Chen X S, Brash A R, Funk C D
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.
Eur J Biochem. 1993 Jun 15;214(3):845-52. doi: 10.1111/j.1432-1033.1993.tb17988.x.
A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His)6-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a Vmax of 2-4 mumol min-1 mg protein-1, Km of 10 microM and kcat of approximately 250 min-1. linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5-8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.
构建了一种杆状病毒表达载体，其由与人类血小板12 - 脂氧合酶cDNA相邻的编码六个组氨酸标签的序列组成，并置于多角体蛋白启动子的控制之下。重组12 - 脂氧合酶杆状病毒用于感染草地贪夜蛾昆虫细胞（Sf9）。感染后54小时，达到最大12 - 脂氧合酶活性和蛋白水平；通过镍离子螯合色谱法一步将该酶纯化至表观均一，其中带有（His）6标签的12 - 脂氧合酶用100 mM咪唑洗脱。纯化的酶几乎只将花生四烯酸代谢为12 - 氢过氧二十碳四烯酸，几乎没有环氧醇或还原产物，如果有的话也是极少，其Vmax为2 - 4 μmol min-1 mg蛋白-1，Km为10 μM，kcat约为250 min-1。另一方面，亚油酸以约为以花生四烯酸为底物时所得速率2%的速率转化为（13S）-13 - 氢过氧 - 十八碳二烯酸，但具有相同的Km。该酶在pH 7.5 - 8之间活性最高，并且在0.006%吐温 - 20存在下活性显著增强。产生了针对重组酶的多克隆抗体，通过免疫印迹和免疫沉淀方法发现其在血小板、人红白血病细胞和12 - 脂氧合酶杆状病毒感染的Sf9细胞中识别一条单一的75 kDa条带。12 - 脂氧合酶蛋白占血小板制剂中总可溶性蛋白的0.1%。在免疫荧光实验中，在感染的昆虫细胞和人巨核母细胞DAMI细胞系的细胞质中观察到了12 - 脂氧合酶。大量纯人类血小板12 - 脂氧合酶的分离应有助于详细的生化结构/功能研究。
