CYP2A6*6, a Novel Polymorphism in Cytochrome P450 2A6, Has a Single Amino Acid Substitution (R128Q) That Inactivates Enzymatic Activity*

Kyoko KitagawaDagger , Naoki KunugitaDagger §, Masatoshi Kitagawa, and Toshihiro KawamotoDagger ||

From the Dagger  Department of Environmental Health, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 and the  Department of Biochemistry 1, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan

Received for publication, October 16, 2000, and in revised form, February 15, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

By using the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP), a novel polymorphism of CYP2A6, CYP2A6*6, was detected in 0.4% of the Japanese population. To study the enzymatic properties of the CYP2A6.6 protein with a single amino acid substitution of arginine 128 to glutamine, both this isozyme and the CYP2A6.1 protein (wild-type) were produced in insect cells using a baculovirus system. Coumarin 7-hydroxylation, which reflects CYP2A6 activity, was significantly reduced (one-eighth of normal) in cell lysate from CYP2A6*6-transfected Sf9 cells compared with that lysate from CYP2A6*1-transfected cells. To clarify the mechanism of inactivation of the CYP2A6.6 enzyme, the heme content and reduced CO difference spectrum were examined. Although CYP2A6.6 retained about one-half the heme content of CYP2A6.1, the reduced CO-bound Soret peak was completely lost. These results suggest that the inactivation of CYP2A6.6 is mainly due to disordering of the holoprotein structure rather than a failure of heme incorporation.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytochrome P450 (CYP)1 is a superfamily of hemoproteins, many of which can metabolize xenobiotics such as procarcinogens, drugs, and environmental pollutants. CYP2A62 is a major hepatic member of the family, which metabolizes pharmaceutical agents such as coumarin and activates some procarcinogens, including 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine (1, 2). CYP2A6 also metabolizes nicotine to cotinine via C-oxidation (3-5). It was previously reported that subjects with a CYP2A6 homozygous gene deletion had significantly impaired nicotine metabolism (6, 7). In addition to a deletion type allele (CYP2A6*4), three more genetic polymorphisms of CYP2A6, CYP2A6*2, *3, and *5, were reported to result in enzymatic inactivation (8-10). This genetic polymorphism of CYP2A6 is suspected to be a major cause of interindividual variation in enzymatic activity for various CYP2A6 substrates, and it is thereby critical to characterize the enzymatic properties caused by the polymorphism.

In our previous work, characterizing the CYP2A6 genotypes among 252 Japanese subjects, a new variant was detected (6). In the present study, this novel variant was more extensively studied using the PCR technology combined with restriction enzyme fragment length polymorphism (PCR-RFLP) that we previously established (6). The PCR product from the previously unknown variant was found to have a single nucleotide mutation in exon 3, resulting in an amino acid substitution of arginine (R) 128 by glutamine (Q); therefore, the variant was designated CYP2A6*6. The arginine 128 of CYP2A6 is highly conserved in this superfamily, and this position is thought to be part of a heme binding site (11-13). Because glutamine is potentially a neutral residue in contrast to the positively charged arginine, such a substitution might affect the electron transfer and/or the tertiary structure and thereby cause a critical alteration in the catalytic function of CYP2A6.

To clarify the properties of this variant protein, CYP2A6.6, both CYP2A6.1 (wild-type) and CYP2A6.6 were produced in insect cells using a baculovirus system.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Study Subjects-- Genotyping analysis was carried out for 894 healthy Japanese individuals (748 men and 146 women) who lived in Fukuoka Prefecture, including the 252 individuals reported previously (6). Genomic DNA from peripheral blood was prepared using a DNA extracter (Applied Biosystems, model-340A). All subjects gave their informed consent.

Genotyping of CYP2A6 and Sequencing of Novel Variants-- To identify the CYP2A6 genotypes, PCR-RFLP analysis was performed as described previously (6). Briefly, the region around exon 3 was amplified using Kd1F and E3R primers, and the PCR products were digested with the restriction enzymes, MspI, XcmI, and DdeI. The digested fragments were analyzed in a 4% agarose gel stained with ethidium bromide.

