Kawashima H, Sequeira D J, Nelson D R, Strobel H W
Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77225, USA.
J Biol Chem. 1996 Nov 8;271(45):28176-80. doi: 10.1074/jbc.271.45.28176.
We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5'-extension, and we posit in this report by analysis of a genomic clone that this 5'-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.
我们之前报道过分离出两个cDNA克隆，命名为2d - 29和2d - 35，它们具有相同的开放阅读框，编码一种属于CYP2D亚家族的新型脑细胞色素P - 450（P - 450），并指出克隆2d - 35的mRNA似乎在脑中表达而不在肝脏中表达(1)。尽管这些克隆推导的氨基酸序列与肝脏CYP2D4的氨基酸序列仅在C末端区域有5个氨基酸不同，但这个新的P - 450 cDNA克隆包含一个独特的5'延伸，并且我们在本报告中通过对一个基因组克隆的分析推测这个5'非翻译序列来自一个与CYP2D4不同的基因。因此，根据推荐的命名法(2)，这种新型P - 450被命名为P - 450 2D18。使用网织红细胞系统进行体外翻译证实了该cDNA的可表达性，并使用COS - M6细胞进行了蛋白质表达。免疫印迹分析显示在预测大小范围内有一条与抗P - 450 2D6抗血清发生交叉反应的条带，在对照细胞中未见到。此外，表达CYP2D18的COS细胞裂解物对丙咪嗪显示出N - 去甲基化活性，而另一种表达脑P - 450 CYP4F6的COS细胞裂解物显示出10 - 羟化活性。这是首次将脑中的一种特定P - 450同工酶与抗抑郁药丙咪嗪的特定代谢改变联系起来的报告。
