Department of Pharmacology, College of Pharmacy, The University of Toledo, Toledo, OH 43614, USA.
Aquat Toxicol. 2011 Mar;102(1-2):18-23. doi: 10.1016/j.aquatox.2010.12.016. Epub 2010 Dec 29.
To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5'- and 3'-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template, the cDNA encoding the zebrafish COMT was PCR-amplified. The recombinant zebrafish COMT protein was subsequently expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. Upon enzymatic characterization, purified COMT displayed methylating activity toward dopamine, dopa, and catecholestrogens, as well as three representative catechol drugs, methyldopa, dobutamine, and isoproterenol. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.
为了将斑马鱼建立为研究药物代谢甲基化途径的模型，我们着手对斑马鱼儿茶酚氧位甲基转移酶（COMT）进行分子克隆。通过在 GenBank 数据库中搜索，鉴定出了一个编码斑马鱼 COMT 的核苷酸序列。根据序列信息，我们设计并合成了与该斑马鱼 COMT 的 5'和 3'编码区相对应的寡核苷酸。使用从 3 个月大的成年雌性斑马鱼分离的总 RNA 反转录的第一链 cDNA 作为模板，通过 PCR 扩增了编码斑马鱼 COMT 的 cDNA。随后，将含有斑马鱼 COMT cDNA 的 pGEX-2TK 表达载体转化的 BL21（DE3）大肠杆菌细胞中表达并纯化重组斑马鱼 COMT 蛋白。经过酶学特性分析，纯化的 COMT 对多巴胺、多巴和儿茶酚雌激素以及三种代表性儿茶酚药物（甲基多巴、多巴酚丁胺和异丙肾上腺素）具有甲基化活性。反转录聚合酶链反应（RT-PCR）分析显示，在胚胎发育过程中和幼体阶段直至成熟期间，斑马鱼 COMT 的表达具有发育阶段依赖性。这些结果为研究 COMT 介导的甲基化在保护儿茶酚类药物和其他外来儿茶酚类化合物免受发育过程中不良影响的作用提供了基础。
