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Journal of Lipid Research, Vol. 41, 1532-1538, September 2000
Copyright © 2000 by Lipid Research, Inc.

Methods

A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids: application to N-acylethanolamine formation in vitro

Correspondence to: Harald S. Hansen

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 ZrOCl₂, 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., K. D. Chapman, and H. S. Hansen. A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids: application to N-acylethanolamine formation in vitro. J. Lipid Res. 2000. 41: 1532;–1538.

Supplementary key words: N-acylphosphatidylethanolamine, N-acylethanolamine, anandamide, N-acyllysophosphatidylethanolamine, glycerophospho(N-acyl)ethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, zirconyl chloride, zirconium oxide chloride

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