Isolation of partially purified P450 2D18 and characterization of activity toward the tricyclic antidepressants imipramine and desipramine

被引:10
作者
Thompson, CM
Kawashima, H
Strobel, HW
机构
[1] Univ Texas, Sch Med, Dept Biochem & Mol Biol, Houston, TX 77225 USA
[2] Osaka City Univ, Sch Med, Dept Urol, Osaka 5458586, Japan
关键词
cytochrome P450; CYP2D18; CYP2D6; CYP2D4; protein expression; baculovirus; enzyme purification; imipramine; desipramine; tricyclic antidepressants; GBR-12935; brain drug metabolism;
D O I
10.1006/abbi.1998.0892
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Previous reports have shown that rat brain microsomes are capable of metabolizing tricyclic antidepressants such as imipramine, Subsequent studies have shown that the protein products of several clones isolated from rat brain cDNA libraries are capable of metabolizing imipramine to both its active metabolite, desipramine, and its inactive hydroxylated metabolites. We report here the overexpression and partial purification of P450 2D18 using the baculovirus expression system and the incorporation of a C-terminal [His](4) tag,P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparent K-M values for P450 2D18 toward imipramine and desipramine were 374 and 314 mu M, respectively. While apparent K-M values were similar, P450 2D18 was shown to have a five-fold increased V-max (2.2 nmol/min/nmol P450) for imipramine compared to desipramine (0.44 nmol/min/nmol P450), suggesting a primary involvement in the activation of imipramine to desipramine. We also examined the effect of the CYP2D6 inhibitor quinidine, the CYP3A inhibitor ketoconazole, and the dopamine reuptake inhibitor GBR-12935 for their ability to inhibit P450 2D18-mediated metabolism of imipramine. These results, when compared with previous studies using rat brain microsomes, suggest that P450 2D18 may play an important role in the conversion of imipramine to its active metabolite desipramine in the rat brain. (C) 1998 Academic Press.
引用
收藏
页码:115 / 121
页数:7
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