PCR products from CYP2A6*1/*6 heterozygotes were subcloned into pCR vector (Invitrogen) for sequencing. Kd1F/E3R PCR-RFLP analysis was then performed again to select the clones containing the CYP2A6*6 allele. The positive clones were sequenced using an Applied Biosystems 373S DNA sequencer following the protocol provided by the manufacturer.

For the alignment study, amino acid sequences of CYP2 family were referenced from online information (Human Cytochrome P450 Allele Nomenclature Committee).

Construction of Expression Plasmids and the Baculovirus Expression System-- The full-length CYP2A6*1 cDNA, in PUC18 (a kind gift from F. J. Gonzalez) was digested with EcoRI and SalI and subcloned into the EcoRI and XhoI sites of pBacPAK9 (CLONTECH). CYP2A6*6 cDNA was generated by PCR using KOD DNA polymerase (Toyobo).

Three kinds of pBacPAK9 vectors, without CYP2A6 (negative control), with CYP2A6*1, or with CYP2A6*6 cDNA, were transfected into Sf9 cells to obtain recombinant baculoviruses. Hemin (Sigma Chemical Co.) was added to the culture medium of Sf9 cells at a final concentration of 4 µg/ml 24 h after infection with baculoviruses. After incubation for 48 h, the cells were harvested, washed with PBS(-), and stored at -80 °C until each analysis.

Immunoblot Analysis-- The cells were lysed with a buffer comprising 50 mM Tris (pH 7.4), 1 mM dithiothreitol, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. To quantify apoprotein levels, 0.4 µg of lysate protein was separated on SDS-polyacrylamide gel electrophoresis (8%), immunoblotted with monoclonal anti-human CYP2A6 antibody (Gentest), applied to a horseradish peroxidase-conjugated anti-mouse IgG polyclonal preparation (Promega), and then visualized using an enhanced chemiluminescence system (NEN Life Science Products). 2.5 µg of commercially available cell lysate containing recombinant CYP2A6 (Gentest) was used as a positive control.

Coumarin 7-Hydroxylase Activity-- Coumarin 7-hydroxylase activity was measured in 125-µl reaction mixtures comprising 50 µg of lysate protein, 1.3 mM NADP+ (Sigma), 3.3 mM glucose 6-phosphate (Sigma), 0.4 unit/ml glucose-6-phosphate dehydrogenase (Sigma), 3.3 mM magnesium chloride, 23 nmol of P450 reductase (Gentest), 0.93 µg of cytochrome b5 (Panvera), and 0.4 mM coumarin (Nakarai) in 50 mM Tris (pH 7.4). After incubation at 37 °C for 20 min, the reaction was stopped by the addition of 50 µl of 20% trichloroacetic acid followed by centrifugation at 10,000 × g for 5 min. Fifty microliters of the supernatant fraction was diluted with 950 µl of 100 mM Tris (pH 9.0), and the fluorescence was determined at wavelengths of 368 nm for excitation and 456 nm for emission using a Hitachi F4010 fluorescence spectrophotometer.

Heme Content Measurement-- Heme content in CYP2A6 was quantified by the high-performance liquid chromatography (HPLC) method of Sato et al. (14). Hemin, used as a standard for heme determination, was dissolved in diisopropylamine:methanol (25:975, DM) at a concentration of 100 µg/ml facilitated by ultrasonication (30 min). After dilution with DM, hemin was injected onto an RSpak DS-613 column (Showa Denko) in a system using an L-6000 pump, an L-7500 Chromato Integrator and an L-4200 UV-visible detector (Hitachi) at 398 nm. The mobile phase was a mixture of diisopropylamine:water:methanol (25:100:9000), and the flow rate was 1.0 ml/min. Concentrations of heme were determined by measuring the height of the peak with a retention time of ~2.0 min. Commercially available CYP2A6 lysate diluted with 100 mM Tris (pH 7.4) served as a standard. Twenty-microliter aliquots of cell lysate were extracted using 980 µl of DM, centrifuged at 1600 × g for 20 min, filtered through a Millipore membrane (type FH, 0.5-µm pores), and immediately analyzed by HPLC. Reagents were all of analytical grade (Wako).

