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At least 19 recordsLinked to original sources
PubMed1996

Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

Antibodies↗
PubMed2006

QM/MM modeling of compound I active species in cytochrome P450, cytochrome C peroxidase, and ascorbate peroxidase.

QM/MM calculations provide a means for predicting the electronic structure of the metal center in metalloproteins. Two heme peroxidases, Cytochrome c Peroxidase (CcP) and Ascorbate Peroxidase (APX), have a structurally very similar active site, yet have active intermediates with very different electronic structures. We review our recent QM/MM calculations on these systems, and present new computational data. Our results are in good agreement with experiment, and suggest that the difference in electronic structure is due to a large number of small differences in structure from one protein to another. We also discuss recent QM/MM calculations on the active species of cytochrome P450, in which a similar sensitivity of the electronic structure to the environment is found. However, this does not appear to explain different catalytic profiles of the different drug-metabolizing isoforms of this class of enzyme.

Ascorbate Peroxidases↗
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↗
PubMed1994

Probing the putative cytochrome P450- and cytochrome c-binding sites on NADPH-cytochrome P450 reductase by anti-peptide antibodies.

Two regions (amino acid residues 110-130 and 204-218) of NADPH-cytochrome P450 reductase (reductase) have been shown to be the putative binding sites for the interaction with cytochrome P450 or cytochrome c. To obtain further insight into the molecular mechanism of protein-protein interaction between these proteins, three anti-peptide antibodies (1A, 2A, and 3A) were generated against the peptides corresponding to these two regions on rat reductase to study the interaction between the reductase and cytochrome P450 or cytochrome c. All three anti-peptide antibodies have high affinity for their peptide antigens on ELISA (titre > 1 x 10(-6) g/L), and they also bind to rat reductase on ELISA under both denatured and native conditions, suggesting that these regions are on the surface of the protein. 1A and 3A also bind to rabbit and human reductase, though 1A binds to human reductase with lower affinity. Antibody 2A does not bind to rabbit or human reductase. Western blot analysis using these anti-peptide antibodies showed similar results. Antibodies 1A and 3A inhibit both cytochrome P4501A1-dependent ethoxycoumarin hydroxylation activity and P4502B1-dependent pentoxyresorufin dealkylation activity, but the inhibition by 1A and 3A was not additive. Antibodies 1A and 3A also have inhibitory effects on the activity of P4501A1-dependent ethoxycoumarin hydroxylation reconstituted with reductase from rabbit and human. However, none of the three anti-peptide antibodies inhibits cytochrome c reduction by rat reductase. These data suggest that reductases from rat, rabbit, and human share similar structure in at least two regions which appear to be on the surface of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Examining the mechanism of stimulation of cytochrome P450 by cytochrome b5: the effect of cytochrome b5 on the interaction between cytochrome P450 2B4 and P450 reductase.

Dissociation constants K(d) for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b(5) (b(5)) stimulates metabolism of this substrate and dramatically decreases the substrate-related K(d) values. Experiments are performed to test if the b(5)-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b(5) (Mn b(5)) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b(5) stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b(5) is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b(5), with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.

Anaerobiosis↗
PubMed1996

Impact of tamoxifen on peripubertal androgen imprinting of rat hepatic cytochrome P450 2C11, cytochrome P450 3A2, and steroid 5 alpha-reductase.

Expression of sex-dependent rat hepatic cytochromes P450 and steroid 5 alpha-reductase is regulated mainly by the sex-specific pattern of growth hormone (GH) secretion and is subject to androgen imprinting. Since tamoxifen suppresses GH pulse amplitude and nadir levels, we investigated the effect of tamoxifen on peripubertal testosterone imprinting of hepatic CYP2C11, CYP3A2, CYP2A1, and steroid 5 alpha-reductase. Prepubertal tamoxifen administration (5 mg once daily s.c. on days 28 and 29 of age) to non-ovariectomized female Sprague-Dawley rats did not affect hepatic microsomal CYP2C11-dependent testosterone 2 alpha-hydroxylase, CYP3A-mediated testosterone 6 beta-hydroxylase, CYP2A1-dependent testosterone 7 alpha-hydroxylase, or steroid 5 alpha-reductase activity in adult rats. Testosterone treatment (5 mumol/kg, s.c., once daily) of intact female rats during either puberty (days 35-49 of age) or adult life (days 69-77 of age) had no effect on these enzyme activities in adult (78-day-old) female rats, but the same treatment given during both of these periods induced the male-specific testosterone 2 alpha- and 6 beta-hydroxylase activities and suppressed the female-predominant testosterone 7 alpha-hydroxylase and steroid 5 alpha-reductase activities, indicating that peripubertal testosterone administration imprints the adult androgen responsiveness but not the basal levels of these enzyme activities in non-ovariectomized female rats. However, peripubertal androgen imprinting of the basal levels of testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities was observed in female rats administered tamoxifen prepubertally. Tamoxifen pretreatment also enhanced testosterone imprinting of the adult androgen responsiveness of testosterone 2 alpha- and 6 beta-hydroxylase and steroid 5 alpha-reductase activities. The enhanced testosterone hydroxylase activities were, however, not associated with an increase in microsomal NADPH-cytochrome P450 reductase activity, but were accompanied by elevated hepatic CYP2C11 and CYP3A2 protein levels. Overall, the present study indicates that prepubertal tamoxifen administration does not interfere with the normal sex differentiation of the gender-dependent hepatic cytochromes P450 and steroid 5 alpha-reductase, but this drug modulates peripubertal androgen imprinting of CYP2C11, CYP3A2, and steroid 5 alpha-reductase in adult female rats.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c.

