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

Sphingosine-1-phosphate (S1P) lyase (SPL) cata- lyzes 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 v(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. En- zyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after sepa- ration 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 sen- sitivity 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 hexa- decenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.—Bandhuvula, P., H. Fyrst, and J. D. Saba. A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity. J. Lipid Res. 2007. 48: 2769-2778.