STUDIES ON ACTIVATION MECHANISM AND STRUCTURE OF PHOSPHOLIPASE D

磷脂酶D激活机制及结构研究

基本信息

批准号：
06454652
负责人：
KANAHO Yasunori
金额：
$ 4.61万
依托单位：
TOKYO INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

[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.

[I]磷脂酶D的激活机制：低细胞量的GTP结合蛋白（小G蛋白），ADP-核糖基化因子（ARF），最近已被鉴定为磷脂酶D（PLD）活化剂。我们发现，钙结合蛋白钙调蛋白（CAM）也使PLD在链霉素蛋白蛋白蛋白O-渗透性的，细胞质缺失的兔腹膜腹膜中性粒细胞中。 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 分数。 Rhoa和ARF再次协同激活了PLD。曲线，建议RhoA的翻译后修饰（Elanylgelanyly）需要RhoA激活PLD.IN的能力。 PLD体外的蛋白质激活。几种证据表明，PLD转移到含有PIP_2和PLD底物（磷脂酰胆碱）的磷脂囊泡上，由于与PIP_2的特异性相互作用，然后被小囊泡上的小G蛋白激活，从而从高度磷脂的磷脂上进行了水解。或兔脑膜分数。然而，目前，PLD是从大鼠脑膜中部分纯化的。此外，Hommand等人从HELA细胞中克隆了ARF敏感的PLD的cDNA。我们正在研究是否存在使用ARF敏感PLD的cDNA作为探针，是否存在PLD同工酶。