Ma Jixiang, Bradbury J Alyce, King Lorraine, Maronpot Robert, Davis Linda S, Breyer Matthew D, Zeldin Darryl C
Division of Intramural Research, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Building 101, Research Triangle Park, NC 27709, USA.
Biochem Pharmacol. 2002 Nov 15;64(10):1447-60. doi: 10.1016/s0006-2952(02)01393-x.
A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72-94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450nm and the appearance of a 420nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze-thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55-57kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.
从小鼠肝脏文库中克隆出一个编码新型细胞色素P450的cDNA。序列分析表明，这个2046 bp的cDNA编码一个501个氨基酸的多肽，该多肽与其他CYP2J亚家族P450的同源性为72 - 94%，被命名为CYP2J6。Northern分析显示，CYP2J6转录本在小肠中丰富，在其他小鼠组织中水平较低。原位杂交表明，CYP2J6 mRNA存在于胃肠道黏膜的腔面上皮细胞中。利用杆状病毒在Sf9细胞中表达CYP2J6 cDNA。异源表达的CYP2J6蛋白呈现典型的P450 CO差光谱；然而，该蛋白不稳定，当表达CYP2J6的细胞通过机械匀浆、超声处理或冻融破坏时，450nm处的Soret峰最大值消失，出现420nm峰即可证明。用与人CYP2J2 IgG交叉反应的抗鼠CYP2J抗体免疫印迹小鼠微粒体，结果表明在各种组织中存在多种不同的鼠CYP2J免疫反应性蛋白。用针对CYP2J6特异性肽的抗体进行免疫印迹，在表达重组CYP2J6的Sf9细胞提取物中检测到一种突出的55 - 57kDa蛋白，但在小鼠小肠微粒体中未检测到类似分子量的蛋白。混合实验表明，重组CYP2J6在小肠微粒体存在的情况下迅速降解，这与在高敏感位点的蛋白水解一致。同时表达CYP2J6和NADPH - P450氧化还原酶的Sf9细胞代谢苄非他明，但不代谢花生四烯酸。我们得出结论，CYP2J6是一种不稳定的P450，它在苄非他明的代谢中具有活性，但在花生四烯酸的代谢中无活性。
