Abstract
Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that Vmax and Vmax/Km of all three variants were less than 3 and 1% of the WT, respectively, although the Km value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.
Footnotes
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Y.S., N.H., and K.M. contributed equally to this work.
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This study was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences, Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare, and a Research Grant from the Japan Health Sciences Foundation.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.005819.
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ABBREVIATIONS: P450, cytochrome P450; SNP, single nucleotide polymorphism; WT, wild type; RT, reverse-transcription; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Received May 30, 2005.
- Accepted September 8, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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