INHIBITION OF MACROPHAGE CA2+-INDEPENDENT PHOSPHOLIPASE A(2) BY BROMOENOL LACTONE AND TRIFLUOROMETHYL KETONES
INHIBITION OF MACROPHAGE CA2+-INDEPENDENT PHOSPHOLIPASE A(2) BY BROMOENOL LACTONE AND TRIFLUOROMETHYL KETONES
复制标题
溴烯醇内酯和三氟甲基酮对巨噬细胞 CA2+-独立磷脂酶 A(2) 的抑制
DOI：
10.1074/jbc.270.1.445
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发表时间：
1995-01-06
影响因子：
4.8
通讯作者：
DENNIS, EA
中科院分区：
文献类型：
--
作者：
ACKERMANN, EJ;CONDEFRIEBOES, K;DENNIS, EA
最近从鼠巨噬细胞样细胞系P388 D（1）中纯化了一种新的不依赖Ca 2+的磷脂酶A（2）（PLA（2））（Ackermann，E. J.，Kempner，E.美国，和Dennis，E. A.（1994）J.Biol.Chem.269，9227-9233）。目前已证实，这种酶可被棕榈酰三氟甲基酮（PACOCF（3））、花生四烯酰三氟甲基酮（AACOCF（3））和溴烯醇内酯（EEL）抑制。发现PACOCF（3）和AACOCF（3）均以浓度依赖性方式抑制巨噬细胞PLA（2），发现PACOCF（3）的效力比AACOCF（3）高4倍，IC 50值分别为3.8 μ M（0.0075摩尔分数）和15 μ M（0.028摩尔分数）。在任一抑制剂存在下的反应进程曲线被发现是线性的，并且PACOCF（3）.PLA（2）复合物在稀释时迅速解离。BEL也被发现以浓度依赖性方式抑制巨噬细胞PLA（2），在40 ℃预孵育5分钟后在60 nM处观察到半最大抑制，在将酶广泛稀释到测定缓冲液中后抑制没有逆转，用EEL处理PLA（2）导致线性的、时间依赖性的活性失活，并且在PACOCF（3）存在下，这种失活的速率降低。此外，用[H-3]BEL处理的PLA（2）导致在M（r）80，000处的主带的共价标记。在用[H-3]BEL处理之前用5，5 '-二硫代双（2-硝基苯甲酸）灭活PLA（2）导致几乎完全缺乏标记，这与酶的共价不可逆自杀抑制一致。用[H-3]BEL处理后，标记的M（r）80，000条带而不是M（r）40，000条带将巨噬细胞Ca 2+非依赖性PLA（2）与先前鉴定的心肌Ca 2+非依赖性PLA（2）区分开来，并为M（r）80，000蛋白是催化亚基提供了强有力的证据。
A novel Ca2+-independent phospholipase A(2) (PLA(2)) has recently been purified from the murine macrophage-like cell line P388D(1) (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF(3)), arachidonyl trifluoromethyl ketone (AACOCF(3)), and a bromoenol lactone (EEL). Both PACOCF(3) and AACOCF(3) were found to inhibit the macrophage PLA(2) in a concentration-dependent manner, PACOCF(3) was found to be similar to 4-fold more potent than AACOCF(3), with IC50 values of 3.8 mu M (0.0075 mol fraction) and 15 mu M (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF(3).PLA(2) complex rapidly dissociated upon dilution.BEL was also found to inhibit the macrophage PLA(2) in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40 degrees C, Inhibition was not reversed after extensive dilution of the enzyme into assay buffer, Treatment of the PLA(2) with EEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF(3). In addition, PLA(2) treated with [H-3]BEL resulted in the covalent labeling of a major band at M(r) 80,000. Inactivation of the PLA(2) by 5,5'-dithiobis(2-nitrobenzoic acid) prior to treatment with [H-3]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a M(r) 80,000 band rather than a M(r) 40,000 band upon treatment with [H-3]BEL distinguishes the macrophage Ca2+-independent PLA(2) from a previously identified myocardial Ca2+-independent PLA(2) and provides strong evidence that the M(r) 80,000 protein is the catalytic subunit.