Isolation of partially purified P450 2D18 and characterization of activity toward the tricyclic antidepressants imipramine and desipramine.
Isolation of partially purified P450 2D18 and characterization of activity toward the tricyclic antidepressants imipramine and desipramine.
复制标题
部分纯化的 P450 2D18 的分离以及对三环抗抑郁药丙咪嗪和地昔帕明的活性表征。
DOI：
10.1006/abbi.1998.0892
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发表时间：
1998
影响因子：
3.9
通讯作者：
Strobel,HW
中科院分区：
文献类型：
--
作者：
Thompson,CM;Kawashima,H;Strobel,HW
先前的报告表明，大鼠脑微粒体能够代谢三环类抗抑郁药，例如丙咪嗪。随后的研究表明，从大鼠脑 cDNA 文库中分离的几个克隆的蛋白质产物能够将丙咪嗪代谢为其活性代谢物地昔帕明及其无活性的羟基化代谢物。我们在这里报告了使用杆状病毒表达系统和 C 端 [His]4 标签的掺入 P450 2D18 的过度表达和部分纯化。 P450 2D18 部分纯化至 4.8 nmol/mg 蛋白质的特定含量，并显示为电泳纯。 P450 2D18 对丙咪嗪和地昔帕明的表观 KM 值分别为 374 和 314 μM。虽然表观 KM 值相似，但与地昔帕明 (0.44 nmol/min/nmol P450) 相比，P450 2D18 丙咪嗪的 Vmax (2.2 nmol/min/nmol P450) 增加了五倍，表明主要参与丙咪嗪向地昔帕明的活化。我们还检查了CYP2D6抑制剂奎尼丁、CYP3A抑制剂酮康唑和多巴胺再摄取抑制剂GBR-12935抑制P450 2D18介导的丙咪嗪代谢的能力。与之前使用大鼠脑微粒体的研究相比，这些结果表明 P450 2D18 可能在大鼠脑中丙咪嗪转化为其活性代谢物地昔帕明的过程中发挥重要作用。
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]4tag. P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparentKMvalues for P450 2D18 toward imipramine and desipramine were 374 and 314 μM, respectively. While apparentKMvalues were similar, P450 2D18 was shown to have a fivefold increasedVmax(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 theCYP2D6 inhibitor quinidine, theCYP3A 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.