PMID: 3755302May 1, 1986Paper

A rapid assay for activity of phospholipase A2 using radioactive substrate

Analytical Biochemistry
M KatsumataA S Goldman

Abstract

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.

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Citations

Sep 18, 1990·Biochimica Et Biophysica Acta·T YasudaK Saito
Dec 20, 2003·Bioorganic & Medicinal Chemistry Letters·Bing Bing LinHui-Ling Chen
Aug 26, 1998·Journal of Biochemical and Biophysical Methods·Y YaoC C Wang
Dec 17, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Melanie Schmitt, Matthias Lehr
Jul 28, 2012·The Journal of Surgical Research·Maw-Shung LiuYuefang Zhou
Nov 1, 1993·Biological Psychiatry·M NoponenJ Rotrosen
Feb 1, 1991·Gastroenterologia Japonica·M KitagawaY Nakae
Jun 6, 2020·Toxicon : Official Journal of the International Society on Toxinology·Kristina B M StillJeroen Kool
Aug 1, 1988·Analytical Biochemistry·C S LaiJ Joseph
May 1, 1988·Analytical Biochemistry·N H Tan, C S Tan

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