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Research ArticleArticle

Enhancement of Cytochrome P-450 3A4 Catalytic Activities by Cytochrome b 5 in Bacterial Membranes

Hiroshi Yamazaki, Miki Nakajima, Mami Nakamura, Satoru Asahi, Noriaki Shimada, Elizabeth M. J. Gillam, F. Peter Guengerich, Tsutomu Shimada and Tsuyoshi Yokoi
Drug Metabolism and Disposition September 1999, 27 (9) 999-1004;

Abstract

Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia colitransformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6β-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b5(b5) and apolipoproteinb5 further enhanced the testosterone 6β-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b5). Apparent maximal activities of testosterone 6β-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b5 was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6β-hydroxylation, nifedipine oxidation, and midazolam 4- and 1′-hydroxylation activities in CYP3A4/NPR membranes plusb5 systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containingb5 are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.

Multiple forms of cytochrome P-450 (P-450)1 exist in mammals, and these P-450 enzymes play important roles in the oxidation of structurally diverse xenobiotic chemicals and endobiotics (Guengerich and Shimada, 1991; Guengerich, 1995; Nelson et al., 1996). P-450s are not self-sufficient enzymes, and the microsomal enzymes require a NADPH-P-450 reductase (NPR) as an electron carrier to function as monooxygenases. In human livers, levels of each of the P-450 forms are different and roles in various substrate oxidations vary. CYP3A4 is the major P-450 enzyme involved in the oxidation of a large number of compounds (Wrighton and Stevens, 1992; Gonzalez and Gelboin, 1994; Shimada et al., 1994).

Recently, recombinant P-450 enzymes from different sources—e.g., microsomes of human lymphoblastoid cells (Gonzalez et al., 1991;Crespi, 1995), yeast (Renaud et al., 1993; Imaoka et al., 1996), and insect cells infected with baculovirus systems (Buters et al., 1994;Lee et al., 1995) and reconstitution systems containing purified P-450 enzymes from Escherichia coli membranes (Gillam et al., 1993; Shaw et al., 1997) have been used widely for drug metabolism research. We have shown that a 1:2:1 molar ratio of CYP3A4/NPR/cytochrome b5(b5) is optimal in the reconstitution of drug oxidation (Yamazaki et al., 1996b; Shimada and Yamazaki, 1998) using purified CYP3A4. However, the marker activities or kinetic parameters of CYP3A4 reported from different research laboratories and commercial enzymes manufacturers are not always similar. In this study, we have determined optimal catalytic activities for testosterone 6β-hydroxylation, nifedipine oxidation, and midazolam 4- and 1′-hydroxylation by CYP3A4/NPR membranes (obtained using a bacterial bicistronic CYP3A4 expression system) fortified withb5 or NPR and compared the activities of several types of recombinant CYP3A4 preparations before and after addition of b5 and/or NPR. Enhancement of CYP3A4-dependent activities in membranes by addition ofb5 from different sources, apolipoproteinb5 (apob5), and kinetic parameters for testosterone 6β-hydroxylation are also reported.

Materials and Methods

Chemicals.

Midazolam and its metabolites were kindly donated by Yamanouchi Pharmaceuticals Co., Ltd. (Tokyo, Japan). Testosterone, nifedipine, and their metabolites and reagents used in this study were obtained from sources described previously or were of the highest qualities commercially available (Yamazaki et al., 1996b; Shimada and Yamazaki, 1998).

Enzyme Preparations.

