title: 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 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.
authors.affiliation: Department of Pharmacy, Shiga University of Medical Science Hospital, Otsu City, Shiga 520-2192, Japan. smorita@belle.shiga-med.ac.jp
authors.forename: Shin-ya
authors.initials: SY
authors.lastname: Morita
authors.forename: Sachimi
authors.initials: S
authors.lastname: Shirakawa
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Keiko
authors.initials: K
authors.lastname: Nakamura
authors.forename: Reiko
authors.initials: R
authors.lastname: Teraoka
authors.forename: Shuji
authors.initials: S
authors.lastname: Kitagawa
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Terada
mesh_terms: D002138:Calibration; D002855:Chromatography, Thin Layer; D005470:Fluorometry / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D019884:Nitrogenous Group Transferases / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D019884:Nitrogenous Group Transferases; C022370:phospholipid serine base exchange enzyme
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