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首页> 外文期刊>Journal of Molecular Biology >Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits
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Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits

机译：白细胞型12-脂氧合酶和网织细胞型15-脂氧合酶在兔中的同时表达

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摘要

In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. Ln fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. Ln the 3'-untranslated region of the novel mRNA a C + U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis. (C) 1998 Academic Press Limited. [References: 52]
机译：在兔网状细胞中，花生四烯酸15-脂氧合酶（15-LOX）以高产量表达。重新筛选兔网织红细胞cDNA文库中的15-LOX转录本，分离出编码新型脂氧合酶的全长cDNA。该酶的预测氨基酸序列与网织细胞型15-脂氧合酶具有高度的同一性（99％）。在两种酶的六个氨基酸残基中，在353位检测到Phe-Leu交换。最近，定点诱变研究表明，该氨基酸交换将15-脂氧合酶转化为12-脂氧合酶。实际上，当该新酶在大肠杆菌中表达时，主要观察到花生四烯酸的12-脂氧合。重组酶表现出相当广泛的底物特异性。各种C-18和C-20聚烯酸脂肪酸，甚至复杂的底物（如生物膜）都被有效地氧化。因此，该新酶可以分类为白细胞型12-脂氧合酶。白细胞型12-脂氧合酶基因3'区域的基因组聚合酶链反应表明，内含子10至13与15-脂氧合酶基因的相应序列相差约10％，尽管它们的大小和内含子-外显子组织非常类似。在新型mRNA的3'非翻译区中，发现了富含C + U，20倍的重复元件，这似乎与15-脂氧合酶mRNA的分化控制元件高度相关。用正常兔制备的各种细胞和组织进行的活性分析表明，只有外周单核细胞才能大量表达该酶，这表明组织对基因表达的特异性调节。这些数据首次表明在一个物种中网织细胞型15-脂氧合酶和白细胞型12-脂氧合酶的两个单独基因的共表达。这对于这两种酶在红细胞发育和动脉粥样硬化中的意义至关重要。 （C）1998 Academic Press Limited。 [参考：52]

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