Membranes were prepared from E. coli into which CYP3A4 and NPR cDNAs had been introduced as described previously (Parikh et al., 1997). Briefly, single transformed colonies of E. colistrains DH5α and JM109 were used to inoculate starter Luria-Bertani medium (LB)/ampicillin (25 μg/ml) cultures. The starter cultures were incubated for 7 to 15 h at 37°C, with shaking at 170 rpm, and then diluted 1:100 into Terrific Broth (TB)/ampicillin (100 μg/ml) medium containing additives (0.5 mM δ-aminolevulinic acid, 1.0 mM isopropyl β-d-thiogalactoside, trace salts, and 1.0 mM thiamine; Guengerich et al., 1996). The expression cultures (100 ml) were grown at 30°C with shaking at 120 to 180 rpm for 24 to 32 h in 500-ml triple-baffled flasks. Membrane fractions were prepared from the bacterial pellets by a series of fractionation and high-speed centrifugation steps (Guengerich et al., 1996) and suspended in one volume of 10 mM Tris-HCl buffer (pH 7.4) containing 0.10 mM EDTA and 20% glycerol (v/v). Recombinant monocistronic CYP3A4, CYP1A2, and NPR were purified from membranes as described elsewhere (Gillam et al., 1993; Dong et al., 1996; Parikh et al., 1997). Rabbit NPR (Guengerich et al., 1981) and rabbit (Strittmatter et al., 1978), rat, and humanb5 (Shimada et al., 1986) were purified from liver microsomes by the methods described. Recombinant human NPR and b5 were purified using similar methods. Apo b5 was prepared from rabbitb5 as described previously (Yamazaki et al., 1996a). Horse heart cytochrome c was purchased from Sigma. Human liver microsomes were prepared in 10 mM Tris-HCl buffer (pH 7.4) containing 0.10 mM EDTA and 20% glycerol (v/v) as described previously (Guengerich, 1994; Inoue et al., 1997).

Recombinant CYP3A4 expressed in microsomes of insect cells infected with baculovirus containing human P-450 and rabbit or human NPR cDNA inserts were obtained from PanVera (Madison, WI) or Gentest (Woburn, MA). Recombinant CYP3A4 in microsomes from lymphoblastoid cells coexpressing NPR was obtained from Gentest. The P-450 contents were used as described in the data sheets provided by the manufacturers.

Enzyme Assays.

Testosterone hydroxylation, nifedipine oxidation, and midazolam hydroxylation activities were determined as described (Kronbach et al., 1989; Brian et al., 1990) with slight modifications (Shimada and Yamazaki, 1998). The standard incubation mixture (final volume of 0.25 ml) contained 0.010 μM recombinant CYP3A4, 0.020 μM NPR, 0.010 μMb5, and 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system consisting of 0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 U of glucose 6-phosphate dehydrogenase/ml, and 200 μM testosterone (or nifedipine) or 100 μM midazolam. In some cases, human liver microsomes were used at the same CYP3A4 concentrations as the recombinant system. Incubations were carried out at 37°C for 10 min and terminated by adding 1.5 ml of CH2Cl2 and 0.3 M NaCl. Reactions with nifedipine were incubated at 37°C for 5 min and terminated by adding 1.5 ml of CH2Cl2, 0.2 M NaCl, and 0.1 M Na2CO3. Organic phases were evaporated under a nitrogen stream, and product formation was determined by HPLC with a C18 (5-μm) analytical column (4.6 × 150 mm). Reactions with midazolam were incubated at 37°C for 5 min and terminated by adding 0.25 ml of CH3OH. The elution was conducted with a mixture of 64% CH3OH/36% H2O (v/v) at a flow rate of 1.2 ml/min, and the detection was by UV absorbance at 240 nm (testosterone) and 254 nm (nifedipine). The elution of midazolam metabolites was conducted with a mixture of 27% CH3OH/18% CH3CN/55% 10 mM potassium phosphate buffer (pH 7.4) (v/v) at a flow rate of 1.5 ml/min, and detection was by UV absorbance at 220 nm.

Other Assays.

Concentrations of P-450 and b5 and protein were estimated spectrally by the described methods (Lowry et al., 1951;Omura and Sato, 1964). NADPH-cytochrome c reduction activities were determined as described (Williams and Kamin, 1962;Yasukochi and Masters, 1976) using Δε550 = 21.1 mM−1 cm−1 and an assumed specific activity of 3.0 μmol reduced cytochromec/min/nmol NPR based on purified human and rabbit NPR preparations (Parikh et al., 1997). The contents of CYP3A4 in human liver microsomes were estimated by coupled SDS-polyacrylamide gel electrophoresis/immunochemical development (Western blotting) (Guengerich et al., 1982).

Kinetic analyses for substrate oxidations by P-450 enzymes were estimated from the fitted curves using a computer program (KaleidaGraph program; Synergy Software, Reading, PA) designed for nonlinear regression analysis.

