Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.
Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.
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在不存在和存在白细胞因子的情况下纯化的重组人 5-脂氧合酶的活性表征。
DOI：
10.1016/s0021-9258(19)67757-4
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Mark Abramovitz
中科院分区：
文献类型：
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作者：
Deschenes Denis;J. Falgueyret;D. Riendeau;Mark Abramovitz
用纯化的重组人5-脂氧合酶研究了该蛋白在白细胞刺激因子存在和不存在情况下的催化性能。重组人5-脂氧合酶通过ATP-琼脂糖层析从高表达杆状病毒系统中纯化到明显的均匀性（95-99%），每100 ml培养（2 × 10(8)个细胞）的蛋白质产量为0.6 mg，在ATP、Ca2+和磷脂酰胆碱存在的情况下，每mg蛋白质的比活性为3-6 μ mol的5-氢过氧二碳四烯酸（5-HPETE）。在没有白细胞因子的情况下，纯化后的重组酶催化的反应最大生成5-HPETE的半衰期为0.5 ~ 0.7 min，对5-脂氧合酶选择性抑制剂BW755C （IC50 = 13微米）和L-656,224 （IC50 = 0.8微米）敏感。花生四烯酸氧化的反应产物为5-HPETE和白三烯A4 （LTA4）的非酶解产物6-反式白三烯B4和12-epi-6-反式白三烯B4，表明纯化后的蛋白同时表达5-加氧酶和白三烯A4合成酶活性（比例为6:1）。当检测到ATP、Ca2+和磷脂酰胆碱存在时，经超声处理的人白细胞的微粒体部分和60-90%硫酸铵沉淀部分不增加分离酶的产物形成。这些因子被发现在酶与试验混合物在37℃预孵育期间稳定5-脂氧合酶，但在预孵育期结束时加入它们未能刺激酶活性。结果表明，人5-脂氧合酶可以以催化活性形式分离出来，并且来自白细胞的蛋白质因子可以防止酶失活，但不是酶活性所必需的。
Purified recombinant human 5-lipoxygenase was used to investigate the catalytic properties of the protein in the presence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 x 10(8) cells) and a specific activity of 3-6 mumol of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to the selective 5-lipoxygenase inhibitors BW755C (IC50 = 13 microM) and L-656,224 (IC50 = 0.8 microM). The reaction products of arachidonic acid oxidation were 5-HPETE and 6-trans- and 12-epi-6-trans-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4), indicating that the purified protein expressed both the 5-oxygenase and leukotriene A4 synthase activities (ratio 6:1). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+, and phosphatidylcholine. These factors were found to stabilize 5-lipoxygenase during preincubation of the enzyme at 37 degrees C with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect against enzyme inactivation but are not essential for enzyme activity.