Direct inhibition of in vitro PLD activity by 4-(2-aminoethyl)-benzenesulfonyl fluoride

While conducting a purification protocol of phospholipase D (PLD) from huma n granulocytes, we observed that PLD activity was inhibited by a commonly-u sed protease inhibitor cocktail. Of the six inhibitors present in the cockt ail, the serine protease inhibitor, 4-(2-aminoethyl)-benezensulfonyl fluori de (AEBSF), was found to be the sole inhibitor of PLD. AEBSF caused a loss of neutrophil and purified plant PLD activities in vitro, but not in intact cells at the concentrations used, nor did it affect the related phospholip ases A, and C, that were utilized as specificity controls. The compound AEB SNH(2), which has the fluoride replaced by an -NH2 group, failed to affect PLD activity as did other compounds structurally related to AEBSF with know n protease inhibitory capabilities. Finally, basal- and agonist-stimulated PLD activity was inhibited in phosphatidylcholine-specific anti-PLD immunop recipitates (IC50 = 75 mu M). These results suggest that AEBSF, in an effec t probably unrelated to its anti-proteolytic ability, directly interferes w ith PLD enzymatic activity, making it a significant compound to bean analyz ing the role of PLD in mammalian cell signaling. (C) 2000 Academic Press.