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Role of phospholipase D in signal transduction of neutrophils and activation mechanism of the enzyme

磷脂酶D在中性粒细胞信号转导中的作用及其激活机制

基本信息

批准号：
02808033
负责人：
KANAHO Yasunori
金额：
$ 0.96万
依托单位：
Gifu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

(1). Chemoattractants such as N-formyl-Met-Leu-Phe (fMLP), leukotriene B_4, and platelet-activating factor, stimulated phospholipase D(PLD) activity as well as enzyme release of rabbit peritoneal neutrophils. In the presence of ethanol, enzyme release and phosphatidic acid production by PLD in response to these chemoattractants were inhibited. However, the extent of the inhibition of the former was less than that of the latter. These results indicate that PLD activation is, at least in part, involved in the enzyme release from neutrophils.(2). PLD activation by fMLP of rabbit neutrophils was augmented by the pretintment with protein Kinase C(PKC) inhibitors, staurosporine and H-7. PKC activation by treatment with PMA inhibited PLD activation by fMLP, NaF and ionomycin. The inhibitory effect of PMA was abolished by staurosporine treatment prior to the PMA treatment. These results conclude that in rabbit neutrophils PKC rather negatively regulates PLD activity.(3). PLD activation by fMLP and ionomycin of neutrophils absolutely required Ca^<2+>. The activation by these stimuli was inhibited by inhibitors of calmodulin (CaM) and of myosin light chain kinase (MLCK), W-7 and ML-7, respectively. From these results, it is suggested that fMLP stimulates influx of extracellular Ca^<2+> to activate CaM/MLCK pathway, thereby activating PLD.(4). We have published that 0.1-0.2 % of Triton X- 1 00 activates PLD of rat and rabbit brain membranes and higher concentrations (0.5-1 %) of Triton X- 1 00 solubilizes more than 70% of PLD activity from membranes. These results indicate that Triton X- 100 may be useful to purify PLD from membranes. However, PLD was completely inactivated 2-3 days after solubilization by Triton X-100. Therefore, it is necessary to purify the enzyme under conditions that PLD is stable.

（一）.化学引诱剂如N-甲酰基-蛋氨酸-亮氨酸-苯丙氨酸（fMLP）、白三烯B_4和血小板活化因子（PAF）可刺激兔腹腔中性粒细胞磷脂酶D（PLD）活性和酶释放。在乙醇的存在下，酶的释放和磷脂酸的生产由PLD在响应这些化学引诱剂被抑制。但前者的抑制程度小于后者。这些结果表明，PLD激活，至少在一定程度上，参与从中性粒细胞的酶释放。（二）、用蛋白激酶C（PKC）抑制剂staurosporine和H-7预处理可增强兔中性粒细胞fMLP对PLD的激活作用。PMA处理的PKC激活抑制了fMLP、NaF和离子霉素对PLD的激活。PMA的抑制作用被PMA处理前的星形孢菌素处理所消除。这些结果表明，在兔中性粒细胞PKC，而不是负调控PLD活性。（三）、fMLP和离子霉素对中性粒细胞PLD的激活绝对需要Ca^<2+>。这些刺激的激活被钙调蛋白（CaM）和肌球蛋白轻链激酶（MLCK）的抑制剂，W-7和ML-7，分别抑制。从这些结果可以看出，fMLP刺激细胞外Ca^2+内流，激活CaM/MLCK通路，从而激活PLD。（四）、我们已经公开了0.1- 0.2%的Triton X-100激活大鼠和兔脑膜的PLD，并且更高浓度（0.5- 1%）的Triton X-100从膜溶解超过70%的PLD活性。这些结果表明Triton X- 100可用于从膜中纯化PLD。然而，PLD被Triton X-100溶解后2-3天完全失活。因此，有必要在PLD稳定的条件下纯化酶。