Site-directed mutagenesis of the acidic clusters 207Asp-Asp-Asp209 and 213Glu-Glu-Asp215 of NADPH-cytochrome P450 oxidoreductase demonstrates that both cytochrome c and cytochrome P450 interact with this region; however, the sites and mechanisms of interaction of the two substrates are clearly distinct. Substitutions in the first acidic cluster did not affect cytochrome c or ferricyanide reductase activity, but substitution of asparagine for aspartate at position 208 reduced cytochrome P450-dependent benzphetamine N-demethylase activity by 63% with no effect on KP450m or KNADPHm. Substitutions in the second acidic cluster affected cytochrome c reduction but not benzphetamine N-demethylase or ferricyanide reductase activity. The E213Q enzyme exhibited a 59% reduction in cytochrome c reductase activity and a 47% reduction in KCyt cm under standard conditions (x0.27 M potassium phosphate, pH 7.7), as well as a decreased KCyt cm at every ionic strength and a shift of the salt dependence of cytochrome c reductase activity toward lower ionic strengths. The E214Q substitution did not affect cytochrome c reductase activity under standard conditions, but shifted the salt dependence of cytochrome c reductase activity toward higher ionic strengths. Measurements of the effect of ionic strength on steady-state kinetic properties indicated that increasing ionic strength destabilized the reductase-cytochrome c3+ ground state and reductase-cytochrome c transition state complexes for the wild-type, E213Q, and E214Q enzymes, suggesting the presence of electrostatic interactions involving Glu213 and Glu214 as well as additional residues outside this region. The ionic strength dependence of kcat/KCyt cm for the wild-type and E214Q enzymes is consistent with the presence of charge-pairing interactions in the transition state and removal of a weak ionic interaction in the reductase-cytochrome c transition-state complex by the E214Q substitution. The ionic strength dependence of the E213Q enzyme, however, is not consistent with a simple electrostatic model. Effects of ionic strength on kinetic properties of E213Q suggest that substitution of glutamine stabilizes the reductase-cytochrome c3+ ground-state complex, leading to a net increase in activation energy and decrease in kcat. Glu213 is also involved in a repulsive interaction with cytochrome c3+. Cytochrome c2+ Ki for the wild-type enzyme was 82.4 microM at 118 mM ionic strength and 10.8 microM at 749 mM ionic strength; similar values were observed for the E214Q enzyme. Cytochrome c Ki for the E213Q enzyme was 17.6 microM at 118 mM and 15.7 microM at 749 mM ionic strength, consistent with removal of an electrostatic repulsion between the reductase and cytochrome c2+.

Amino Acid Sequence↗
PubMed1986

Magnetic resonance study of the structure and functions of cytochrome P450.

Cytochrome P450 is a membrane-bound enzyme providing oxidation of numerous organic compounds in organisms. The objective of this review is to show the wide possibilities that are provided by Electron Spin Resonance (ESR) and Nuclear Magnetic Resonance (NMR) techniques to the study of the structure and functions of this unique enzyme. High sensitivity of ESR spectra of cytochrome P450 to its functional state and interaction with substrates and inhibitors is illustrated. NMR and proton relaxation make it possible to obtain unique information about the structure of the active center of cytochrome P450 under physiological conditions. ESR and NMR methods allow one to obtain structural data on location of substrates, inhibitors, and their spin-labeled analogs with respect to Fe3+ ions in the enzyme-active center. Of special interest seems to be coupling of ESR with the affinity modification method. For this purpose, the spin-labeled analogs of cytochrome P450 substrates containing alkylating groups were used. As a result, an important datum has been obtained on the structure of active centers of cytochrome P450 in microsomes and in a highly purified state. In conclusion, the problems of the structure and functions of cytochrome P450, which can be most efficiently resolved with the use of magnetic resonance methods, are discussed.

Affinity Labels↗
PubMed1992

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.

Aniline Compounds↗
PubMed1996

Genetic analysis of factors controlling high-level expression of cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities in LPR house flies, Musca domestica.

