TY - JOUR
T1 - A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids
T2 - Application to N-acylethanolamine formation in vitro
AU - Petersen, G.
AU - Hansen, Harald S.
AU - Chapman, K.D.
PY - 2000/1/1
Y1 - 2000/1/1
N2 - 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, 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.
AB - 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, 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.
UR - http://www.scopus.com/inward/record.url?scp=0033805809&partnerID=8YFLogxK
M3 - Journal article
AN - SCOPUS:0033805809
SN - 0022-2275
VL - 41
SP - 1532
EP - 1538
JO - Journal of Lipid Research
JF - Journal of Lipid Research
IS - 9
ER -