Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids.
Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids.
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DOI：
10.1016/s0021-9258(18)66623-2
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发表时间：
1986-12
期刊：
影响因子：
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通讯作者：
Chieko Yokoyama;F. Shinjo;Tanihiro Yoshimoto;Shozo YamamotoS;John A. Oatese;Alan;Brash
中科院分区：
文献类型：
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作者：
Chieko Yokoyama;F. Shinjo;Tanihiro Yoshimoto;Shozo YamamotoS;John A. Oatese;Alan;Brash
用抗花生四烯酸12-脂氧合酶的单抗，通过硫酸铵分级沉淀、DEAE-纤维素层析和免疫亲和层析，从猪白细胞胞浆组分中纯化出接近均一的花生四烯酸12-脂氧合酶。纯化的酶是不稳定的(在4℃下半衰期约为24小时)，但在亚铁离子存在下或在没有空气的情况下储存，可显着防止失活。酶反应开始前的滞后期被12-羟基过氧基-5，8，10，14-二十碳四烯酸所消除。花生四烯酸和其他活性底物对底物有明显的抑制作用，但在0.03%吐温20存在下底物浓度曲线归一化。花生四烯酸转化为omega-9氧化产物12-羟基过氧基-5Z，8Z，10Z，14Z-二十碳四烯酸。5-羟基和5-羟基二十碳四烯酸也发生了C-12氧化，最大速率分别是花生四烯酸的60%和150%。十八碳烯酸也是很好的底物。γ-亚麻酸在omega-9位氧化(C-10)，而亚油酸和α-亚麻酸在omega-6位氧化(C-13)。以15-羟基过氧基-5，8，11，13-二十碳四烯酸为底物的反应要复杂得多。与花生四烯酸的反应速率为70%。通过紫外吸收光谱、高效液相色谱、气相色谱-质谱法对产物进行了鉴定。其中，(8S，15S)-二氢过氧基-5Z，9E，11Z，13E-二十碳四烯酸和(14R，15S)-赤基-二氢过氧基-5Z，8Z，10E，12E-二十碳五烯酸的生成量分别大于(8R)-和(14S，15S)-苏氨酸异构体，这些产物归因于15-羟基过氧酸的8-和14-氧化反应。此外，14，15-白三烯A4的形成是从其由等量的(8R，15S)-和(8S，15S)-dihydroxy-5Z，9E，11E，13E-eicosatetraenoi c酸以及少量的(14R，15S)-赤霉酸和(14S，15S)-threo-dihydroxy-5Z，8Z，10E，12E-eicosate曲烯酸组成的特征模式推断的。因此，用猪白细胞12-脂氧合酶的均一制剂证明了脂氧合酶和白三烯合成酶的活性。
Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.