Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including K_m of 90.4 µM and V_max of 2.63 IU were determined for LOXe. The enzyme with 0.1 nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.

中文翻译：

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大肠杆菌可溶性膜结合型氨比索表皮脂氧合酶的纯化及其对人胎包皮成纤维细胞的生长作用。

脂加氧酶是催化脂质加氢过氧化的非血红素铁脂质双加氧酶。墨西哥x（Ambystoma mexicanum）是该酶的重要来源，其四肢具有再生能力。已经表明，用表皮脂氧合酶（LOXe）转染的人骨肉瘤和角质形成细胞具有增加的细胞迁移速率。在本研究中，LOXe是一种外周膜蛋白，是在大肠杆菌中产生的。使用不同的去污剂，阴离子溶液和凝胶过滤色谱法纯化酶。使用花生四烯酸作为底物，通过光谱法进行酶活性的动力学测定。最后，对该酶进行了表征，并通过MTT活力测定法检测了其对人成纤维细胞的生长作用。确定了LOXe的酶动力学参数，其中K _m为90.4 µM，V _max为2.63 IU。终浓度为0.1 nM的酶可促进5000个细胞/孔的人类成纤维细胞生长达11％（P <0.01）。在本研究中，我们介绍了一种大肠杆菌 表达系统产生过量的可溶性LOXe，以及有效的纯化方法以提供可溶性和活性形式的LOXe，其有效刺激人成纤维细胞的增殖。