A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids: application to n-acylethanolamine formation in vitro.
Activation of phospholipase D (PLD) is involved in a number of signal transduction pathways in eukaryotic cells. The most common method for determination of PLD activity in vitro involves incubation with a radiolabeled substrate and lipid extraction followed by thin-layer chromatography in order to separate and quantify substrate and product(s). A more rapid assay can be used when utilizing phosphatidylcholine as a substrate because one of the products, choline, is water soluble and therefore easily separated from the substrate. However, this separation principle is not applicable in evaluating N-acylphosphatidylethanolamine (NAPE)-hydrolyzing PLD activity, which produces two lipophilic products, N-acylethanolamine (NAE) and phosphatidic acid. Therefore, we developed a rapid assay for the routine detection of NAPE-hydrolyzing PLD activity. This assay is based on precipitation of radiolabeled substrate (NAPE) in the presence of ZrOCl(2), followed by quantification of radiolabeled NAE released into a methanolic supernatant. The precipitation involves a chemical reaction of the zirconyl cation with the phosphate anion. Conditions were optimized for the complete precipitation of NAPE, whereas N-acyllysophosphatidylethanolamine and glycerophospho(N-acyl)ethanolamine were precipitated at least 95%. Furthermore, this precipitation method can be extended to assays of other anionic phospholipid-hydrolyzing PLD activities by selecting an optimal pH of the precipitation solution. For example, 98;-99% precipitation of phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine was achieved.Consequently, this new assay allows for a convenient examination of PLD activities toward a variety of phospholipid substrates, and in particular allows for the analysis of NAE formation from NAPE in vitro, a feature that will facilitate a more complete biochemical characterization of this anandamide-generating enzyme.
Petersen
G
G
Department of Pharmacology, Royal Danish School of Pharmacy, DK-2100 Copenhagen, Denmark.
Chapman
K D
KD
Hansen
H S
HS
D000818:Animals; D000838:Anions / Q000302:isolation & purification; D002250:Carbon Radioisotopes; D002417:Cattle; D011232:Chemical Precipitation; D002855:Chromatography, Thin Layer / Q000379:methods; D004983:Ethanolamines / Q000032:analysis / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D019308:Palmitic Acid / Q000378:metabolism; D010740:Phospholipases / Q000032:analysis* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D015040:Zirconium
D000838:Anions; D002250:Carbon Radioisotopes; D004983:Ethanolamines; D010743:Phospholipids; D019308:Palmitic Acid; D015040:Zirconium; D010740:Phospholipases