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Proceedings of the National Academy of... Dec 1993Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the...
Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids; such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5 for the 6 beta-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.
Topics: Base Sequence; Cloning, Molecular; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Cytochromes b5; DNA Primers; Erythromycin; Escherichia coli; Humans; Hydroxylation; Kinetics; Liver; Mixed Function Oxygenases; Molecular Sequence Data; Mutagenesis; NADPH-Ferrihemoprotein Reductase; Nifedipine; Plasmids; Polymerase Chain Reaction; Recombinant Fusion Proteins; Substrate Specificity; Testosterone
PubMed: 8265621
DOI: 10.1073/pnas.90.24.11748 -
Journal of Biochemistry Nov 1986Three forms of cytochrome P-450, tentatively designated P-450(M-1), P-450(M-2), and P-450(M-3), and one form of cytochrome P-450, P-450(F-1), were purified from the... (Comparative Study)
Comparative Study
Three forms of cytochrome P-450, tentatively designated P-450(M-1), P-450(M-2), and P-450(M-3), and one form of cytochrome P-450, P-450(F-1), were purified from the liver microsomes of untreated male and female rats, respectively. Each purified form of the cytochrome showed a single protein band on SDS-polyacrylamide gel electrophoresis, and gave a minimum molecular weight of 51,000 for P-450(M-1), 48,000 for P-450(M-2), 49,000 for P-450(M-3), and 50,000 for P-450(F-1). The carbon monoxide-difference spectra of reduced P-450(M-1), P-450(M-2), P-450(M-3), and P-450(F-1) showed an absorption maximum at 451, 451, 448, and 449 nm, respectively. Judging from the absolute absorption spectra, the four forms of cytochrome P-450 were of low-spin type in the oxidized forms. The antibodies against P-450(M-2) did not crossreact with the other forms in the Ouchterlony double diffusion test, whereas the immunodiffusion test showed immunocrossreactivity between P-450(M-1) and P-450(F-1), P-450(M-1) and P-450(M-3), and P-450(M-3) and P-450(F-1). The NH2-terminal amino acid sequences of the four forms confirmed that they were different molecular species, although significant homology was noticed among P-450(M-1), P-450(M-3), and P-450(F-1). The quantitation of P-450(M-1) and P-450(F-1) in liver microsomes by quantitative immunoprecipitation confirmed that these two forms of cytochrome P-450 were developmentally induced in male and female rats, respectively. P-450(M-2) was also developmentally induced in male rats. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, P-450(M-1) oxidized benzphetamine at a high rate, whereas the other forms had low activity toward benzphetamine. None of the four forms showed high activity toward benzo(a)pyrene. P-450(M-1) catalyzed the hydroxylation testosterone at the 16 alpha and 2 alpha positions, whereas P-450(M-2) catalyzed the 15 alpha hydroxylation of the same substrate.
Topics: Aging; Amino Acid Sequence; Animals; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Epitopes; Female; Immunodiffusion; Male; Microsomes, Liver; Molecular Weight; Peptide Fragments; Rats; Rats, Inbred Strains; Sex Characteristics; Spectrophotometry; Substrate Specificity
PubMed: 2434473
DOI: 10.1093/oxfordjournals.jbchem.a121842 -
The Journal of Biological Chemistry Jun 1985A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular...
A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 49,000. Absolute spectrum of the oxidized form indicates that this isozyme is essentially all in the low spin state. The maximum of the reduced CO complex is at 449 nm. Amino-terminal partial amino acid sequence and amino acid composition are different from those of RLM3 and RLM5, two other native forms of cytochrome P-450 previously reported from this laboratory as well as other forms reported in the literature. RLM2 is capable of oxidizing a variety of drug substrates, like benzphetamine and aminopyrine, and to a lesser extent ethoxycoumarin. With the steroid substrate multiple isomeric products are formed differentially. Progesterone is preferentially hydroxylated at the 15-position (15 beta-hydroxylation (34%) and 15 alpha-hydroxylation (13%) of the total) and at the 6 beta-position (21%). The major metabolite when testosterone was the substrate, 15 alpha-hydroxytestosterone, comprised 43% of the total, while a modest amount of 6 beta-hydroxytestosterone (12%) is formed. Another major metabolite (31%) has yet to be unequivocally identified, but is suggested to be 7 beta-hydroxytestosterone. Examination of the substrate dependence of major and minor isomeric metabolites provides evidence for a single substrate-binding site on RLM2. Regardless of the position hydroxylated, a common Km value was obtained. It is suggested that differences in formation of the isomeric and epimeric products relate to differences in distance from the active oxygen center and the position of attack.
