人多形核白细胞中花生四烯酸5-脂氧合酶和亚油酸ω-6脂氧合酶（花生四烯酸15-脂氧合酶）的表征与分离

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes.

作者信息

Soberman R J, Harper T W, Betteridge D, Lewis R A, Austen K F

出版信息

J Biol Chem. 1985 Apr 10;260(7):4508-15.

PMID:3920219

Abstract

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.

摘要

用60%硫酸铵沉淀的人多形核白细胞胞质部分，能从[14C]花生四烯酸产生5-脂氧合酶产物，从[14C]亚油酸以及在较小程度上从[14C]-和[3H]花生四烯酸产生ω-6脂氧合酶产物。通过薄层层析放射自显影法和反相高效液相色谱法（RP-HPLC）鉴定了源自[14C]花生四烯酸的花生四烯酰基5-脂氧合酶产物5-氢过氧-6,8,11,14-二十碳四烯酸（5-HPETE）和5-羟基-6,8,11,14-二十碳四烯酸（5-HETE），以及源自[14C]亚油酸的ω-6脂氧合酶产物13-氢过氧-9,11-十八碳二烯酸（13-OOH亚油酸）和13-羟基-9,11-十八碳二烯酸（13-OH亚油酸），还有源自[14C]-和[3H]花生四烯酸并鉴定出的15-羟基过氧-5,8,11,13-二十碳四烯酸（15-HPETE）和15-羟基-5,8,11,13-二十碳四烯酸（15-HETE）。通过对从重复薄层层析板上刮下的样品进行计数以及通过测定RP-HPLC期间的积分光密度对产物进行定量。花生四烯酰基5-脂氧合酶的最适pH为7.5，在Ca2+浓度为0.05 mM时活性达到最大活性的50%；从花生四烯酸产生5-HPETE/5-HETE的Km为12.2±4.5 μM（平均值±标准差，n = 3），Vmax为2.8±0.9 nmol/min×mg蛋白质（平均值±标准差，n = 3）。ω-6亚油酸脂氧合酶的最适pH为6.5，在5 mM乙二醇双四乙酸（EGTA）存在下，Ca2+浓度为0.1 mM时活性达到最大活性的50%。当通过二乙氨基乙基葡聚糖（DEAE-Sephadex）离子交换色谱法分离花生四烯酰基5-脂氧合酶和ω-6脂氧合酶时，ω-6脂氧合酶将亚油酸转化为13-OOH/13-OH亚油酸的Km为77.2 μM，Vmax为9.5 nmol/min×mg蛋白质（平均值，n = 2），从花生四烯酸形成15-HPETE/15-HETE的Km为63.1 μM，Vmax为5.3 nmol/min×mg蛋白质（平均值，n = 2）。