title: Determination of sphingosine-1-phosphate lyase activity by gas chromatography coupled to electron impact mass spectrometry.
abstract: Sphingosine-1-phosphate lyase (SGPL1) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldolic cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. In this article we report on an easy and sensitive procedure to determine SPL activity. The assays uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Acquisition in selected ion monitoring mode allows very high sensitivity, with a limit of detection of 281fmol. The assay is linear with both protein concentration and incubation time up to 20μg and 40min, respectively. The K(m) value obtained (6μM) is similar to that for the natural substrate sphingosine-1-phosphate. Using this method, FTY720 and deoxypyridoxine phosphate inhibited SPL with similar potencies to those reported.
authors.affiliation: Dept. of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), Barcelona, Spain.
authors.forename: Ester
authors.initials: E
authors.lastname: Reina
authors.forename: Luz
authors.initials: L
authors.lastname: Camacho
authors.forename: Josefina
authors.initials: J
authors.lastname: Casas
authors.forename: Paul P
authors.initials: PP
authors.lastname: Van Veldhoven
authors.forename: Gemma
authors.initials: G
authors.lastname: Fabrias
mesh_terms: D000446:Aldehyde-Lyases / Q000032:analysis / Q000378:metabolism*; D000447:Aldehydes / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D008246:Lysophospholipids / Q000378:metabolism; D051379:Mice; D012680:Sensitivity and Specificity; D013107:Sphingolipids / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000447:Aldehydes; D008246:Lysophospholipids; D013107:Sphingolipids; C060506:sphingosine 1-phosphate; C003061:pentadecanal; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D013110:Sphingosine