Abstract
1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, Km and Vmax for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (Vmax/Km) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in Km without a difference in Vmax. 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, Km2 and Vmax2 of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.
| Original language | English |
|---|---|
| Pages (from-to) | 140-147 |
| Number of pages | 8 |
| Journal | Xenobiotica |
| Volume | 39 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Feb 2009 |
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Keywords
- CYP3A4
- Function
- Non-synonymous single nucleotide polymorphism (SNP)
- Substrate dependency
ASJC Scopus subject areas
- Pharmacology
- Toxicology
- Biochemistry
- Health, Toxicology and Mutagenesis
Cite this
Functional characterization of CYP3A4.16 : Catalytic activities toward midazolam and carbamazepine. / Maekawa, K.; Yoshimura, T.; Saito, Y.; Fujimura, Y.; Aohara, F.; Emoto, C.; Iwasaki, K.; Hanioka, N.; Narimatsu, S.; Niwa, T.; Sawada, J.
In: Xenobiotica, Vol. 39, No. 2, 02.2009, p. 140-147.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Functional characterization of CYP3A4.16
T2 - Catalytic activities toward midazolam and carbamazepine
AU - Maekawa, K.
AU - Yoshimura, T.
AU - Saito, Y.
AU - Fujimura, Y.
AU - Aohara, F.
AU - Emoto, C.
AU - Iwasaki, K.
AU - Hanioka, N.
AU - Narimatsu, S.
AU - Niwa, T.
AU - Sawada, J.
PY - 2009/2
Y1 - 2009/2
N2 - 1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, Km and Vmax for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (Vmax/Km) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in Km without a difference in Vmax. 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, Km2 and Vmax2 of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.
AB - 1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, Km and Vmax for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (Vmax/Km) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in Km without a difference in Vmax. 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, Km2 and Vmax2 of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.
KW - CYP3A4
KW - Function
KW - Non-synonymous single nucleotide polymorphism (SNP)
KW - Substrate dependency
UR - http://www.scopus.com/inward/record.url?scp=61649088626&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=61649088626&partnerID=8YFLogxK
U2 - 10.1080/00498250802617746
DO - 10.1080/00498250802617746
M3 - Article
C2 - 19255940
AN - SCOPUS:61649088626
VL - 39
SP - 140
EP - 147
JO - Xenobiotica
JF - Xenobiotica
SN - 0049-8254
IS - 2
ER -