Results

Recovery of CYP3A4 and NPR in Membranes of E. coli.

CYP3A4 and NPR were coexpressed in E. coli from a bicistronic vector using six different culture conditions (Table1). With regard to CYP3A4 expression levels, long incubation for both LB and TB cultures with vigorous shaking resulted in good yields (Table 1, lot F). On the other hand, the final yield of NPR was highest when the culture time in LB was 7 h, followed by 32 h with mild shaking, in TB medium (Table1, lot B). Catalytic activities of CYP3A4/NPR membranes for testosterone 6β-hydroxylation were higher in samples B and A.

View this table:
Table 1

Catalytic activities for testosterone 6β-hydroxylation by several preparations of CYP3A4/NPR membranes obtained from E. coli transformed with a bicistronic CYP3A4/NPR expression vector1-a

Effects of Exogenous b5 and NPR on Catalytic Rates of CYP3A4/NPR Membranes.

Because a 1.1:1 molar ratio of NPR/CYP3A4 in membrane preparations appeared to give highest catalytic activities for CYP3A4-dependent testosterone 6β-hydroxylation among the conditions tested (Table 1), the effects of b5 on activity were investigated mainly using this preparation. The effect ofb5 was shown to be concentration-dependent (Fig. 1A). Both humanb5 in E. coli membranes and purified recombinant human b5 enhanced the testosterone hydroxylation activities about 2-fold; a 1 to 2:1 molar ratio of b5/CYP3A4 produced the highest activities.

Figure 1
Figure 1

Concentration dependence of the effects of human b5 and NPR in membranes on testosterone 6β-hydroxylation by CYP3A4/NPR membranes.

A, testosterone (200 μM) was incubated with CYP3A4/NPR membranes (Table 1, preparation B, 0.010 μM CYP3A4) fortified with membranes expressing human NPR (final, 0.020 μM) in the presence of recombinant human b5 added in E. colimembranes (▪) or as purified b5 (▴). B, testosterone (200 μM) was incubated with CYP3A4/NPR membranes containing CYP3A4 (0.010 μM) and recombinant human NPR (0.011 μM) that were fortified further with different amounts of NPR, added in bacterial membranes to the final ratios shown, in the absence (○) or presence of added, purified human b5 (▴) or b5 in membranes (▪) (0.010 μM). At the “zero” molar ratio of NPR to CYP3A4, E. colimembranes expressing only CYP3A4 were used.

The effects of adding recombinant human NPR (in membranes) were studied. Rates of testosterone 6β-hydroxylation by recombinant CYP3A4 (lot B) were increased 2-fold by the supplementation with recombinant human NPR to an 8-fold excess of NPR over CYP3A4 in membranes in the absence of b5 (Fig. 1B). In the presence ofb5, apparent optimal activities were observed after the addition of a 2:1 final molar ratio of NPR to CYP3A4 (Fig. 1B).

Stimulating effects of b5 also were observed upon addition of either rabbit, rat, or humanb5 purified from liver microsomes as well as recombinant human b5 (Table2). Apo b5and recombinant CYP1A2 also enhanced the catalytic activities of CYP3A4/NPR as well as b5, but cytochromec did not.

View this table:
Table 2

Effects of source of b5 and other proteins on testosterone 6β-hydroxylation activities by CYP3A4/NPR membranes

Recombinant human NPR, purified from E. coli membranes, catalyzed NADPH-dependent cytochrome c reduction and supported CYP3A4-dependent testosterone 6β-hydroxylation (Table3); however, purified human NPR-supported CYP3A4 activity for testosterone hydroxylation was lower in a reconstituted system containing a 1:2:1 molar ratio of CYP3A4/NPR/b5 than was that supported by native rabbit NPR. The effects of supplementation with purified native rabbit NPR and cholate on CYP3A4 expressed in membranes were investigated (Fig. 2). Cholate enhanced dose-despondently the exogenous rabbit NPR-supported activities of CYP3A4/NPR membranes in the presence of b5(Fig. 2A). When purified native rabbit NPR was used, testosterone 6β-hydroxylation was improved in the presence of bothb5 and cholate (Fig. 2B). The apparent maximal activities were similar after addition of purified rabbit NPR (∼80 min−1) (Fig. 2B) and recombinant human NPR (in membranes) (Fig. 1B).

