that a peroxide-induced reaction might proceed readily with P450 1A1, whereas P450 1A2 mainly functions in NADPH-induced lipid peroxidation via generation of an active oxygen species. It is furthermore indicated that the difference in the effect of SOD in NADPH-induced lipid peroxidation depends on the P450 used.

Animals↗
PubMed1996

Tetrahydronaphthalenes: influence of heterocyclic substituents on inhibition of steroid enzymes P450 arom and P450 17.

In search of new leads for selective inhibition of estrogen and androgen biosynthesis, respectively, heterocyclic substituted 2-(arylmethylene)-1-tetralones (1-4, 9-17), 2-(aryl-hydroxymethyl)-1-tetralones (5-8), exo-1a,2,3,7b-tetrahydro-1H-cyclopropa[alpha] naphthalenes (18-24), and 3-alkyl substituted 4,5-dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized and tested for inhibitory activity toward four steroidogenic enzymes (P450 arom, P450 17, P450 18, and P450 scc, as well as another P450 enzyme, thromboxane A(2) (TXA(2)) synthase. The test compounds inhibited human placental P450 arom, showing a wide range of inhibitory potencies. (Z)-4-Imidazolyl compound 17 was the most potent inhibitor, with a relative potency (rp) of 110 [rp of aminoglutethimide (AG) = 1), rp of fadrozole = 359]. A competitive type of inhibition was shown by the (E)-4-imidazolyl compound 16(rp = 71). On the other hand some of these compounds inhibited rat testicular P450 17. Maximum activity was shown by the 3-pyridyl compound 20 (rp = 10, ro of ketoconazole = 1). 20 was the only compound which exhibited a marked inhibition of TXA(2) synthase (IC(50) = 14.5 microM; IC(50) of dazoxiben = 1.1 microM). Regarding selectivity toward the steroidogenic enzymes, compound 16 was relatively selective toward P450 arom, whereas compound 20 was relatively selective toward P450 17. (P450 arom: K(m) testosterone = 42 nM, K(i)16 = 33 nM, K(i)20 = 3 microM. P450 17: K(m)progesterone = 7 microM, K(i)16 = 9 microM, K(i)20 = 80 nM). 17 and 24 were not selective since they showed strong inhibition of P450 arom (K(i)17 = 26 nM, K(i)24 = 0.12 microM) and P450 17 (K(i) 17 = 0.7 microM, K(i)24 = 0.11 microM).

Aminoglutethimide↗
PubMed1997

Hydroperoxide-mediated cytochrome P450-dependent 8-anilino-1-naphthalenesulfonic acid destruction, product formation and P450 modification.

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 home directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of lambda max of ANS. Moreover, a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable.

Anilino Naphthalenesulfonates↗
PubMed1990

Ontogenesis of rabbit liver cytochrome P450. Evidence for a cytochrome P450-IIE (3a)-related form prevailing during the post-natal period.

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post-partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post-natal period. Total P450 as well as isozymes 2, 3b, 3c, 4 and 6 were measured. The evolution of these proteins with ageing, together with qualitative modification of an electrophoretic profile, produced evidence of an early developing P450 prevailing from one week to three weeks after birth. We isolated and characterized a cytochrome, called P450 2y, from two-week liver microsomes. It is closely related to P450 3a, an adult form of rabbit P450 induced by ethanol. They have similar molecular masses, the same lambda max of CO-reduced spectrum and exhibit immunological cross-reactivity. However, we cannot conclude that the two proteins are identical from N-terminal amino acid analysis or the two-dimensional gel electrophoresis pattern. These results, as well as the recent evidence of two different genes coding for the P450 3a family, strengthen the idea that P450 2y and 3a are distinct proteins. P450 2y seems to be an early developing form abundant soon after birth, while P450 3a is a delayed form appearing like most P450 isozymes during the fourth post-natal week. Besides the quantitative development during perinatal life, there is an important qualitative modification of liver cytochrome P450 content.

Aging↗
PubMed2001

Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads.