Spectral Analysis-- The reduced CO difference spectrum was obtained as described by Omura and Sato (15). Cells were solubilized in 100 mM Tris buffer (pH 7.4) containing 20% (v/v) glycerol and 0.2% Emulgen 913 (kindly supplied from Kao Chemical Co., Japan), and the insoluble fraction was removed by centrifugation at 10,000 × g for 10 min at 4 °C. The soluble fraction was then divided into reference and sample cuvettes, CO was gently bubbled into the sample cuvette for 30 s, and sodium dithionite (Wako) was dissolved in both cuvettes. CO difference spectra were recorded using a Beckman DU70 spectrophotometer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Genotyping of CYP2A6 and Sequencing of Novel Variants-- CYP2A6 genotyping by PCR-RFLP was carried out for 894 healthy Japanese subjects. A homozygous deletion was found in 3.7% of subjects, however, no individual with either CYP2A6*2 or CYP2A6*3 was detected. These findings were consistent with those of a previous study (16). Six subjects showed two kinds of unknown enzyme digestion patterns. Four individuals (0.4%) showed a single MspI digestion site heterozygosity, and the another two (0.2%) showed a single DdeI recognition site heterozygosity (Fig. 1, A and C). The PCR products from these individuals were subcloned, sequenced from exon 3, and compared with CYP2A6*1 (Fig. 1B). The difference found in those individuals was one of two single nucleotide substitutions, either G to A at 383 or C to A at 406, resulting in amino acid substitution R128Q (CGG to CAG) or a silent mutation at R136 (CGG to AGG), respectively (Table I). This R128Q polymorphism was designated CYP2A6*6.


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Fig. 1.   CYP2A6 genotyping and sequence analysis of novel variants. A, human genomic DNA from healthy individuals was subjected to PCR-RFLP for CYP2A6 genotyping. PCR product (lane 1) was digested by three kinds of restriction enzyme, MspI, DdeI, and XcmI, and then analyzed using 4% agarose gel electrophoresis. The enzyme digestion pattern of the PCR product of CYP2A6*1 is shown in lanes 2-4. Heterozygotes carrying the novel variants, CYP2A6*1/*6 (lanes 6-8) and CYP2A6*1/silent mutant (lanes 9-11), were identified. Lane 5 represents 100-bp DNA ladder size marker (GenSura). B, DNA sequences of Kd1F/E3R PCR products were compared among CYP2A6*1, CYP2A6*6, and the silent mutation. Dots indicate the identical nucleotides to CYP2A6*1. Both variants are distinguished from wild-type (CYP2A6*1) by the boxed restriction enzyme recognition sites. C, the restriction sites of MspI and the fragment sizes are shown. Horizontal arrows (Kd1F and E3R) show PCR primer annealing sites and orientation of the extension. D, the core structure of P450 and the alignment of amino acid sequence in the C-helix of CYP2A subfamily are shown. The striped boxes (A through L) and the solid box indicate the helices and the heme binding site, respectively. The region coded in exon 3 is also indicated. Asterisks show amino acid sequences conserved in the CYP2 family. The box corresponds to R128Q, which is the amino acid substitution in CYP2A6.6.

                              
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Table I
CYP2A6 genotypes in a healthy Japanese population determined by Kd1F/E3R PCR-RFLP analysis

The P450 superfamily has highly conserved structural elements, including the A- to L-helices (Fig. 1D). The positively charged arginine residue at 128 in the C-helix is especially highly conserved (11, 17, 18). Because glutamine is either neutral or weakly basic, it is suspected that this substitution of R128Q would probably not retain the native structure or enzymatic activity of CYP2A6.

To carry out further analysis of the CYP2A6.6 protein, a recombinant version of CYP2A6.1 was expressed in insect cells using a baculovirus system.

Preparation of Recombinant CYP2A6 Proteins-- The expression of CYP2A6 in Sf9 cells was confirmed by immunoblot analysis using a monoclonal CYP2A6 antibody. A single band of the same size as commercially available CYP2A6 was detected in lysates of both CYP2A6.1- and CYP2A6.6-expressing cells, whereas no such band was detected in the lysate from cells infected with empty virus (Fig. 2A). The band size was consistent with the mass of CYP2A6, 56.5 kDa. These results showed minimum endogenous production of CYP2A6 in Sf9 cells. The same band was also detected when CYP2A6.1 was expressed in Sf9 cells without exogenously added hemin, indicating that the presence or absence of hemin in the culture medium did not influence the level of CYP2A6 expression. The level of each CYP2A6 apoprotein in 0.4 µg of the positive lysate was higher than that in 2.5 µg of commercially available lysate. These results indicate that all recombinant CYP2A6 constructs were expressed efficiently, resulting in similar levels of CYP2A6 apoprotein content.


