HPLC fluorescence assay for measuring the activity of NAPE-PLD and the action of inhibitors affecting this enzyme

Abstract

N-Acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) is the major enzyme for the biosynthesis of the endocannabinoid anandamide. The role of NAPE-PLD in various physiological and pathophysiological conditions is currently under investigation. For example, the enzyme might be involved in the control of neuronal activity, embryonic development and pregnancy, and prostate cancer. We synthesized a novel NAPE-PLD substrate with a fluorogenic pyrene substituent at the N-acyl residue as tool compound for studying this enzyme. As shown by HPLC with fluorescence detection, in rat brain microsomes the substrate was transformed into the expected pyrene-labeled N-acylethanolamine (NAE), but minor amounts of three by-products could also be detected. In the presence of pan-serine hydrolase and secretory phospholipase A₂ inhibitors, the generation of these compounds, whose identity was verified using reference substances, was abolished. Based on these results, a method for determining the activity of NAPE-PLD was developed, validated, and applied to evaluate the action of known inhibitors of this enzyme. With human sperm, it was shown that the fluorescent substrate can also be used to study NAPE metabolism in intact cells.

Keywords: Fluorescence assay; HPLC; Inhibitor; NAPE-PLD; Pyrene-labeled substrate.