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

(1) n -甲酰基met -亮氨酸（fMLP）、白三烯B_4和血小板活化因子等化学引诱剂可刺激兔腹膜中性粒细胞的磷脂酶D（PLD）活性和酶释放。在乙醇存在的情况下，PLD对这些化学引诱剂的酶释放和磷脂酸产生受到抑制。然而，前者的抑制程度小于后者。这些结果表明，PLD激活至少部分参与了中性粒细胞的酶释放(2)。蛋白激酶C（PKC）抑制剂、staurosporine和H-7可以增强fMLP对兔中性粒细胞PLD的激活作用。PMA对PKC的激活抑制了fMLP、NaF和离子霉素对PLD的激活。在PMA治疗前，施陶孢素可消除PMA的抑制作用。这些结果表明，在兔中性粒细胞中PKC负调控PLD活性。中性粒细胞的fMLP和离子霉素激活PLD绝对需要Ca^<2+>。钙调素（CaM）和肌球蛋白轻链激酶（MLCK）、W-7和ML-7抑制剂分别抑制了这些刺激的激活。这些结果表明，fMLP刺激细胞外Ca^<2+>内流激活CaM/MLCK通路，从而激活PLD(4)。我们已经发表了0.1- 0.2%的Triton X- 100可以激活大鼠和家兔脑膜的PLD，更高浓度（0.5- 1%）的Triton X- 100可以溶解膜上70%以上的PLD活性。这些结果表明Triton X- 100可用于从膜中纯化PLD。然而，在Triton X-100增溶后2-3天，PLD完全失活。因此，有必要在PLD稳定的条件下纯化酶。

(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.