绵羊CYP2J的分子克隆与酶学特性分析
Molecular cloning and enzymatic characterization of sheep CYP2J.
作者信息
Messina A, Nencioni S, Gervasi P G, Gotlinger K H, Schwartzman M L, Longo V
机构信息
Istituto di Fisiologia Clinica, Consiglio Nazionale delle Ricerche, Via Moruzzi 1, Pisa, Italy.
出版信息
Xenobiotica. 2010 Feb;40(2):109-18. doi: 10.3109/00498250903410590.
Cytochrome P450 (CYP) 2Js have been studied in various mammals, but not in sheep, as an animal model used to test veterinary drug metabolism. Sheep CYP2J was cloned from liver messenger RNA (mRNA) by RACE. The cDNA, after modification at its N- and C-terminals, was expressed in Escherichia coli and the sheep CYP2J protein, purified by chromatography, was 80% homologous to human and monkey CYP2J2. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that CYP2J mRNA was expressed in liver, cortex, respiratory and olfactory mucosa, heart, bronchi, lung, spleen, small intestine and kidney. The purified enzyme was catalytically active towards aminopyrine, all-trans-retinoic acid, and particularly arachidonic acid forming 20-HETE, 19-HETE, and 18-HETE (about 86% of the total) and 14,15-, 11,12-, 8,9-, and 5,6-EETs (cis-epoxyeicosatrienoic acids; about 14% of total), with a regioselectivity similar to that shown by the mammalian CYP2J2s.
细胞色素P450（CYP）2J已在多种哺乳动物中进行了研究，但尚未在绵羊中进行研究，绵羊是用于测试兽药代谢的动物模型。通过RACE从肝脏信使核糖核酸（mRNA）中克隆出绵羊CYP2J。在其N端和C端进行修饰后的cDNA在大肠杆菌中表达，通过色谱法纯化的绵羊CYP2J蛋白与人和猴的CYP2J2具有80%的同源性。逆转录聚合酶链反应（RT-PCR）实验表明，CYP2J mRNA在肝脏、皮质、呼吸和嗅觉黏膜、心脏、支气管、肺、脾脏、小肠和肾脏中表达。纯化后的酶对氨基比林、全反式维甲酸具有催化活性，尤其对花生四烯酸具有催化活性，可生成20-羟基二十碳四烯酸（20-HETE）、19-羟基二十碳四烯酸（19-HETE）和18-羟基二十碳四烯酸（18-HETE）（约占总量的86%）以及14,15-、11,12-、8,9-和5,6-环氧二十碳三烯酸（顺式环氧二十碳三烯酸；约占总量的14%），其区域选择性与哺乳动物CYP2J2所表现出的相似。
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