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Table 3

Cytochrome c reduction and CYP3A4-dependent testosterone 6β-hydroxylation by purified recombinant human and native rabbit NPR

Figure 2
Figure 2

Concentration dependence of the effects of cholate and purified NPR on testosterone 6β-hydroxylation by CYP3A4/NPR membranes.

A, cholate (0–1.0 mM) was added to CYP3A4/NPR membranes containing CYP3A4 (0.010 μM) and human NPR (0.011 μM), which were fortified further with exogenous rabbit NPR (0.010 μM) with or without purified human b5 (0.010 μM). These components were mixed and allowed to stand 10 min at room temperature, followed by addition of other reaction components. Testosterone 6β-hydroxylation activities (200 μM testosterone) were determined. B, exogenous rabbit NPR (0–0.080 μM) was added to CYP3A4/NPR membranes containing CYP3A4 (0.010 μM) and human NPR (0.011 μM), which were fortified with purified human b5 (0.010 μM) and/or cholate (0.25 mM). Testosterone 6β-hydroxylation activities were determined as described in A.

Comparison of Bacterial CYP3A4/NPR Membranes with Other Recombinant Proteins and Human Liver Microsomes.

To compare testosterone 6β-hydroxylation activities among recombinant CYP3A4 systems, we determined the rates of other recombinant CYP3A4 systems and human liver microsomes after addition of purifiedb5 and/or NPR with cholate (Table4). Catalytic activities of microsomes of lymphoblastoid cells were increased 3-fold by the addition of a 2-fold excess of b5 over CYP3A4. However, the rates of this system fortified with NPR were lower than those obtained with CYP3A4 expressed using the bacterial CYP3A4/NPR membranes. The activities of one of the microsomal systems from insect cells with baculovirus systems (Baculosomes; PanVera) containing a 1:4.6 molar ratio of CYP3A4 to NPR were improved by the addition of a 2-fold excess of b5 to CYP3A4. The activities of another baculovirus system containing CYP3A4/NPR/b5(1:12:16) (Supersomes; Gentest) were not affected by further addition of b5 and/or NPR. Human liver microsomes containing 71% CYP3A4 (of total P-450) also were used. Based on CYP3A4 contents, the testosterone 6β-hydroxylation activity of this sample was 85 nmol/min/nmol CYP3A4. The molar ratio of CYP3A4/NPR/b5 in this human liver microsomal preparation was 1:0.06:0.52, and testosterone 6β-hydroxylation was only minimally affected by the addition of recombinant b5 or NPR.

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Table 4

Effects of exogenous NPR and b5 on testosterone 6β-hydroxylation activities in CYP3A4/NPR membranes, microsomes containing recombinant CYP3A4/NPR, and human liver microsomes

Nifedipine and midazolam oxidation activities of recombinant CYP3A4 systems and human liver microsomes also were determined (Table5). In addition to a reconstituted system, the same enzyme sources as in Table 4 were used after addition of purified b5 and NPR, with cholate added. Activities of nifedipine oxidation and midazolam 4- and 1′-hydroxylation of CYP3A4/NPR membranes plusb5 and in the reconstituted system were similar to those of human liver microsomes (∼30, ∼10, and ∼20 min−1, respectively), based on CYP3A4 contents. Catalytic activities of microsomes of lymphoblastoid cells were lower, and nifedipine oxidation activities of a baculovirus system that coexpressed b5 and NPR were higher than those of human liver microsomes.

View this table:
Table 5

Nifedipine and midazolam oxidation activities in a reconstitution system, CYP3A4/NPR membranes, microsomes containing recombinant CYP3A4/NPR, and human liver microsomes5-a

Kinetic Analysis of Activity Catalyzed by CYP3A4/NPR Membranes Plusb5 and Human Liver Microsomes.

