Advanced lipid extraction method for the determination of the phospholipase D activity

Abstract

Phospholipase D is a ubiquitous enzyme that plays an important role in various lipid mediated cellular signaling pathways and produces rare phospholipids, phosphatidylethanol or phosphatidylbutanol, instead of phosphatidic acid with unique catalytic activity transphosphatidylation in the presence of primary alcohols. The reaction products, phosphatidylethanol or phosphatidylbutanol are used as markers of in vitro phospholipase D activity in many studies. For the sensitive detection of the phospholipase D products, we developed an advanced lipid extraction method that facilitates recovery of the compounds. With the new method, the activity change of phospholipase D by agonists could be detected more easily and the recovery rate was also increased. The increase of detected enzyme activity change was about double fold compared to the conventional lipid extraction method. This method provides selective force for the phospholipase D products in the extraction procedure.

STAT3 is involved in phosphatidic acid-induced Bcl-2 expression in HeLa cells

“…PA is converted into lyso-PA (LPA) by PLA2 or DAG by PA-phosphohydrolase (Yon et al, 2003). Therefore, we checked the effect of propranolol, a well known PAP inhibitor, and mepacrine, a PLA2 inhibitor, on mRNA and protein expression of Bcl-2.…”

The detection of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol in human dried blood spots

“…23,24 Previous reports in the literature utilize high-pressure liquid chromatography (HPLC) with an evaporative lightscattering detector (ELSD). [23][24][25][26][27][28] The ELSD detects all PEth isomers but has limited sensitivity when compared to a tandem mass spectrometry (MSMS) method that is focused on a single isomer. 24 A comparison of ELSD and MSMS PEth whole blood methods was reported with excellent correlation at lower concentrations but diverged as the concentrations exceeded 2000 ng mL À1 .…”