Cloning and expression of an airway epithelial 12-lipoxygenase.
Cloning and expression of an airway epithelial 12-lipoxygenase.
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DOI：
10.1152/ajplung.1992.263.6.1-a
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发表时间：
1992
期刊：
影响因子：
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通讯作者：
N. D. Marzo;D. Sloane;S. Dicharry;Ella Highland;Elliott Sigal
中科院分区：
文献类型：
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作者：
N. D. Marzo;D. Sloane;S. Dicharry;Ella Highland;Elliott Sigal
花生四烯酸12-脂氧合酶产生的代谢产物可以调节气道功能。为了进一步表征这种酶，我们使用人网织红细胞15-脂氧合酶cDNA作为探针，从牛气管上皮cDNA文库中分离出对应于12-脂氧合酶的cDNA。所得的2.9-kb cDNA，其同一性通过在大肠杆菌中表达活性催化功能而得到证实，具有编码75，000 kDa蛋白质的2.0-kb开放阅读框架，并包括5 bp的5 ′-非翻译区和0.9 kb的3 ′-非翻译区。在北方杂交中，12-脂氧合酶cDNA与牛气管上皮RNA的一条带（3.5 kb）杂交。识别人气管15-脂氧合酶的多克隆抗体在免疫印迹上与表达的牛气管12-脂氧合酶发生交叉反应。此外，推导的氨基酸序列是86%相同（93%相似）的人15-脂氧合酶，但64%相同的人血小板12-脂氧合酶，这表明牛气管酶是同源的人15-脂氧合酶。这是第一个来自任何物种的上皮脂氧合酶序列。牛序列与其他脂肪氧合酶序列的比较表明，在12-脂肪氧合酶和15-脂肪氧合酶之间只有4个氨基酸是保守的差异。我们推测，这四个氨基酸可能是负责酶的位置特异性。
Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region. On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA. Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase. Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase. This is the first sequence of an epithelial lipoxygenase from any species. A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase. We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme.