权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：一种低分子量的GTP结合蛋白（小G蛋白），ADP-核糖基化因子（ARF），最近被鉴定为磷脂酶D（PLD）激活剂。我们发现，钙结合蛋白，钙调素（CaM），也sctivates PLD在链球菌溶血素O-通透，细胞溶质耗尽兔腹膜中性粒细胞。此外，我们还发现，当CaM和ARF与透性中性粒细胞同时重组时，它们协同激活PLD，我们还证明了RhoA，这是一个小G蛋白超家族的成员，并被肉毒梭菌C3外切酶特异性地ADP-核糖基化，激活ARF敏感的PLD部分纯化的大鼠脑膜组分。RhoA和ARF再次协同激活PLD，并且RhoA的翻译后修饰（gelanylgelanylation）是RhoA激活PLD所必需的，除了上述PLD的蛋白激活剂外，磷脂酰肌醇4，5-二磷酸（PIP_2）被鉴定为PLD的小G蛋白激活的辅助因子。有证据表明，PLD与PIP_2发生特异性相互作用后，可转位到含有PIP_2的磷脂囊泡中，并被囊泡上的小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的克隆：我们试图从大鼠或兔的脑膜组分中高度纯化PLD。然而，目前PLD是从大鼠脑膜中部分纯化的，最近Hommand等从HeLa细胞中克隆了ARF敏感PLD的cDNA，我们正在用ARF敏感PLD的cDNA作为探针研究是否存在PLD同工酶。