Abstract
Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific 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 quantified 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 TLC-phosphorus 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, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.
Highlights
Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions
We developed an enzyme-based, fluorometric method for measuring PS. This new procedure can provide simple, sensitive, specific, and high-throughput quantification of PS. Using this novel enzymatic assay, we examined the relationship between cell density and PS content in HEK293 cells, and we evaluated the effect of PS synthase 1 (PSS1) overexpression on the cellular contents of PS, PC, and PE
PS measurement We developed a new method for the enzymatic measurement of PS
Summary
Shin-ya Morita,1,* Sachimi Shirakawa,† Yukiko Kobayashi,† Keiko Nakamura,† Reiko Teraoka,† Shuji Kitagawa,† and Tomohiro Terada*.
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