Identification, characterization, and ontogenic study of a catechol O-methyltransferase from zebrafish.
Identification, characterization, and ontogenic study of a catechol O-methyltransferase from zebrafish.
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DOI：
10.1016/j.aquatox.2010.12.016
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发表时间：
2011-03
影响因子：
4.5
通讯作者：
Liu, Ming-Cheh
中科院分区：
文献类型：
--
作者：
Alazizi, Adnan;Liu, Ming-Yih;Williams, Frederick E.;Kurogi, Katsuhisa;Sakakibara, Yoichi;Suiko, Masahito;Liu, Ming-Cheh
为了建立斑马鱼作为研究药物代谢甲基化途径的模型，我们对斑马鱼儿茶酚O-甲基转移酶（COMT）进行了分子克隆。通过检索GenBank数据库，确定了一个编码假定COMT的斑马鱼核苷酸序列。根据序列信息，我们设计并合成了对应于该斑马鱼COMT的5 '和3'编码区的寡核苷酸。以从3月龄成年雌性斑马鱼的总RNA中反转录的第一链cDNA为模板，PCR扩增了编码斑马鱼COMT的cDNA。重组斑马鱼COMT蛋白随后在用含有斑马鱼COMT cDNA的pGEX-2 TK表达载体转化的BL 21（DE 3）大肠杆菌细胞中表达并纯化。酶的特性，纯化的COMT显示甲基化活性对多巴胺，多巴，和儿茶酚雌激素，以及三个代表性的儿茶酚类药物，甲基多巴，多巴酚丁胺，异丙肾上腺素。逆转录-聚合酶链反应（RT-PCR）分析显示，发育阶段依赖的表达斑马鱼COMT在胚胎发育过程中，并在整个幼虫阶段成熟。这些结果为研究COMT介导的甲基化在发育过程中对儿茶酚类药物和其他外源性儿茶酚的不良影响的保护作用提供了基础。
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.
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