ROLE OF PHOSHOLIPASE D IN SIGNAL TRANSDUCTION AND ACTIVATION MECHANISM IN RABBIT PERITONEAL NEUTROPHILS

最近有报道称，在各种类型的哺乳动物细胞中，许多激动剂通过质膜上的受体激活磷脂酶D。该酶活化的机制尚不清楚。为了研究磷脂酶D在兔腹膜中性粒细胞中的活化机制，本实验对大鼠脑制备的渗透中性粒细胞和细胞质进行了重组，并在不同条件下研究了磷脂酶D的活化作用。[1]含渗透性中性粒细胞和细胞质的重组体系中磷脂酶D的活化-用[^3H]lyso血小板活化因子在37ºC下预标记1-组微米-烷基-磷脂酰胆碱2小时后，用1 U/ml的37ºC溶链素渗透细胞20 mn，去除细胞质。将通透化的细胞在不同条件下于1%乙醇中孵育，然后测定[^3H]磷脂酰乙醇（PEt），这是磷脂酶D的明确产物。用渗透细胞重建大鼠脑制备的胞浆，GTPgammaS/Ca^<2+>/Mg-ATP显著刺激[^3H]PEt的形成。在没有胞质溶胶的情况下，没有观察到这种刺激。由于磷脂酶D位于质膜上，这些结果表明磷脂酶D的激活需要细胞质因子。[II]磷脂酶D激活因子：由于Ca^<2+>依赖性因子可能至少部分参与兔腹膜中性粒细胞的磷脂酶D激活，因此研究了钙调素（CaM）是否参与上述重组系统中的磷脂酶D激活。采用氟非那嗪固定亲和柱层析法制备了无cam细胞质溶胶。用EGTA溶液洗脱后，CaM与色谱柱结合。在不含cam的细胞质重组的渗透中性粒细胞中，GTPgammaS/Ca^<2+>/Mg-ATP对磷脂酶D的刺激程度远低于有细胞质重组的系统。当从色谱柱中提取的CaM加入到含有无CaM细胞质的重组体系中时，GTPgammaS/Ca^<2+>/Mg-ATP对磷脂酶D的激活明显增强。从牛脑细胞醇中纯化的CaM与亲和柱洗脱的CaM具有相同的效果。这些结果表明，CaM至少部分参与了兔腹膜中性粒细胞中磷脂酶D的激活。少

It has recetly been reported that, in vaious types of mammalian cells, many agonists activates phospholipase D thrugh receptors on plasma membranes. The mechanism f the enzyme activation is nt yet been clarified. To investigate the activation mechanism f phospholipase D in rabbit peritoneal neutrophls, permeabilized neutrophils and cytosol prepared from rat brain were reconstituted and the phoshlipase D activation was investigated under various conditions.[I] Phosholipase D ctivation in reconstituted system containing permeabilized neutrohils and cytosol - After prelabelng 1-omicron-alkyl-phoshatidylcholine in rabbit peritoneal neutrophils with [^3H]lyso platelet-activating factor at 37ﾟC for 2 hr, the cells were permeabilized with 1 U/ml of streptolysin Oat 37ﾟC for 20 mn to remove cytosol. The permeabilized cells were incubated in the presence of 1% ethanol under vaius conditions, then [^3H]phsphatidylethanl (PEt), an unambiguous product of phospholipase D, were determined. When cyto … More sol prepared from rat brain were reconstituted with permeabilized cells, GTPgammaS/Ca^<2+>/Mg-ATP significantly stimulted [^3H]PEt formation. The stimulation was not observed in the absence of cytosol. Since phospholipase D locates on plasma membranes, these results suggest that phospholipase D activation requires cytosolic factor(s).[II] Phospholipase D activation factor : Since it is likely that a Ca^<2+>-dependent factor is, at least in part, involved in phospholipase D activation in rabbit peritoneal neutrophils it was investigated whether or not calmodulin (CaM) is involved in phospholipase D activation in the reconstituted system described above. CaM-free cytosol was prepared by flufenazine-immobilized affinity column chromatography. CaM bound to the column as eluted with EGTA slution. In permeabilized neutrophils reconstituted with CaM-free cytosol, GTPgammaS/Ca^<2+>/Mg-ATP stimulated phospholipase D to much less extent than in the reconstitute dsystem with cytosol. When CaM euted frm the column was added to the reconstituted system with CaM-free cytosol, phospholipase D activation by GTPgammaS/Ca^<2+>/Mg-ATP was maredly augmented. The urified CaM from bovine brain cytosl had the same effect with CaM eluted from the affinity column. From these results, it is suggested that CaM ia, at least in part, involved in phospholipase D activation as a cytosolic factor in rabbit peritoneal neutrophils. Less