Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria

Presence of phospholipase D-like (PLD) activity in the intestinal mitochondria was identified using endogenous phospholipids as substrate. The enzyme had a pH optimum of 6.5, did not show trans-phosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by high-performance liquid chromatography and analysis of composition of the PA formed which gave phosphate/fatty acid ratio of 1:2. PLD-like activity was further confirmed by the formation of ethanolamine and choline as products of enzyme action. This activity was stimulated by various metal ions; when stimulated by Mg²⁺ and Ba²⁺, it hydrolyzed both phosphatidylcholine and phosphatidylethanolamine, and when stimulated by Ca²⁺, it preferentially hydrolyzed phosphatidylethanolamine. There was no requirement for sodium oleate for the PLD-like activity in mitochondria. These results suggest that intestinal mitochondria have an active PLD-like enzyme which differs in certain properties from phospholipase D from other tissues.