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.
| Original language | English |
|---|---|
| Pages (from-to) | 1905-1910 |
| Number of pages | 6 |
| Journal | Drug Metabolism and Disposition |
| Volume | 33 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - Dec 2005 |
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ASJC Scopus subject areas
- Pharmacology
- Toxicology
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Functional analysis of three CYP1A2 variants found in a Japanese population. / Saito, Yoshiro; Hanioka, Nobumitsu; Maekawa, Keiko; Isobe, Takashi; Tsuneto, Yumi; Nakamura, Ryosuke; Soyama, Akiko; Ozawa, Shogo; Tanaka-Kagawa, Toshiko; Jinno, Hideto; Narimatsu, Shizuo; Sawada, Jun Ichi.
In: Drug Metabolism and Disposition, Vol. 33, No. 12, 12.2005, p. 1905-1910.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Functional analysis of three CYP1A2 variants found in a Japanese population
AU - Saito, Yoshiro
AU - Hanioka, Nobumitsu
AU - Maekawa, Keiko
AU - Isobe, Takashi
AU - Tsuneto, Yumi
AU - Nakamura, Ryosuke
AU - Soyama, Akiko
AU - Ozawa, Shogo
AU - Tanaka-Kagawa, Toshiko
AU - Jinno, Hideto
AU - Narimatsu, Shizuo
AU - Sawada, Jun Ichi
PY - 2005/12
Y1 - 2005/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=28144435129&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=28144435129&partnerID=8YFLogxK
U2 - 10.1124/dmd.105.005819
DO - 10.1124/dmd.105.005819
M3 - Article
C2 - 16174806
AN - SCOPUS:28144435129
VL - 33
SP - 1905
EP - 1910
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
SN - 0090-9556
IS - 12
ER -