Topics: Amino Acid Sequence; Amino Acids; Animals; Chromatography, Ion Exchange; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Gas Chromatography-Mass Spectrometry; Isoenzymes; Male; Microsomes, Liver; Progesterone; Rats; Testosterone
PubMed: 3997859
DOI: No ID Found -
Journal of Biochemistry Jun 1985A total of nine forms of cytochrome P-450 were purified to homogeneity from liver microsomes of male Wistar rats. They were P-451 I and P-451 II from untreated rats,...
A total of nine forms of cytochrome P-450 were purified to homogeneity from liver microsomes of male Wistar rats. They were P-451 I and P-451 II from untreated rats, P-450 II and P-450 III from phenobarbital-treated rats, MC-P-448 L and MC-P-448 H from 3-methylcholanthrene-treated rats, and P-452, P-448 L, and P-448 H from 3,4,5,3',4'-pentachlorobiphenyl-treated rats. Among them, MC-P-448 L and MC-P-448 H were indistinguishable from P-448 L and P-448 H, respectively, with regard to electrophoretic, spectral, catalytic and immunochemical properties, and thus seven forms were distinct hemoproteins. The minimal molecular weight of each form was as follows: P-451 I (49,000), P-451 II (52,000), P-450 II (52,000), P-450 III (53,500), P-452 (48,000), P-448 L (56,000), P-448 H (54,000). Judging from the oxidized absolute spectra, P-448 H was a high-spin form and the others were of low-spin type. In a reconstituted system, N-demethylations of benzphetamine and aminopyrine were catalyzed by most of the forms at comparable rates. On the other hand, the activities for the oxidations of benzo[a]pyrene, 7-ethoxycoumarin, biphenyl, and estradiol-17 beta varied greatly among the forms of cytochrome P-450. The most efficient catalysts were as follows: P-448 L and P-451 II for benzo[a]pyrene 3-hydroxylation; P-448 L for 7-ethoxycoumarin O-deethylation; P-448 L, P-451 II, and P-448 H for biphenyl 4-hydroxylation; P-448 L and P-448 H for biphenyl 2-hydroxylation; and P-451 II and P-448 H for estradiol 2-hydroxylation. P-451 I, P-450 II, and P-450 III were somewhat poorer catalysts in metabolizing all the substrates except for benzphetamine and aminopyrine, but their substrate specificities were still distinguishable from one another. Of all the purified cytochrome P-450's, P-452 showed the least ability to metabolize all the substrates. Judging from the properties, it appears that six forms in male Wistar rats correspond to the distinct forms of cytochrome P-450 in Long-Evans and/or Sprague-Dawley rats reported by other workers, but P-451 I is a new constitutive isozyme in Wistar rats.
Topics: Animals; Catalysis; Chromatography; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Immunodiffusion; Isoenzymes; Male; Microsomes, Liver; Molecular Weight; Rats; Rats, Inbred Strains
PubMed: 3928617
DOI: 10.1093/oxfordjournals.jbchem.a135234 -
The Journal of Biological Chemistry Mar 1982Two constitutive forms of cytochrome P-450 isozymes, designated RLM3 and RLM5, have been purified from untreated rat liver microsomes. RLM3 and RLM5 have minimum...