Because recombinant CYP3A4 (expressed in bacterial membranes using the bacterial bicistronic system) premixed at a molar ratio of 1:2:1 of CYP3A4 to human NPR to human b5 appeared to be a suitable model for human liver microsomal CYP3A4 with regard to catalytic activities, kinetic parameters were compared with those of human liver microsomes. Testosterone 6β-hydroxylation was dependent on CYP3A4 concentration (Fig. 3); linearity of product formation was obtained in a narrow range (0–0.010 μM). Kinetic analysis for testosterone 6β-hydroxylation over a substrate concentration range of 10 to 500 μM yieldedKm and Vmaxvalues of 80 ± 23 μM and 105 ± 12 nmol/min/nmol CYP3A4 for the recombinant bacterial CYP3A4 system plusb5 (1:2:1 molar ratio of CYP3A4/NPR/b5) and 49 ± 14 μM and 105 ± 10 nmol/min/nmol P-450 for human liver microsomes, respectively.

Figure 3
Figure 3

Effects of concentration of CYP3A4/NPR membranes premixed with b5 on testosterone 6β-hydroxylation.

Testosterone (200 μM) was incubated with different concentrations of CYP3A4/NPR membranes (0.005–0.160 μM CYP) premixed with human NPR and b5 (1:2:1 molar ratio of CYP3A4/NPR/b5).

Discussion

A number of studies have shown that CYP3A4 is a major P-450 enzyme involved in the oxidation of many clinically used drugs in human liver microsomes (Guengerich, 1995; Wilkinson, 1996; Li et al., 1997). Prediction of microsomal oxidation of drugs in human livers has been studied recently using activities or kinetic parameters obtained from recombinant systems (Iwatsubo et al., 1997; Ito et al., 1998). A “relative activity factor” was proposed by Crespi (1995), and calculations have been reported with other factors (Kobayashi et al., 1997; Nakajima et al., 1998; Venkatakrishnan et al., 1998). Omeprazole oxidation by human liver microsomes was predicted by using a combination of liver microsomal contents of CYP2C19 and CYP3A4 (immunochemically determined) and kinetic parameters obtained from experiments using recombinant CYP2C19 and CYP3A4 (Yamazaki et al., 1997b), using the methods outlined by Iwatsubo et al. (1997). In these studies, catalytic activities and kinetic parameters of marker drug oxidations catalyzed by recombinant P-450 enzymes are very important for prediction; however, different activities have been reported using several expression systems for CYP3A4 (Shaw et al., 1997; Yamazaki et al., 1997b). Drug oxidation activities of purified CYP3A4 have been studied extensively, and it has been shown that some CYP3A4 activities are dependent on b5, specific lipid mixtures, cholate, and buffer and salt compositions (Yamazaki et al., 1996a; Shimada and Yamazaki, 1998). We have shown (using the purified recombinant CYP3A4 obtained from a monocistronic bacterial expression system) that a 1:2:1 molar ratio of CYP3A4/NPR/b5 was suitable for reconstitution of drug oxidation (Yamazaki et al., 1996b).

The present results indicate that a 1:2:1 molar ratio of CYP3A4/NPR/b5 gives apparently optimal activities for testosterone 6β-hydroxylation, nifedipine oxidation, and midazolam 4- and 1′-hydroxylations by recombinant CYP3A4 expressed in bacterial membranes using a bicistronic system. The molar ratio of the three proteins in membranes was the same as in a reconstituted system containing purified CYP3A4. Enhancing effects of exogenousb5 were observed with eitherb5 purified from human, rabbit, or rat liver microsomes, with recombinant human b5added in bacterial membranes or with apob5, devoid of heme. Effects ofb5 on CYP3A4 activities also were observed in microsomes from lymphoblastoid cells and insect cells (lacking endogenous b5). Cholate (0.25 mM) was needed to enhance rates when purified NPR was added to CYP3A4 in bacterial membranes; however, cholate appeared not to be necessary when NPR was supplemented from bacterial membranes. Testosterone 6β-hydroxylation (∼80 min−1), nifedipine oxidation (∼30 min−1), and midazolam 4- and 1′-hydroxylation (∼10 and 20 min−1) activities were similar among human liver microsomes and/or baculovirus systems based on CYP3A4 contents. These results indicate that the bacterial CYP3A4/NPR membranes plus b5 should be a simple and suitable model of drug oxidation study for microsomal CYP3A4-dependent reactions in human livers.

