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 × g. In parallel reactions with exogenous 1-oleoyl-2-[ 3H]oleoyl-PC ( sn-2-[ 3H] DOPC) and phosphatidyl[ 3H]choline ([ choline- 3 H]PC), [ 3H]DAG was formed by a reaction pathway in which [ 3H]choline was the only product derived from [ choline- 3 H]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 V max and 7-fold lower apparent K m 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.
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Schedule a callMore From: Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism
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