STUDIES ON ACTIVATION MECHANISM AND STRUCTURE OF PHOSPHOLIPASE D

[I]磷脂酶D的激活机制：一种低分子量的GTP结合蛋白(小G蛋白)，ADP核糖化因子(ARF)，最近被确定为磷脂酶D(PLD)的激活剂。我们发现钙结合蛋白，钙调素(CaM)，在链溶素O通透性、胞浆耗竭的兔腹膜中性粒细胞中也激活PLD。此外，我们还发现，当CaM和ARF与渗透性中性粒细胞同时重组时，它们可以协同激活PLD。我们还证明了小G蛋白超家族成员RhoA可以激活ARF敏感的PLD部分纯化的大鼠脑膜部分。RhoA和ARF再次协同激活PLD。此外，RhoA激活PLD的能力需要RhoA的翻译后修饰。此外，在体外，磷脂酰肌醇4，5-二磷酸(PIP_2)被确定为激活PLD小G蛋白的辅因子。一些证据表明，由于PLD与PIP_2的特异性相互作用，PLD被转移到含有PIP_2和PLD底物(磷脂酰胆碱)的磷脂小泡上，然后被小泡上的小G蛋白激活，从而水解磷脂酰胆碱。[II)磷脂酶D的纯化及其cDNA克隆：我们试图从大鼠或兔脑膜组分中高纯度地纯化PLD。最近，Hommand等人从HeLa细胞中克隆了对ARF敏感的PLD基因。我们正在使用ARF敏感的PLD的cDNA作为探针来研究是否存在PLD同工酶。

[I] Activation mechanism of phospholipase D : A low mocelcular weight GTP-binding protein (small G protein), ADP-ribosylation factor (ARF), has recently been identified as a phospholipase D (PLD) activator. We found that the calcium-binding protein, Calmodulin (CaM), also sctivates PLD in the streptolysin O-permeabilized, cytosol-depleted rabbit peritoneal neutrophils. Furthermore, it was found that CaM and ARF synergistically activate PLD when they are simulteneously reconstituted with the permeabilized neutrophils.We also demonstrated that RhoA,which is a member of small G protein superfamily and specificaly ADP-ribosylated by Clostridium botulinum C3 exoenzyme, activates the ARF-sensitive PLD partially purified rat brain membrane fraction. RhoA and ARF again synergistically activated the PLD.Furthermore, it was suggested that the post-translational modification of RhoA (gelanylgelanylation) is required for the ability of RhoA to activate the PLD.In addition to the protein activators of PLD described above, phosphatidylinositol 4,5-bisphosphate (PIP_2) is identified as a cofactor for the small G protein activation of PLD in vitro. Several lines of evidence suggested that PLD translocates to phospholipid vesicles containing PIP_2 and the PLD substrate (phosphatidylcholine), due to the specific interaction with PIP_2, then is activated by the small G proteins on the vesicles, thereby hydrolyzing phosphatidylcholine.[II] Purification of phospholipase D and its cDNA cloning : We are trying to highly purify PLD from rat or rabbit brain membrane fraction. At present, however, PLD is partially purified from rat brain membranes.Recently, cDNA of the ARF-sensitive PLD has been cloned from HeLa cells by Hommand et al. We are investigating whether or not there exist PLD isozymes by using cDNA of the ARF-sensitive PLD as a probe.