NPR is essential to P-450-dependent drug oxidation, and many catalytic activities of CYP3A4 require b5 for optimal rates. We have shown that b5 can stimulate some CYP3A4-catalyzed oxidations by complexing with CYP3A4 in a synthetic phospholipid mixture, cholate, and MgCl2 and enhancing its reduction by NPR without directly transferring electrons to P-450 (Yamazaki et al., 1996a). In the present study, purified b5, membraneb5 as well as apob5 enhanced the catalytic activities of CYP3A4 in bacterial membranes. Exogenous b5also was effective when added to microsomal CYP3A4 systems from lymphoblastoid and insect cells (lacking coexpressedb5). This suggested that insertion of rabbit, rat, or human b5 into the phospholipid bilayers is facile, and b5 can make a suitable complex with CYP3A4 and NPR for efficient electron transfer. Similarly, the membrane-associated NPR was able to mix with membranes containing CYP3A4 and enhance catalytic activities; however, catalytic function of exogenous-purified NPR was dependent on the presence of added b5 and cholate. Both cholate and b5 may be necessary for insertion of purified NPR into phospholipid membranes or for complexation of purified NPR with CYP3A4. Cholate may not be an essential component for all of the activities catalyzed by CYP3A4 in reconstituted system; however, it sometimes has been helpful for reconstitution (Yamazaki et al., 1997a). Similar enhancing effects of cholate were observed at the same concentrations (0.01% w/v, or 0.25 mM) on catalytic activities of CYP1B1 in a reconstituted system (Shimada et al., 1998).

In vitro assays are used to predict in vivo drug metabolism in humans. It has been reported that assay conditions, such as different buffers, salts, ionic strengths, detergent level, and components of NADPH-generating systems may affect the CYP3A4-mediated reactions (Yamazaki et al., 1997a; Mäenpää et al., 1998;Shimada and Yamazaki, 1998). Because effects of buffer and salt concentrations are likely to be more significant in reconstituted systems containing CYP3A4 than in human liver microsomes (Yamazaki et al., 1996b), we used 100 mM potassium phosphate buffer (pH 7.4) for drug metabolism reactions with CYP3A4/NPR membranes in this study.

In conclusion, the present study suggested that CYP3A4 coexpressed with NPR in bacterial membranes supplemented with additional NPR plusb5 is a very useful model for prediction of the rates for microsomal CYP3A4-dependent drug oxidations in human livers. The ratio of NPR to CYP3A4 in human liver microsomes is low and very different from that found to give apparent maximal activities using the bacterial CYP3A4/NPR membranes. We reported previously that CYP3A4-dependent testosterone 6β-hydroxylation is stimulated by CYP1A2 in reconstitution systems (Yamazaki et al., 1997a). In this study, CYP1A2 also enhanced catalytic activities of CYP3A4/NPR membranes. These findings indicate the potential for one P-450 enzyme to influence the catalytic characteristics of another P-450 enzyme when a low amount of NPR is present in human liver microsomes. Further studies are necessary for optimization of recombinant P-450 enzyme systems for use as human liver models.

Footnotes

  • Send reprint requests to: Hiroshi Yamazaki, Ph.D., Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail: yamazak{at}kenroku.kanazawa-u.ac.jp

  • This work was supported in part by grants from the Japanese Ministries of Education, Science, Sports, and Cultures; and Health and Welfare.

  • Abbreviations used are::
    P-450
    cytochrome P-450
    NPR
    NADPH-P-450 reductase
    CYP3A4/NPR membranes
    membranes prepared from bacteria coexpressing CYP3A4 and NPR from a bicistronic vector
    apo b5
    apolipoprotein cytochromeb5
    LB
    Luria-Bertani medium
    TB
    Terrific Broth
    • Received March 9, 1999.
    • Accepted June 2, 1999.

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Enhancement of Cytochrome P-450 3A4 Catalytic Activities by Cytochrome b 5 in Bacterial Membranes

Hiroshi Yamazaki, Miki Nakajima, Mami Nakamura, Satoru Asahi, Noriaki Shimada, Elizabeth M. J. Gillam, F. Peter Guengerich, Tsutomu Shimada and Tsuyoshi Yokoi
Drug Metabolism and Disposition September 1, 1999, 27 (9) 999-1004;
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