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Characterization of monkey cytochrome P450, P450 CMLd, responsible for S-mephenytoin 4-hydroxylation in hepatic microsomes of cynomolgus monkeys.

We isolated a new form of cytochrome P450 (P450) which was able to catalyze S-mephenytoin 4'-hydroxylation from hepatic microsomes of cynomolgus monkeys. The final preparation (referred to as P450 CMLd) was apparently homogenous judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the estimated minimum molecular weight of this protein was 53 kDa. The N-terminal amino acid sequence of P450 CMLd (identified 16 residues) was identical with that of protein encoded by P450 2C9 cDNA. P450 CMLd was cross-reactive with both antibodies raised against P450 2C11 and P450 2C9 which were purified from hepatic microsomes of male rats and humans, respectively. In hepatic microsomes of cynomolgus monkeys, both antibodies recognized two proteins showing different mobilities on SDS-PAGE (50 and 53 kDa). P450 CMLd was a good catalyst for S-mephenytoin 4'-hydroxylation in a reconstituted system. Anti-P450 2C9 antibody inhibited the activity of S-mephenytoin 4'-hydroxylase, but not the activities of R-mephenytoin 4'-hydroxylase and R- and S-mephenytoin N-demethylases in liver microsomes from cynomolgus monkeys. From these lines of evidence we conclude that P450 CMLd is classified into the P450 2C subfamily and acts as one of the S-mephenytoin 4'-hydroxylases in hepatic microsomes of cynomolgus monkeys.

Amino Acid Sequence↗
PubMed1992

Effects of testosterone and estrogen on hepatic levels of cytochromes P450 2C7 and P450 2C11 in the rat.

The effects of exogenous hormone treatment on the expression of cytochromes P450 2C7 and P450 2C11 were studied in neonatally gonadectomized and sham-operated male and female rats. Hepatic levels of cytochrome P450 2C7 were found to be two- to threefold higher in intact adult female versus male rats. Neonatal gonadectomy resulted in a reversal of the relative cytochrome P450 2C7 levels in male and female animals at maturity. Expression of this isozyme was restored in ovariectomized females by estradiol treatment. Furthermore, neonatal and/or pubertal administration of estradiol to intact male rats induced cytochrome P450 2C7 to adult female levels. On the other hand, administration of testosterone at all times examined had no effect in intact female rats, but decreased cytochrome P450 2C7 to normal levels in neonatally castrated males treated during adulthood. Neonatal testosterone treatment also increased hepatic cytochrome P450 2C7 content in both ovariectomized females and intact males. These results indicate that estrogen is required for full expression of cytochrome P450 2C7 while the effect of testosterone is ambiguous. In comparison, neonatal gonadectomy of male rats abolished the adult expression of cytochrome P450 2C11. Normal levels were restored only by treatment with testosterone during adulthood. Neonatal testosterone treatment did not induce cytochrome P450 2C11 levels in gonadectomized rats of either sex. In contrast, neonatal estrogen treatment suppressed cytochrome P450 2C11 expression in intact adult male rats to the same extent as neonatal castration. These results indicate that androgen exposure during the adult, and not the neonatal, phase is essential for full expression of cytochrome P450 2C11.

Aging↗
PubMed1995

Identification of a common cytochrome P450 epitope near the conserved heme-binding petide with antibodies raised against recombinant cytochrome P450 family 2 proteins.

