IDENTIFICATION AND FUNCTIONAL-CHARACTERIZATION OF LEUKOTRIENE-B4 20-HYDROXYLASE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES
IDENTIFICATION AND FUNCTIONAL-CHARACTERIZATION OF LEUKOTRIENE-B4 20-HYDROXYLASE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES
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DOI：
10.1073/pnas.82.8.2292
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
AUSTEN, KF
中科院分区：
文献类型：
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作者：
SOBERMAN, RJ;HARPER, TW;AUSTEN, KF
1.5μM(5S,12R)-二羟基-6,14-顺式-8,10-反式-[3H]二十碳四烯酸(白三烯B4，LTB4)在37℃下孵育30分钟期间形成单一反应产物。 C在具有100μM NADPH和150,000×的10mM磷酸钾缓冲液(pH 7.5)中。 g 超声处理的人多形核白细胞（PMN）的上清液。反应产物在反相 HPLC [高效液相色谱] (RP-HPLC) 和 TLC 上表现出与标准 20-羟基-LTB4 (20-OH-LTB4) 相同的迁移率。当[3H]LTB4的Ω-氧化产物从Sep-Pak中洗脱、通过RP-HPLC解析并通过GC/MS[气相色谱/质谱法]分析时，其结构被确定为仅为20-OH-LTB4。 [3H]LTB4 的 20-羟化酶在其最佳 pH 值 7.5 下的 Km 为 0.22.+/-。 0.88μM（平均值±SD，n=4）并且Vmax为48±SD。 11 pmol/分钟.cntdot。蛋白质毫克（平均值.+-.SD，n = 4）。当[ 3 H]LTB4的浓度固定在1.5μM时，NADPH的Km为1.01±。 0.59μM（平均值±SD，n=3）。位置在150,000次。 LTB4 20-羟化酶的g上清液将其与肝、肺和肾微粒体的细胞色素P-450系统以及人PMN的NADPH氧化酶-细胞色素b-245系统区分开来。 LTB4 20-羟化酶是一种独特的细胞色素 P-450 或其他单加氧酶。
A single reaction product was formed during the incubation of 1.5 .mu.M (5S,12R)-dihydroxy-6,14-cis-8,10-trans-[3H]icosatetraenoic acid (leukotriene B4, LTB4) for 30 min at 37.degree. C in 10 mM potassium phosphate buffer (pH 7.5) with 100 .mu.M NADPH and the 150,000 .times. g supernatant of sonicated human polymorphonuclear leukocytes (PMN). The reaction product exhibited the same mobility on reversed-phase HPLC [high performance liquid chromatography] (RP-HPLC) and TLC as standard 20-hydroxy-LTB4 (20-OH-LTB4). When the .omega.-oxidation product of [3H]LTB4 was eluted from a Sep-Pak, resolved by RP-HPLC, and analyzed by GC/MS [gas chromatography/mass spectrometry], its structure was determined to be solely 20-OH-LTB4. The Km of the 20-hydroxylase for [3H]LTB4 at its optimal pH of 7.5 was 0.22 .+-. 0.88 .mu.M (mean .+-. SD, n = 4) and the Vmax was 48 .+-. 11 pmol/min .cntdot. mg of protein (mean .+-. SD, n = 4). When the concentration of [3H]LTB4 was fixed at 1.5 .mu.M, the Km for NADPH was 1.01 .+-. 0.59 .mu.M (mean .+-. SD, n = 3). The location in the 150,000 .times. g supernatant of the LTB4 20-hydroxylase distinguishes it from the cytochrome P-450 system of liver, lung and kidney microsomes and from the NADPH oxidase-cytochrome b-245 system of the human PMN. The LTB4 20-hydroxylase is either a unique cytochrome P-450 or other monooxygenase.