title: Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity.
abstract: A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.
authors.affiliation: Department of Biomedical Sciences, University of Teramo, Teramo, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Fezza
authors.forename: Valeria
authors.initials: V
authors.lastname: Gasperi
authors.forename: Cinzia
authors.initials: C
authors.lastname: Mazzei
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D063388:Endocannabinoids; D051379:Mice; D008810:Mice, Inbred C57BL; D010714:Phosphatidylethanolamines / Q000737:chemistry* / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D012588:Scintillation Counting; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; C109143:N-arachidonylphosphatidylethanolamine; D010714:Phosphatidylethanolamines; D053284:Polyunsaturated Alkamides; D010739:Phospholipase D; C078814:anandamide