title: Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells.
abstract: 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.
authors.affiliation: Department of Pathology, St. Louis University School of Medicine, MO 63104.
authors.forename: T W
authors.initials: TW
authors.lastname: Martin
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis*; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000648:ultrastructure; D005989:Glycerides / Q000096:biosynthesis*; D005997:Glycerylphosphorylcholine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000378:metabolism; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010767:Phosphorylcholine / Q000378:metabolism; D011651:Pulmonary Artery; D051381:Rats
chemical_list: D004075:Diglycerides; D005989:Glycerides; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010767:Phosphorylcholine; D005997:Glycerylphosphorylcholine; D010740:Phospholipases; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; C017251:1,2-oleoylphosphatidylcholine; D002794:Choline