Direct Inhibition of in Vitro PLD Activity by 4-(2-Aminoethyl)-Benzenesulfonyl Fluoride

• Published in: Biochemical and biophysical research communications Vol. 273; no. 1; pp. 302 - 311
• Main Authors: Andrews, Brooke, Bond, Kristina, Lehman, Jason A., Horn, Jeffrey M., Dugan, Amy, Gomez-Cambronero, Julia
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.06.2000
• Subjects: Brassica - cytology; Brassica - drug effects; Brassica - enzymology; Cells, Cultured; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - isolation & purification; Enzyme Inhibitors - pharmacology; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; neutrophils; Neutrophils - cytology; Neutrophils - drug effects; Neutrophils - enzymology; Phosphatidylcholines - metabolism; Phospholipase D - antagonists & inhibitors; Phospholipase D - isolation & purification; Phospholipase D - metabolism; Phospholipases A - metabolism; PLD; Precipitin Tests; protease inhibitor; signal transduction; Signal Transduction - drug effects; Substrate Specificity; Sulfones - chemistry; Sulfones - isolation & purification; Sulfones - pharmacology; Time Factors; Type C Phospholipases - metabolism; PLD; protease inhibitor; signal transduction; neutrophils
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.2938

While conducting a purification protocol of phospholipase D (PLD) from human granulocytes, we observed that PLD activity was inhibited by a commonly-used protease inhibitor cocktail. Of the six inhibitors present in the cocktail, the serine protease inhibitor, 4-(2-aminoethyl)-benezensulfonyl fluoride (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 phospholipases A2 and C, that were utilized as specificity controls. The compound AEBSNH2, which has the fluoride replaced by an -NH2 group, failed to affect PLD activity as did other compounds structurally related to AEBSF with known protease inhibitory capabilities. Finally, basal- and agonist-stimulated PLD activity was inhibited in phosphatidylcholine-specific anti-PLD immunoprecipitates (IC50 = 75 μM). These results suggest that AEBSF, in an effect probably unrelated to its anti-proteolytic ability, directly interferes with PLD enzymatic activity, making it a significant compound to begin analyzing the role of PLD in mammalian cell signaling.