Two constitutive forms of cytochrome P-450 isozymes, designated RLM3 and RLM5, have been purified from untreated rat liver microsomes. RLM3 and RLM5 have minimum molecular weights of 50,000 and 51,000, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Absolute oxidized spectra indicate that RLM3 is essentially all in the low spin state, whereas RLM5 contains some high spin component. The CO-reduced difference spectral maximum of RLM3 is at 449 nm and RLM5 is at 451 nm. RLM3 preferentially hydroxylates testosterone at 6 beta and 7 alpha positions and forms an even higher amount of an as yet unidentified polar metabolite, whereas RLM5 hydroxylates testosterone preferentially at 2 beta and 16 alpha positions, and exhibits 6 beta-hydroxylase activity similar to RLM3. RLM5 metabolizes aminopyrine, ethylmorphine, and benzphetamine all faster than does RLM3. When RLM3 and RLM5 were digested with proteolytic enzymes, peptide maps showed that these two cytochrome P-450 isozymes have different primary structures. Cyanogen bromide digestion breaks both isozymes into fragments too small to compare on polyacrylamide gels. Amino acid analyses indicate that RLM3 and RLM5 have very similar but not identical compositions. NH2-terminal residues were determined by Edman degradation and high pressure liquid chromatography separation of phenylthiohydantoin-derivatives. The first four residues of both enzymes are identical, NH2-Met-Asp-Pro-Val, but the fifth residue of RLM5 is -Leu-while that of RLM3 is -Val-. These sequences differ dramatically from all other P-450 isozymes reported to date.
Topics: Amino Acids; Animals; Carbon Monoxide; Cytochrome P-450 Enzyme System; Isoenzymes; Male; Microsomes, Liver; Molecular Weight; Peptide Fragments; Protein Binding; Rats; Spectrophotometry
PubMed: 7061428
DOI: No ID Found -
Molecular Pharmacology Oct 2008Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of...
Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-alpha-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.
Topics: Alkynes; Aryl Hydrocarbon Hydroxylases; Benzoxazines; Benzphetamine; Binding Sites; Catalysis; Cyclopropanes; Cytochrome P-450 CYP2B1; Cytochrome P-450 CYP2B6; Cytochrome P-450 Enzyme System; Cytochromes b5; Escherichia coli; Ethinyl Estradiol; Humans; Kinetics; Molecular Structure; Mutation; NADPH-Ferrihemoprotein Reductase; Oxidants; Oxidoreductases, N-Demethylating; Protein Binding; Spectrophotometry, Ultraviolet; tert-Butylhydroperoxide
PubMed: 18621926
DOI: 10.1124/mol.108.048637 -
Journal of Biochemistry Nov 1979Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's...
Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's procedures )1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16 nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities. In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous conbinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common. Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.
Topics: Amino Acids; Animals; Antibodies; Cytochrome P-450 Enzyme System; Immunoassay; Immunodiffusion; Kinetics; Male; Methylcholanthrene; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Phenobarbital; Rats; Spectrophotometry; Substrate Specificity
PubMed: 118169
DOI: 10.1093/oxfordjournals.jbchem.a132655 -
The Journal of Biological Chemistry Nov 1995Site-directed mutagenesis of the acidic clusters 207Asp-Asp-Asp209 and 213Glu-Glu-Asp215 of NADPH-cytochrome P450 oxidoreductase demonstrates that both cytochrome c and... (Comparative Study)
Comparative Study
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+.
Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Candida; Cytochrome P-450 Enzyme System; Cytochrome c Group; Flavodoxin; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; NADPH-Ferrihemoprotein Reductase; Oligodeoxyribonucleotides; Osmolar Concentration; Rats; Recombinant Proteins; Saccharomyces cerevisiae
PubMed: 7499204
DOI: 10.1074/jbc.270.46.27475 -
The Journal of Biological Chemistry Sep 1975The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were...
The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.
Topics: Anaerobiosis; Animals; Circular Dichroism; Cytochrome P-450 Enzyme System; Dithionite; Electron Transport; Microsomes, Liver; Oxidation-Reduction; Potentiometry; Protein Binding; Protein Conformation; Rabbits; Species Specificity; Spectrophotometry; Spectrophotometry, Ultraviolet
PubMed: 1165247
DOI: No ID Found -
European Journal of Biochemistry Dec 1992The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier...
The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphetamine N-demethylation by liver microsomes of the phenobarbital-treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N-demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH-cytochrome-P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH-cytochrome-P450 reductase and its charged redox partners [Voznesensky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669-14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic strength nor polyol addition affected the rate of electron transfer from NADPH-cytochrome-P450 reductase to an uncharged acceptor 1,4-benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH-cytochrome-P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450 LM2 for NADPH-cytochrome-P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax.
Topics: Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Electric Conductivity; Ethylene Glycol; Ethylene Glycols; Glycerol; Kinetics; Male; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Oxidoreductases, N-Demethylating; Phenobarbital; Rabbits
PubMed: 1483457
DOI: 10.1111/j.1432-1033.1992.tb17475.x