Reconstitution of the Brain Mixed Function Oxidase System: Purification of NADPH-Cytochrome P450 Reductase and Partial Purification of Cytochrome P450 from Whole Rat Brain

NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot techniques. Kinetic studies using cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P450c (P450IA1) as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. Our results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.     

Bioconversion by functional P450 1A9 and P450 1C1 of Anguilla japonica

We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4'-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize.     

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

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

Induction of cytochrome P450-dependent monooxygenase in mouse liver and kidney by rutaecarpine, an alkaloid of the herbal drug Evodia rutaecarpa.

Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.     

Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies

A cDNA of human cytochrome P450IA1 was expressed in yeast Saccharomyces cerevisiae on a multicopy plasmid under the control of the constitutive GAPFL or the inducible PHO5 promoter. Microsomes of transformed yeast contained substantial amounts of the heterologous enzyme as determined by reduced CO-difference spectra (156-68 pmol/mg). Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in a Km value of 92 nM and a Vmax value of 223 pmol/mg/min, which is comparable to data obtained with human liver microsomes. The antimycotic drug ketoconazole (Ki = 22nM) as well as the isozyme specific P450 inhibitor alpha-naphthoflavone (Ki = 1.2 nM) were shown to be strong inhibitors of human P450IA1. Taken together, these data show that heterologous P450 gene expression in yeast is a potent instrument for the study of enzyme specific parameters and might be used to answer further questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities.     

Influence of ligand binding to human cytochrome P-450 1A2: conformational activation and stabilization by alpha-naphthoflavone

Human cytochrome P-450 (P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 (P-420) in the presence of detergents. alpha-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification. However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood. We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K(m) values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased alpha-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5 degrees C increase in thermal stability.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Dose-dependent, mechanism-based inactivation of cytochrome P450 monooxygenases in vivo by 1-aminobenzotriazole in liver, lung, and kidney of untreated, phenobarbital-treated, and beta-naphthoflavone-treated guinea pigs.

The mechanism-based inactivation of the cytochrome P450 (P450) dependent monooxygenase system was studied in vivo in liver, lung, and kidney of untreated, phenobarbital-treated, and beta-naphthoflavone-treated guinea pigs 24 h after administration of 1-aminobenzotriazole (1-100 mg/kg, i.p.). Microsomal isozyme-selective or -specific monooxygenase activities were inhibited in a dose-dependent manner in all three organs. In the liver of untreated and phenobarbital-treated animals, 7-pentoxyresorufin O-depentylation (catalyzed primarily by P450 2Bx, an orthologue of rabbit P450 2B4/rat 2B1) was inhibited more than 7-ethoxyresorufin O-deethylation (P450 1A1), 4-aminobiphenyl N-hydroxylation (P450 1A2), erythromycin N-demethylation (P450 3A), or benzphetamine N-demethylation; in beta-naphthoflavone-treated animals, 4-amino-biphenyl N-hydroxylation activity was preferentially inhibited. In lung, the order of inactivation of monooxygenase activities was 4-aminobiphenyl N-hydroxylation (4Bx, the orthologue of rabbit 4B1) > 7-pentoxyresorufin O-depentylation activity (2Bx) > 7-ethoxyresorufin O-deethylation (1A1; for example 72, 53, and 29% inactivation, respectively, in phenobarbital-treated animals at 100 mg/kg). In all three tissues the loss in spectrally assayed P450 content corresponds quite well to the inhibition of monooxygenase activities. Thus, these studies show that 1-aminobenzotriazole is an effective inactivator of the pulmonary, hepatic, and renal P450 systems in guinea pigs following i.p. administration, and that P450 1A2 (liver) and P450 4Bx (lung), isozymes efficient for the oxidation of primary aromatic amines, are preferentially inactivated.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism

Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.     

Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.     

Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.     

Stimulation of mixed-function oxidation of 7-ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol

Rates of O-deethylation of 7-ethoxycoumarin by perfused livers from fasted, phenobarbital-treated rats were 3.7 mumol X g-1 X h-1. Approximately 50% of the product was conjugated. When rates of 7-ethoxycoumarin O-deethylation were varied by infusing different concentrations of substrate, a good correlation (r = 0.91) was found between rates of O-deethylation of 7-ethoxycoumarin and fluorescence of 7-hydroxycoumarin detected from the liver surface. Micro-light guides (tip diameter 170 microns) placed on periportal and pericentral regions on the liver surface were used to monitor the conversion of nonfluorescent 7-ethoxycoumarin to fluorescent 7-hydroxycoumarin. The O-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin increased fluorescence 64% and 28% in pericentral and periportal regions of the liver lobule, respectively. Rates of 7-ethoxycoumarin O-deethylation estimated from these increases in fluorescence were 5.2 mumol X g-1 X h-1 in pericentral and 2.2 mumol X g-1 X h-1 in periportal regions of the liver. During mixed-function oxidation of 7-ethoxycoumarin, the oxidation:reduction state of NADP(H) was similar in both regions of the liver lobule. Xylitol (2 mM) decreased the NADP+/NADPH ratio and stimulated rates of drug metabolism in both regions of the liver lobule. This indicates that conditions exist where the supply of NADPH is an important rate-determining factor for 7-ethoxycoumarin metabolism in both periportal and pericentral regions of the liver lobule.     