To understand better the biochemical genetics of cytochrome P450 monooxygenase-mediated insecticide resistance, we examined the microsomal monooxygenases in insecticide-susceptible (aabys) and pyrethroid-resistant (LPR) house fly strains, as well as 15 house fly lines derived from crosses of LPR and aabys. In comparison to the aabys strain, LPR had higher levels of total cytochromes P450, cytochrome b5, P450 reductase, CYP6D1, and three P450 monooxygenase activities: 7-ethoxycoumarin O-deethylase (ECOD), methoxyresorufin O-demethylase (MROD), and aromatic hydrocarbon hydroxylase (AHH). The elevated levels of cytochrome b5 were linked to factors on autosomes 1 and 2. This is similar to previous reports on monooxygenase-mediated resistance and is consistent with the idea that elevated cytochrome b5 levels are involved in monooxygenase-mediated resistance in the LPR strain. Linkage of the elevated P450 reductase is different from that of monooxygenase-mediated resistance. Strains having high levels of CYP6D1 (i.e., like LPR) had high levels of P450 reductase, while strains having intermediate levels of CYP6D1 also had high levels of reductase. Therefore, there is no clear evidence that the elevated P450 reductase in the LPR strain is required for the increased monooxygenase activity. Overexpression of total cytochromes P450, CYP6D1 (mRNA and protein), and CYP6D1-mediated monooxygenase activities (MROD and AHH) in LPR microsomes was linked to a combination of factors on autosomes 1 and 2. This demonstrates that increased expression of CYP6D1 in the LPR strain is both cis regulated by a factor(s) on autosome 1 and trans regulated by a factor(s) on autosome 2. The correlation between the overexpression of CYP6D1 mRNA and protein suggests that CYP6D1 expression is regulated transcriptionally. Monooxygenase-mediated resistance in LPR is controlled by factors on autosomes 1 and 2, which supports previous claims that CYP6D1 is responsible for monooxygenase-mediated resistance in the LPR strain.

Animals↗
PubMed1990

Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450.

The role of NADPH--cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP--Fe3(+)-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPH- and ADP--Fe3(+)-dependent lipid peroxidation involves both NADPH--cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-linked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for O2 reduction. The first electron is used for reduction of ADP--Fe3+ and subsequent addition of O2 to the perferryl radical (ADP--Fe3(+)-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with O2 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione.

Adenosine Diphosphate↗
PubMed1997

Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5.

The development of enzyme and buffer premixes for in vitro biotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochrome b5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at -80 degrees C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4 degrees C for 2 weeks showed no significant loss in the rate of testosterone 6 beta-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave different Vmax and K(m) values indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated that Vmax and K(m) could be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays.

Amitriptyline↗
PubMed1998

Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b5. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b5. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.

Aryl Hydrocarbon Hydroxylases↗
PubMed1988

Reconstituted microsomal lipid peroxidation: ADP-Fe3+-dependent peroxidation of phospholipid vesicles containing NADPH-cytochrome P450 reductase and cytochrome P450.

A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30-50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe3+ was not required for lipid peroxidation, distinguishing this system from the one previously described by Pederson and Aust [Biochem. Biophys. Res. Comm. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O2-.-independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe3+ under anaerobic conditions.

Adenosine Diphosphate↗
PubMed2005

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes.

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.

Coenzymes↗
PubMed1981

Effect of bran and citrus pulp on hepatic, small intestinal and colonic HMG CoA reductase, cytochrome P450 and cytochrome b5 levels in rats.

The effect of dietary wheat bran and citrus pulp on hepatic, small intestinal and colonic HMG-CoA reductase activity, cytochrome P450 and cytochrome b5 levels was studied in F344 rats. Colonic and small intestinal HMG-CoA reductase activity was significantly lower in the wheat bran group than the control group. No differences in hepatic HMG-CoA reductase activity occurred between the wheat bran, citrus pulp and control groups. A direct relationship between hepatic cholesterol levels and hepatic Cytochrome P450 levels was demonstrated. A similar trend was exhibited between fecal cholesterol and colonic cytochrome P450 levels in the colon. These results suggest that the effect of certain types of dietary fiber in small intestinal and colonic cholesterogenesis is mediated through alterations in small intestinal and colonic mixed function oxidase activity.

Animals↗
PubMed2000

Drug interactions due to cytochrome P450.

Cytochrome P450 is a family of isozymes responsible for the biotransformation of several drugs. Drug metabolism via the cytochrome P450 system has emerged as an important determinant in the occurrence of several drug interactions that can result in drug toxicities, reduced pharmacological effect, and adverse drug reactions. Recognizing whether the drugs involved act as enzyme substrates, inducers, or inhibitors can prevent clinically significant interactions from occurring. Avoiding coadministration or anticipating potential problems and adjusting a patient's drug regimen early in the course of therapy can provide optimal response with minimal adverse effects.

Journal Article↗
PubMed1999

Nature's universal oxygenases: the cytochromes P450.

Cytochromes P450 are utilized in an enormous diversity of biological reactions, including degradation of xenobiotics, generation of hormones and biosynthesis of a variety of important biological compounds. The cytochrome P450 family is a major participant in nearly all metabolism of pharmaceutical reagents. The presence of different P450 enzymes in various quantities in individuals makes the prediction of drug responses in patients highly complex. A large literature describing mechanistic studies has characterized several intermediates in the oxygenation pathway. It has recently been shown that two or more possible oxygenated forms of the P450 haem can participate in various oxygenations, with some intermediates being highly electrophilic and others being nucleophilic.

Animals↗