Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids.
Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids.
复制标题
从猪白细胞中纯化花生四烯酸 5-脂氧合酶及其与氢过氧二十碳四烯酸的反应性。
DOI：
10.1016/s0021-9258(19)57499-3
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Shozo Yamamotog
中科院分区：
文献类型：
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作者：
Natsuo Ueda;Shuji KanekoS;Tanihiro Yoshimoto;Shozo Yamamotog
使用单克隆抗 5-脂氧合酶抗体，通过免疫亲和层析从 105,000 X g 猪白细胞匀浆上清液中纯化花生四烯酸 5-脂氧合酶，使其接近均质。纯化的酶与花生四烯酸的反应主要产生5-氢过氧-6,8,11,14-二十碳四烯酸，同时形成少量的几种更具极性的化合物。这些次要产物被鉴定为白三烯 A4 的降解产物，即 6-反式白三烯 B4（C-12 处的差向异构体）和 5,6-二羟基-7,9,11,14-二十碳四烯酸的差向异构混合物。这些化合物也是通过酶与 5-氢过氧-二十碳四烯酸的反应产生的。 5-脂氧合酶和白三烯A合酶活性的关联通过多个实验得到证明：酶的热失活、选择性5-脂氧合酶抑制剂的作用、钙离子和ATP的需要以及酶的自催化失活。该酶对 12- 和 15-氢过氧二十碳四烯酸也具有活性，分别产生 (5S,12S)- 和 (5S,15S)-二氢过氧酸。与花生四烯酸（100%，0.6μmol/3分钟/mg蛋白质）相比，这些氢过氧酸的反应最大速度如下：5-氢过氧酸，3.5%，12-氢过氧酸，22%，和15-氢过氧酸，30%。
Arachidonate 5-lipoxygenase was purified to near homogeneity from the 105,000 X g supernatant of porcine leukocyte homogenate by immunoaffinity chromatography using a monoclonal anti-5-lipoxygenase antibody. Reaction of the purified enzyme with arachidonic acid produced predominantly 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid with concomitant formation of several more polar compounds in smaller amounts. These minor products were identified as the degradation products of leukotriene A4, namely, 6-trans-leukotriene B4 (epimeric at C-12) and an epimeric mixture of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids. These compounds were also produced by reaction of the enzyme with 5-hydroperoxy-eicosatetraenoic acid. Association of the 5-lipoxygenase and leukotriene A synthase activities was demonstrated by several experiments: heat inactivation of enzyme, effect of selective 5-lipoxygenase inhibitors, requirements of calcium ion and ATP, and self-catalyzed inactivation of enzyme. The enzyme was also active with 12- and 15-hydroperoxy-eicosatetraenoic acids producing (5S,12S)- and (5S,15S)-dihydroperoxy acids, respectively. Maximal velocities of the reactions with these hydroperoxy acids as compared with that of arachidonic acid (100%, 0.6 mumol/3 min/mg of protein) were as follows: 5-hydroperoxy acid, 3.5%, 12-hydroperoxy acid, 22%, and 15-hydroperoxy acid, 30%.