Martin T W
Department of Pathology, St. Louis University School of Medicine, MO 63104.
Biochim Biophys Acta. 1988 Oct 14;962(3):282-96. doi: 10.1016/0005-2760(88)90258-5.
The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.
在含有0.05% Triton X - 100和2 mM EDTA的条件下，研究了超声处理的内皮细胞及亚细胞组分中磷脂酰胆碱（PC）向二酰基甘油（DAG）的转化。DAG的形成主要发生在以15,000 x g离心沉淀的细胞器组分中。在与外源性1 - 油酰基 - 2 - [3H]油酰基 - PC（sn - 2 - [3H]DOPC）和磷脂酰[3H]胆碱（[胆碱 - 3H]PC）的平行反应中，[3H]DAG通过一种反应途径形成，其中[3H]胆碱是[胆碱 - 3H]PC衍生的唯一产物。[3H]胆碱并非由[3H]甘油磷酸胆碱或[3H]磷酸胆碱二次形成。在与sn - 2 - [3H]DOPC的短时间孵育反应中，分离出少量的[3H]磷脂酸（[3H]PA），并证实存在大量的PA磷酸酶活性。综合这些数据，支持了DAG形成的磷脂酶D - PA磷酸酶途径。动力学数据表明，在与sn - 2 - [3H]DOPC的反应中形成的[3H]PA / [3H]DAG比例较低，是由于PA磷酸酶的Vmax高15倍且表观Km低7倍。通过添加未标记的PA或用Triton X - 100选择性提取磷脂酶D，可提高[3H]PA / [3H]DAG产物比例。磷脂酶D的特性表明其是一种独特酶。在EDTA存在下活性最佳，且几乎完全依赖于Triton X - 100。pH曲线在7.0处出现峰值。特别重要的是其严格的底物特异性。磷脂酰肌醇不被水解，对磷脂酰乙醇胺和鞘磷脂的活性比对PC的活性低至多30至50倍。在多种大鼠组织和其他细胞中鉴定出了磷脂酶D和PA磷酸酶。磷脂酶D在肺和内皮细胞中的活性最高。通过Triton X - 100提取和阴离子交换色谱法从大鼠肺中部分纯化了磷脂酶D。当与PA磷酸酶相连时，磷脂酶D可启动一条对PC具有高度特异性的DAG形成途径。
