Abstract
In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.
Highlights
In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells
PA is deacylated by a phospholipase A activity to form lysophosphatidic acid (LPA), a monoacylated form of PA, which is reconverted to PA by LPA acyltransferase
The 3 steps for the enzymatic measurement of PA are illustrated in Fig. 1: 1) Bacterial lipase hydrolyzes PA to G3P and two FAs, 2) G3P is oxidized by glycerol-3-phosphate oxidase (GPO), which generates hydrogen peroxide and dihydroxyacetone phosphate, and 3) in the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin that can be measured
Summary
Shin-ya Morita,1,* Kazumitsu Ueda,†,§ and Shuji Kitagawa* Laboratory of Pharmaceutical Technology,* Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan; and Laboratory of Cellular Biochemistry,† Division of Applied Life Sciences, Graduate School of Agriculture, and Institute for Integrated Cell-Material Sciences, § Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
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