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Fig. 2.   Enzyme activity of a novel polymorphism CYP2A6*6. A, the three kinds of pBacPAK9 vectors without cDNA or with CYP2A6*1 or CYP2A6*6 cDNA were transfected into Sf9 cells. After incubation of Sf9 cells for 48 h in the absence (-) or presence (+) of hemin, each 0.4 µg of cell lysate was subjected to immunoblot analysis with a monoclonal anti-CYP2A6 antibody. 2.5 µg of commercially available cell lysate containing CYP2A6 protein was also loaded as the positive control for immunoblotting (lane 4). B, coumarin 7-hydroxylase activity of CYP2A6 expressed in Sf9 cells was measured by the fluorescence spectrophotometer at 368-nm excitation and 456-nm emission as described under "Experimental Procedures." Samples were prepared by the same procedure as in A. Each activity is indicated as mean ± S.E. of four experiments from two different sets of samples. C, kinetic study of coumarin 7-hydroxylase activity in Sf9 cell lysate containing 0.12 nmol of CYP2A6.1 or CYP2A6.6 was carried out by Lineweaver-Burk plot. The reactions were performed at final substrate concentration from 5 to 50 µM for CYP2A6.1 and 10 to 100 µM for CYP2A6.6. The amounts of CYP2A6.1 and CYP2A6.6 in cell lysates were calculated from immunoblot analysis data in A, in comparison with commercially available CYP2A6 lysate as a standard.

Coumarin 7-Hydroxylation (COU 7-OH) Activity-- Addition of hemin to the expression system increased COU 7-OH activity in lysates from cells expressing CYP2A6.1 nearly 3-fold, from 322 to 861 pmol/mg/min. The rate observed in lysates from cells expressing CYP2A6.6, even when hemin was added, was only 103 pmol/mg/min, which was about one-eighth of the rate in hemin-activated lysates from cells expressing CYP2A6.1. Enzyme activity in lysates from cells expressing the empty virus was negligible.

The kinetic properties of the recombinant proteins encoded by CYP2A6*1 and CYP2A6*6 were studied further. As shown in Fig. 2C and Table II, the CYP2A6.6 enzyme exhibited about 5-fold higher Km than CYP2A6.1. In addition, the Vmax value of CYP2A6.6 was decreased to about one-tenth that of CYP2A6.1. These results demonstrate that the single amino acid exchange, R128Q, decreased the affinity for coumarin.

                              
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Table II
Kinetic properties of coumarin 7-hydroxylase activity in cells lysate from CYP2A6-transfected Sf9 cells
Km and Vmax are calculated from the Lineweaver-Burk plots in Fig. 2C.

Heme Contents in CYP2A6-- Heme content was analyzed by HPLC as described under "Experimental Procedures." Heme content was quantitated by measuring the height of the peak monitored at 398 nm and eluted at 2.0 min (Fig. 3, A and B), and the peak height was shown to be linear up to 200 µg of commercially available CYP2A6, r = 0.9999 (Fig. 3D). Heme was detected and quantitated in both the commercially available CYP2A6 and in the lysates containing recombinant proteins (Fig. 3C).


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Fig. 3.   Measurement of heme content in CYP2A6 holoprotein. A, wavelength-scanning spectrum of hemin was determined between 375 nm and 450 nm to confirm the peak at 398 nm. Hemin was dissolved in DM (diisopropylamine:methanol = 25:975) at the concentration of 100 µg/ml. The solvent (DM) was subjected to the analysis as was the background. B, as a standard of heme, 5 ng/ml hemin dissolved in DM was subjected to HPLC at 398-nm absorption. C, commercially available CYP2A6 lysate (0, 50, 100, and 200 µg/20 µl) was subjected to HPLC and detected at 398 nm ((1) through (4)) as standards. Four kinds of cell lysates were compared: without hemin and CYP2A6.1, CYP2A6.6 and empty vector with hemin using the same method with the immunoblot analysis in Fig. 2A. Each 20 µl of cell lysate was mixed with 980 µl of DM. After centrifuging and filtration, 150 µl of each was immediately injected into the HPLC. D, a linear relation was recognized between the concentration of commercially available CYP2A6 and peak height at 2.0 min as shown in C (1)-(4). E, heme content of each sample, which was determined in C (5)-(8), was compared. The peak height derived from CYP2A6.1 and CYP2A6.6 were corrected according to the amount of CYP2A6 apoprotein content. The values are indicated as mean ± S.E. of four experiments.

