Enzymatic measurement of phosphatidylserine in cultured cells

Abstract

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fl uorometric method for measuring PS using combinations of specifi c enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 μM) and high (50-1000 μM) concentrations, respectively, and the detection limit was 5 μM (50 pmol in the reaction mixture). This assay quantifi ed PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLCphosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specifi c, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.