Specific inhibition of rat brain phospholipase D by lysophospholipids

Although the importance of phospholipase D (PLD) in signal transduction in mammalian cells is well documented, the negative regulation of PLD is poorl y understood, This is primarily due to a lack of known specific inhibitors of PLD, We herein report that the activity of partially purified rat brain PLD is inhibited by certain lysophospholipids, such as lysophosphatidylinos itol, lysophosphatidylglycerol, and lysophosphatidylserine in a highly spec ific manner Inhibition of PLD by lysophospholipids was dose-dependent: the concentration of lysophosphatidylinositol required for half-maximal inhibit ion was about 3 mu M. An anal-sis of the enzyme-kinetics suggested that lys ophospholipids act as non-competitive inhibitors of PLD activity. As expect ed, PLD activity was stimulated by ADP-ribosylation factor (Arf) and phosph atidylinositol 4,5-bisphosphate (PIP2). The inhibition of PLD by lysophosph olipids, however, was not affected by the presence or absence of Arf or by an increase in PIPE concentration. A protein-binding assay suggested that l ysophospholipids bind directly to PLD, These results indicate that the obse rved inhibition of PLD by lysophospholipids is due to their direct interact ion rather than to an interaction between lysophospholipids and either Arf or PIP2. The present study suggests that certain lysophospholipids are spec ific inhibitors of rat brain PLD in a cell-free system and may provide the new opportunities to investigate mechanisms by which PLD is regulated by ly sophospholipids, presumably liberated by phospholipase A(2) activation, in mammalian cells.