When expressed on a heme/mg of apoprotein basis, the heme content in fortified CYP2A6.1 (CYP2A6.1/hemin+) was found to be about 7-fold higher than that in CYP2A6.1 without exogenous heme (CYP2A6.1/hemin-) (Fig. 3E). When the isoforms were compared under heme fortified conditions, CYP2A6.6/hemin+ was found to contain only about half as much heme as CYP2A6.1/hemin+. Thus, the R128Q substitution appeared to significantly reduce the heme binding capacity, whereas it decreased the catalytic activity 7-fold, as shown in Fig. 2B.

Analysis of CO Difference Spectra of CYP2A6-- Reduced CO difference spectra were examined to test for any detectable conformational changes within the CYP2A6 structure. Commercially available CYP2A6 exhibited a single broad maximum peak in the reduced CO difference spectrum at 447 nm (Fig. 4A). This same absorbance spectrum was obtained when CYP2A6.1 was fortified with hemin (Fig. 4B). The CO difference spectrum for hemin fortified CYP2A6.6, however, was not detectable in the absorbance range from 400 to 500 nm and was not different from that obtained with lysate from cells infected with the empty virus (Vec/hemin+) (Fig. 4B). It should be noted that these spectra were obtained with identical levels of apoprotein as determined by immunoblotting (Fig. 2A). These results demonstrate the disappearance of the hemoprotein Soret peak from CYP2A6.6 even though heme is known to be present. This suggests that the structure of CYP2A6.6 is at least partially destabilized in areas involved in heme binding


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Fig. 4.   Reduced CO difference spectra of recombinant CYP2A6 lysates. Reduced CO difference spectra were determined to study the structural status of CYP2A6 polymorphism. A, commercially available lysate containing CYP2A6 expressed in Sf9 cells was subjected to spectral analysis as a standard. B, lysate with CYP2A6.1 expressed in Sf9 cells was incubated without hemin and, similarly, lysates containing CYP2A6.1, CYP2A6.6, and the empty virus fortified with hemin were also analyzed. The total protein concentration in each cell lysate was about 5 mg/ml. The ratio of CYP2A6 apoprotein level was calculated from the result of immunoblot analysis. The cell lysates were solubilized in Tris buffer containing 20% glycerol and 0.2% Emulgen 913, and the soluble fractions were isolated by centrifuging. Samples were then divided into reference and experimental cuvettes. After bubbling of CO gas into the experimental cuvette for 30 s, sodium dithionite was dissolved in both cuvettes. The difference spectra were recorded using a spectrophotometer.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammalian P450s contain a noncovalently bound heme (protoporphyrin IX), the absence of which results in the loss of enzymatic activity. Catalytically active cytochrome P450 can be expressed at a high level using baculovirus when hemin is added to the culture medium during the course of viral infection. This requirement for exogenous hemin may be due to the inability of the Sf9 cells to synthesize sufficient heme, de novo, to activate the large amount of P450 (19, 20). The present results are consistent with this hypothesis, because both COU 7-OH activity and heme content were significantly reduced when CYP2A6.1 was produced without exogenously added hemin.