Inhibitory effects of 1,4-naphthoquinone derivatives on rat cytochrome P4501A1-dependent monooxygenase activity in recombinant yeast microsomes

We reported previously that various naphthoquinone derivatives inhibited cytochrome P450-dependent monooxygenase of liver and placenta microsomes [Muto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 487-494]. To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P450 species. In the present study, the inhibitory effects of six derivatives of 1,4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P4501A1. Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5,8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition. Judging from the inhibitor constant (K(i)), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P4501A1 is in the order: 2-CH(3)-5-OH-NQ > 2-CH(3)-NQ > NQ > 2-OH-NQ. On binding with P4501A1, 2-CH(3)-5-OH-NQ, 2-CH(3)-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively. These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity.     

N-aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity

The mechanism-based inactivation of hepatic cytochrome P-450 by the suicide inhibitor 1-aminobenzotriazole and two of its derivatives, N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole, was investigated in microsomes from untreated, phenobarbital-induced, and beta-naphthoflavone-induced guinea pigs. Microsomal 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-dealkylase, and benzphetamine N-demethylase activities, and cytochrome P-450 content were determined following incubation with 1-aminobenzotriazole and its analogues. The loss of hepatic cytochrome P-450 content and monooxygenase activity was dependent on inhibitor concentration and required NADPH. N-Benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of monooxygenase activity than the parent compound in microsomes from untreated and phenobarbital-induced guinea pigs. In microsomes from phenobarbital-induced guinea pigs, N-alpha-methylbenzyl-1-aminobenzotriazole (10 microM) was highly selective for the inactivation of the major cytochrome P-450 isozyme catalyzing 7-pentoxyresorufin O-dealkylation (the guinea pig ortholog of P-450IIB1) compared with those isozymes catalyzing 7-ethoxyresorufin O-deethylation or benzphetamine N-demethylation (88 +/- 3% loss of activity vs. 35 +/- 11 and 13 +/- 7%, respectively). N-Benzyl-1-aminobenzotriazole was also selective for the inactivation of 7-pentoxyresorufin O-dealkylase activity, but to a lesser degree (56 +/- 6 vs. 31 +/- 8 and 21 +/- 8%, respectively). In hepatic microsomes from untreated guinea pigs, the two N-substituted analogues were selective for the inhibition of 7-pentoxyresorufin O-dealkylation compared with benzphetamine N-demethylation, but not 7-ethoxyresorufin O-deethylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The specificity and multiplicity of human placental xenobiotic-metabolizing monooxygenase system studied by potential substrates, inhibitors and gel electrophoresis

The specificity of the placental monooxygenase system to metabolize foreign compounds was studied by using different potential substrates and inhibitors and by performing electrophoresis of placental microsomes. Placental preparations from smokers catalyzed benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation and 2,5-diphenyloxazole hydroxylation, but not biphenyl hydroxylation at 2-, 3- or 4-carbon, aldrin epoxidation to dieldrin or coumarin hydroxylation or aminopyrine N-demethylation. Enzyme activities were inhibited by alpha-naphthoflavone, but to a much lesser extent by SKF 525-A or metyrapone. Correlations between the metabolism of benzo(a)pyrene, 7-ethoxycoumarin and 2,5-diphenyloxazole were highly significant. There was a clear difference in Michaelis-Menten constant of 7-ethoxycoumarin O-deethylation between placentas from smokers and nonsmokers. Gel electrophoresis revealed that protein bands of placental microsomes in the region of cytochrome P-450 enzymes were less prominent than those of rat liver microsomes, a finding that accorded with the relative amounts of cytochrome P-450. There were no consistent differences in the electrophoretic pattern between placentas of variable benzo(a)pyrene hydroxylase activities. Results show that the human placental monooxygenase system is restricted in substrate specificity, that there may be a qualitative difference between smokers and nonsmokers and that the increase in several enzyme activities by cigarette smoking cannot be detected by the standard gel electrophoresis.     

Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.     

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.     

Structural characterization of the complex between alpha-naphthoflavone and human cytochrome P450 1B1

The atomic structure of human P450 1B1 was determined by x-ray crystallography to 2.7 Å resolution with α-naphthoflavone (ANF) bound in the active site cavity. Although the amino acid sequences of human P450s 1B1 and 1A2 have diverged significantly, both enzymes exhibit narrow active site cavities, which underlie similarities in their substrate profiles. Helix I residues adopt a relatively flat conformation in both enzymes, and a characteristic distortion of helix F places Phe²³¹ in 1B1 and Phe²²⁶ in 1A2 in similar positions for π-π stacking with ANF. ANF binds in a distinctly different orientation in P450 1B1 from that observed for 1A2. This reflects, in part, divergent conformations of the helix B′-C loop that are stabilized by different hydrogen-bonding interactions in the two enzymes. Additionally, differences between the two enzymes for other amino acids that line the edges of the cavity contribute to distinct orientations of ANF in the two active sites. Thus, the narrow cavity is conserved in both P450 subfamily 1A and P450 subfamily 1B with sequence divergence around the edges of the cavity that modify substrate and inhibitor binding. The conservation of these P450 1B1 active site amino acid residues across vertebrate species suggests that these structural features are conserved.     

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (V_max) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.