Abstract
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.
Highlights
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
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
From the alignment analysis of the P450 superfamily, it has been determined that the arginine residue R128 of CYP2A6 is highly conserved
Summary
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). Genotyping of CYP2A6 and Sequencing of Novel Variants—To identify the CYP2A6 genotypes, PCR-RFLP analysis was performed as described previously (6). 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. PCR products from CYP2A6*1/*6 heterozygotes were subcloned into pCR vector (Invitrogen) for sequencing. Kd1F/E3R PCR-RFLP analysis was performed again to select the clones containing the CYP2A6*6 allele. Amino acid sequences of CYP2 family were referenced from online information
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