The structural analysis of P450 was reported first for P450cam by Poulos et al. (21). From the alignment analysis of the P450 superfamily, it has been determined that the arginine residue R128 of CYP2A6 is highly conserved. Lewis et al. reported that the function of this residue was suspected to be formation of a catalytically essential salt binding site with a heme propionate moiety and cytochrome b5 in P450cam, which was critical to catalytic activity (12). CYP2A7, which is catalytically inactive, shares 94% amino acid sequence identity with CYP2A6.1, and has 38 amino acid substitutions out of a total of 494 residues, including a leucine substitution at amino acid 128 (Fig. 1D) (8, 22). The R128L substitution in CYP2A7 results in loss of a positive charge, like the R128Q substitution of CYP2A6.6. Some groups have also studied the catalytic significance of amino acid substitutions of P450 by the site-directed mutagenesis method (23, 24). For example, the substitution of phenylalanine 429 of CYP2E1 by aspartate, arginine, or leucine resulted in the disappearance of the P450-specific CO difference spectrum (23). Furthermore, the substitution of I172N of P450c21 caused a conformational change and was associated with congenital adrenal hyperplasia (24). CYP2A6.2 and CYP2A6.5, which contain single amino acid substitutions, are also inactive enzymes (8, 10). CYP2A6.5 is a somewhat unstable enzyme. Although CYP2A6.2 is stable as a protein, it possibly fails in correct folding and uptake of the critical cofactor heme or efficient membrane insertion. As mentioned above, enzymatic inactivation by amino acid substitutions in P450 can be explained by a number of scenarios in addition to effects on heme binding, such as failure to assume the correct conformation. It is also possible that the R128Q single amino acid substitution in CYP2A6.6 caused a three-dimensional structural change in the C-helix and a decrease in the heme folding capacity.

In the present study, CYP2A6*6 was detected as a novel polymorphism of CYP2A6, with a low allele frequency in the Japanese population. Further studies of other ethnic populations should be made, because marked inter-ethnic differences in the distribution of CYP2A6 polymorphisms have been reported (10, 16, 25-27).

A significant number of metabolic enzymes, especially, the P450 superfamily, are involved in the metabolism of both endogenous compounds and xenobiotic chemicals. It is therefore possible that qualitative or quantitative variations of specific metabolic enzymes may be associated with interindividual differences in health effects due to consequent variability in handling substrates and/or their metabolites. It has been reported that CYP2A6*2 heterozygotes have lower nicotine C-oxidation activity than CYP2A6*1 homozygotes (16). Coumarin is detoxified by 7-hydroxylation in individuals who have CYP2A6*1. However, coumarin metabolism is altered to 3-hydroxylation in CYP2A6*2 homozygotes, as evidenced by the greatly enhanced excretion of 2-hydroxyphenylacetic acid (28, 29). 2-Hydroxyphenylacetaldehyde, an intermediate metabolite of coumarin to 2-hydroxyphenylacetic acid, can covalently bind microsomal proteins and cause hepatotoxicity in experimental animals (30). Because CYP2A6 is involved in the metabolism of some toxic substrates, as mentioned above, it is suggested that individuals who are CYP2A6*6 heterozygotes may have altered metabolic pathways and may be more susceptible to some adverse effects of chemical compounds such as 2-hydroxyphenylacetaldehyde. Further studies will be required to analyze whether other metabolic pathways may compensate for CYP2A6 variations and whether there are effects on the health of individuals carrying polymorphic alleles.

    ACKNOWLEDGEMENTS

We are very grateful to Dr. Frank J. Gonzalez (National Cancer Institute, National Institutes of Health) and Dr. Takahiko Katoh (Public Health, Miyazaki Medical College) for kindly provided us with CYP2A6*1 cDNA and human genomic DNA, to Dr. Gary L. Foureman (Hazardous Pollutant Assessment Group, National Center for Environmental Assessment, United States Environmental Protection Agency) for his useful discussion and editorial advice, and to Reiko Suenaga and Chihiro Nishiura for their technical assistance.

    FOOTNOTES

* This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF326721.

§ Current address: Dept. of Health Information Science, School of Health Science, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.

|| To whom correspondence should be addressed: Tel.: 81-93-691-7243; Fax: 81-93-691-9341; E-mail: kawamott@med.uoeh-u.ac.jp.

Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M009432200

2 We have used the nomenclature system for CYP2A6 alleles and their products recommended by the Human Cytochrome P450 Allele Nomenclature Committee (available on the Web). According to the rule, the CYP2A6 alleles are indicated as CYP2A6*1, *2, *4, and *6, and these gene products are indicated as CYP2A6.1, .2, and .6, respectively, with the exception of the CYP2A6*4 gene, which is the deleted type.

    ABBREVIATIONS

The abbreviations used are: CYP, cytochrome P450; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; HPLC, high performance liquid chromatography; DM, diisopropylamine:methanol; COU 7-OH, coumarin 7-hydroxylation; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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