The cytochrome P450 (P450) proteins constitute a superfamily of enzymes involved in various oxidations and related activities. Polyclonal antibodies raised against bacterial recombinant human P450s varied in specificity, depending upon the individual rabbits used. Several of the antisera raised against P450s 2C10 and 2E1 recognized a number of P450 family 1, 2, and 3 proteins, and two of the less selective antibodies were used to identify cross-reactive epitopes. P450 2C10 peptides reacting with anti-P450 2E1 and P450 2E1 peptides reacting with anti-P450 2C10 were isolated after electrophoresis/immunoblotting and analyzed by Edman degradation. Several of these were in a region near the highly conserved Cys that is a putative axial ligand to the heme. Peptides corresponding to the most conserved regions in this area were synthesized. Anti-P450 2C10 sera did not recognize 14-mer peptides corresponding to the heme-binding region (2C10 410-423 or 2E1 409-422) or the 14-mer peptides immediately C-terminal to these (2C10 425-438 or 2E1 424-437), but anti-P450 2E1 sera showed weak reaction with the latter two synthetic peptides. A longer peptide (29-mer) of P450 2E1 containing parts of both regions (412-440) reacted with both anti-P450 2C10 and anti-P450 2E1 antisera. Antibodies raised against a conjugate of the 29-mer peptide (with hemocyanin) recognized this antigen, the more C-terminal 14-mer peptides (2C10 425-438 and 2E1 424-437), P450s 2C10 and 2E1, and P450s 1A1, 11A1, and 17A. The 29-mer peptide showed considerable alpha-helix structure as judged by CD spectroscopy, in contrast to any of the 14-mers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Modulation of cyclophosphamide-based cytochrome P450 gene therapy using liver P450 inhibitors.

The sensitivity of tumors to cyclophosphamide (CPA) and other anticancer prodrugs can be substantially enhanced by transduction of tumors with a prodrug-activating mammalian cytochrome P450 (CYP) enzyme in combination with the flavoenzyme P450 reductase. This gene therapy strategy provides for intratumoral prodrug activation, but is also associated with a high level of hepatic prodrug activation, which reduces the extent of intratumoral prodrug activation and contributes to systemic drug toxicity. To address this issue, five P450 inhibitors were tested for their ability to block liver CYP2C-catalyzed CPA activation selectively, i.e., without inhibiting the corresponding intratumoral activation of CPA catalyzed by a transduced CYP2B enzyme. In vitro studies revealed that the P450 inhibitors 1-aminobenzotriazole and DDEP were preferentially inhibitory toward CYP2C-dependent liver microsomal CPA activation, whereas the P450 inhibitor SKF-525A inhibited CYP2C- and CYP2B-dependent CPA activation without P450 form selectivity. By contrast, the P450 inhibitors chloramphenicol and metyrapone preferentially inhibited CYP2B-dependent CPA activation. Rat pharmacokinetic studies confirmed the inhibitory action of these compounds in vivo, with up to a 4-fold decrease in C(max) and a 7-fold increase in apparent half-life of the activated CPA metabolite, 4-hydroxy-CPA, seen in the case of 1-aminobenzotriazole. Although the rate of hepatic CPA activation could thus be decreased substantially by P450 inhibitor treatment, the net extent of hepatic CPA activation was only modestly decreased, as judged by plasma area-under-the-curve values for 4-hydroxy-CPA. Moreover, P450 inhibitor treatment did not decrease CPA's host toxicity and did not enhance the tumor growth delay response to CPA in rats bearing CYP2B1-transduced gliosarcomas. These findings are discussed in the context of P450-based gene therapy strategies and ongoing efforts to enhance anticancer drug activity by increasing the exposure of P450-expressing tumors to the P450-activated prodrug CPA.

Animals↗
PubMed1992

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.

Animals↗
PubMed2003

Determination of the rate of reduction of oxyferrous cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase.

The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1.

Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k(cat), 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (approximately 0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process.

Amino Acid Sequence↗
PubMed2000

Cytochrome P450-dependent monooxygenase activities and their inducibility by classic P450 inducers in the liver, kidney, and nasal mucosa of male adult ring-necked pheasants.

