Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli.

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.

[1]  R. Estabrook,et al.  Interaction of apo-cytochrome b5 with cytochromes P4503A4 and P45017A: relevance of heme transfer reactions. , 2001, Biochemistry.

[2]  R. T. Miller,et al.  The kinetic and spectral characterization of the E. coli-expressed mammalian CYP4A7: cytochrome b5 effects vary with substrate. , 2001, Archives of biochemistry and biophysics.

[3]  H. Yamazaki,et al.  Formation of a dihydroxy metabolite of phenytoin in human liver microsomes/cytosol: roles of cytochromes P450 2C9, 2C19, and 3A4. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[4]  T. Shimada,et al.  Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli. , 2000, Protein expression and purification.

[5]  E. Gillam,et al.  Phenytoin metabolism by human cytochrome P450: involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[6]  F. Arnold,et al.  Formation of indigo by recombinant mammalian cytochrome P450. , 1999, Biochemical and biophysical research communications.

[7]  H. Yamazaki,et al.  Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[8]  H. Yamazaki,et al.  Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome P-450 enzymes. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[9]  H. Yamazaki,et al.  Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[10]  Rodrigues Ad,et al.  Integrated Cytochrome P450 Reaction Phenotyping: Attempting to Bridge the Gap Between cDNA-expressed Cytochromes P450 and Native Human Liver Microsomes , 1999 .

[11]  Kiyoshi Inoue,et al.  Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes , 1999, Archives of Toxicology.

[12]  T. Shimada,et al.  Recombinant human cytochrome P450 1B1 expression in Escherichia coli. , 1998, Archives of biochemistry and biophysics.

[13]  W. Miller,et al.  Cytochrome b 5 Augments the 17,20-Lyase Activity of Human P450c17 without Direct Electron Transfer* , 1998, The Journal of Biological Chemistry.

[14]  F. Guengerich,et al.  Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5. , 1997, Archives of biochemistry and biophysics.

[15]  F. Guengerich,et al.  Drug metabolism by Escherichia coli expressing human cytochromes P450 , 1997, Nature Biotechnology.

[16]  H. Yamazaki,et al.  Reconstitution of recombinant cytochrome P450 2C10(2C9) and comparison with cytochrome P450 3A4 and other forms: effects of cytochrome P450-P450 and cytochrome P450-b5 interactions. , 1997, Archives of biochemistry and biophysics.

[17]  H. Yamazaki,et al.  Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation activities in livers of Japanese and Caucasian populations. , 1997, Pharmacogenetics.

[18]  T Ishizaki,et al.  Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. , 1997, Pharmacology & therapeutics.

[19]  H. Yamazaki,et al.  Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4 , 1996, The Journal of Biological Chemistry.

[20]  T Sakaki,et al.  Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. , 1996, Biochemical pharmacology.

[21]  H. Yamazaki,et al.  Recombinant human cytochrome P450 1A2 and an N-terminal-truncated form: construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPH-cytochrome P450 reductase. , 1996, Archives of biochemistry and biophysics.

[22]  D W Nebert,et al.  P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. , 1996, Pharmacogenetics.

[23]  F. Guengerich,et al.  Purification of functional recombinant P450s from bacteria. , 1996, Methods in enzymology.

[24]  T. Kost,et al.  CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. , 1995, Archives of biochemistry and biophysics.

[25]  C. W. Fisher,et al.  The effects of cytochrome b5, NADPH-P450 reductase, and lipid on the rate of 6 beta-hydroxylation of testosterone as catalyzed by a human P450 3A4 fusion protein. , 1995, Archives of biochemistry and biophysics.

[26]  C. Crespi.  Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research , 1995 .

[27]  K. Korzekwa,et al.  cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus. , 1994, Drug metabolism and disposition: the biological fate of chemicals.

[28]  H. Yamazaki,et al.  Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. , 1994, The Journal of pharmacology and experimental therapeutics.

[29]  P. Beaune,et al.  Recombinant yeast in drug metabolism. , 1993, Toxicology.

[30]  F. Guengerich,et al.  Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. , 1993, Archives of biochemistry and biophysics.

[31]  F. Guengerich,et al.  Human cytochrome P-450 enzymes. , 1992, Life sciences.

[32]  T. Shimada,et al.  Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. , 1991, Chemical research in toxicology.

[33]  F. Gonzalez,et al.  DNA-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment. , 1991, Mutation research.

[34]  F. Guengerich,et al.  Cytochrome P-450 isozyme/isozyme functional interactions and NADPH-cytochrome P-450 reductase concentrations as factors in microsomal metabolism of warfarin. , 1985, European journal of biochemistry.

[35]  F. Guengerich,et al.  Estimation of isozymes of microsomal cytochrome P-450 in rats, rabbits, and humans using immunochemical staining coupled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , 1982, Biochemistry.

[36]  A. Hayes.  Principles and methods of toxicology , 1982 .

[37]  F. Guengerich,et al.  Immunological comparison of rat, rabbit, and human liver NADPH-cytochrome P-450 reductases. , 1981, Biochemistry.

[38]  P. Strittmatter,et al.  [8] Purification of cytochrome b5 , 1978 .

[39]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. , 1964, The Journal of biological chemistry.

[40]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.