A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity

• Main Authors: Padmavathi Bandhuvula, Henrik Fyrst, Julie D. Saba
• Format: Article
• Language: English
• Published: Elsevier 2007-12-01
• Series: Journal of Lipid Research
• Subjects: high-performance liquid chromatography; ω(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro-sphingosine; sphingosine-1-phosphate; dihydrosphingosine-1-phosphate
• Online Access: http://www.sciencedirect.com/science/article/pii/S002222752042927X

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available ω(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20–200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by ∼70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.