In this study, several P450-dependent monoxygenase activities in the liver, kidney, and nasal mucosa of ring-necked pheasants were examined. In addition, the presence and inducibility of P450 isoenzymes in the hepatic and renal tissues of pheasants were examined by using typical substrates and inducers of P450s along with polyclonal antibodies raised against mammalian isoforms. Anti-rat P450 1A1 recognized in microsomes of both pheasant liver and kidney a protein that was markedly induced by beta-naphthoflavone and accompanied by an increase of various monooxygenases, in particular, methoxyresorufin-O-demethylase (MROD) activity. Anti-rat P450 2E1 revealed in microsomes of the pheasant liver but not in kidney an immunoreactive protein that was slightly induced by acetone but not accompanied by an increase of para-nitrophenol hydroxylase activity. On the other hand, acetone treatment caused an induction of other hepatic monoxygenases including MROD, erythromycin N-demethylase, and 6beta-testosterone hydroxylase. These two latter activities, known to be markers for 3A isoenzymes in rodents, were also enhanced in pheasant liver by phenobarbital but not by dexamethasone. The treatment with these two inducers also lacked to point out hepatic and renal proteins immunorelated to P450 3A or 2B subfamily, suggesting that these isoforms may be not expressed in pheasant. On the other hand, anti-rat P450 2C11 recognized two immunorelated proteins in the liver of both control and treated pheasants. The treatment with clofibrate, a mammalian inducer of 4A subfamily, induced both in liver and kidney of pheasant: i) a protein that cross-reacted with anti rat P450 4A1 and ii) the (omega) and (omega-1) lauric acid hydroxylase activities, known to be associated in mammals to this P450 subfamily. In the nasal mucosa of pheasant, a protein immunorelated to P450 2A and some monooxygenase activities (i.e., 7-ethoxycoumarin O-deethylase) linked, in mammals, to this isoform have been found; by contrast a protein immunoreactive with anti P450 2G1 was not found. In conclusion, the immunochemical properties and monooxygenase activities of constitutive and inducible P450s in pheasants were different not only from those of mammals but also from those of chickens. The findings of the present work also suggest that the P450 induction profiles might provide a potential biomarker of pheasant exposure to chemicals or environmental pollutants in the wild-field or in the stock-farm.

Acetone↗
PubMed2001

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

Bacillus subtilis↗
PubMed1992

The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233, WIN 59075) by mouse liver.

SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.

Animals↗
PubMed1998

High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli.

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.

Amino Acid Sequence↗
PubMed2001

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares.

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Mechanism-based inactivation of bovine adrenal cytochromes P450 C-21 and P450 17 alpha by 17 beta-substituted steroids.

A series of progesterone derivatives has been studied as potential inactivators of the bovine adrenocortical cytochromes P450, P450 17 alpha, and P450 C-21. Replacement of the 21-methyl group of progesterone with a difluoromethyl group resulted in a selective inactivator of P450 C-21 in a reconstituted system. The loss of 21-hydroxylase activity caused by this compound exhibits a number of characteristics of mechanism-based inactivation including NADPH dependence, pseudo-first-order kinetics, saturability, irreversibility, and protection by substrate. In addition to the difluoro compound, 21,21-dichloroprogesterone, the acetylenic compound pregn-4-en-20-yn-3-one, and the olefinic compound pregna-4,20-dien-3-one all inactivate P450 C-21. In contrast, the only compound to inactivate the rabbit adrenal progesterone 21-hydroxylase is 21,21-dichloroprogesterone. In binding studies, the 21,21-dihalo steroids produce a greater maximal type I spectral shift of P450 C-21 than the two 17 beta-unsaturated steroids. The dihalo compounds inactivate P450 C-21 by both heme destruction and protein modification as shown by significant decreases in residual 21-hydroxylase activity and spectrally detectable P450 after incubation with P450 C-21 in a reconstituted system. Liquid chromatographic and mass spectral analyses of the organic extracts from these incubations showed that 21-pregnenoic acid is a major metabolite of the dihalo compounds with a partition ratio of 5 nmol of acid produced/nmol of P450 C-21 inactivated. This supports the hypothesis that inactivation proceeds in part through an acyl halide intermediate. In contrast, the acetylenic compound pregn-4-en-20-yn-3-one inactivates P450 C-21 mainly by protein modification, producing an NADPH-dependent irreversible type I spectral shift. The stoichiometry of inactivation is approximately 1.5 nmol of compound bound/nmol of enzyme inactivated, indicating selective modification of the enzyme at or near the substrate binding site.

Adrenal Glands↗