Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells.
Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells.
复制标题
大鼠嗜碱性白血病细胞白三烯 C4 合成酶的分离和表征。
DOI：
10.1073/pnas.82.24.8399
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发表时间：
1985
影响因子：
11.1
通讯作者：
Austen,KF
中科院分区：
文献类型：
--
作者：
Yoshimoto,T;Soberman,RJ;Lewis,RA;Austen,KF
当在谷胱甘肽存在下，将白三烯（LT）A4与超声处理的大鼠嗜碱性白血病（RBL）细胞的亚细胞级分孵育时，在105，000 X g沉淀（微粒体）中发现了产生LTC 4的酶（称为LTC 4合成酶），其比活性比超声处理物高3倍。通过反相HPLC上与合成LTC 4相同的保留时间、[3 H]谷胱甘肽掺入产物、其在放射免疫测定中的反应性及其UV吸收光谱，确认反应产物为LTC 4。相比之下，谷胱甘肽S-转移酶活性（用1-氯-2，4-二硝基苯进行比色测定）主要在105，000 X g上清液（89%）和微粒体（7%）中检出。微粒体谷胱甘肽S-转移酶和LTC 4合成酶用0.4%Triton X-102溶解，并通过DEAE-Sephacel色谱分离;前者出现在流出液中，后者在平衡缓冲液中加入0.16 M NaCl后出现在洗脱液中。可溶性微粒体谷胱甘肽S-转移酶被S-己基谷胱甘肽抑制，IC 50为36 μ M，在40 ℃下稳定5分钟，而LTC 4合成酶仅受到轻微抑制（IC50，部分纯化的LTC 4合成酶显示出1.34 +/-的比活性，0.51 nmol的LTC 4/10 min/mg蛋白质（平均值+/- SD，n = 9），代表从超声处理中纯化10倍，并催化LTA 4和谷胱甘肽产生剂量和时间依赖性LTC 4。通过Lineweaver-Burk图估计LTA 4和谷胱甘肽的表观Km值分别为5-10 μ M和3-6 mM。这些结果表明，LTA 4与谷胱甘肽结合形成LTC 4是由一种独特的微粒体酶催化的。
When leukotriene (LT) A4 was incubated with subcellular fractions of sonicated rat basophilic leukemia (RBL) cells in the presence of glutathione, the enzyme producing LTC4, designated LTC4 synthetase, was found in the 105,000 X g pellet (microsomes) with a 3-fold enrichment in specific activity over that of the sonicate. The identification of the reaction product as LTC4 was confirmed by its identical retention time on reverse-phase HPLC to that of synthetic LTC4, the incorporation of [3H]glutathione into the product, its reactivity in a radioimmunoassay, and its UV absorption spectrum. In contrast, glutathione S-transferase activity, measured spectrophotometrically with 1-chloro-2,4-dinitrobenzene, was detected predominantly in the 105,000 X g supernatant (89%) and also in the microsomes (7%). The microsomal glutathione S-transferase and LTC4 synthetase were solubilized with 0.4% Triton X-102 and separated by DEAE-Sephacel chromatography; the former appeared in the effluent and the latter in the eluate after the addition of 0.16 M NaCl to the equilibration buffer. Solubilized, microsomal glutathione S-transferase was inhibited by S-hexylglutathione with an IC50 of 36 microM and was stable at 40 degrees C for 5 min, whereas LTC4 synthetase was only slightly inhibited (IC50, 2.3 mM) by S-hexylglutathione and retained no activity after incubation at 40 degrees C for 5 min. The partially purified LTC4 synthetase showed a specific activity of 1.34 +/- 0.51 nmol of LTC4 per 10 min per mg of protein (mean +/- SD, n = 9), representing a 10-fold purification from the sonicate and catalyzed the dose- and time-dependent production of LTC4 from LTA4 and glutathione. The apparent Km values for LTA4 and glutathione were estimated by Lineweaver-Burk plots to be 5-10 microM and 3-6 mM, respectively. These results indicate that the conjugation of LTA4 with glutathione to form LTC4 is catalyzed by a unique microsomal enzyme.