J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.D700010-JLR200 on September 13, 2007

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Journal of Lipid Research, Vol. 48, 2769-2778, December 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

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

Padmavathi Bandhuvula1, Henrik Fyrst1 and Julie D. Saba2

Children's Hospital, Oakland Research Institute, Oakland, CA 94609

Published, JLR Papers in Press, September 13, 2007.

1 P. Bandhuvula and H. Fyrst contributed equally to this work.

2 To whom correspondence should be addressed. e-mail: jsaba{at}chori.org

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 {omega}(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.

Supplementary key words high-performance liquid chromatography • {omega}(7-nitro-2-1,3-benzoxadiazol-4-yl)-D-erythro-sphingosine • sphingosine-1-phosphate • dihydrosphingosine-1-phosphate

Abbreviations: CLAP, chymostatin, leupeptin, antipain, and pepstatin A; DHS1P, dihydrosphingosine-1-phosphate; 2,4-DNPH, 2,4-dinitrophenylhydrazine; GFP, green fluorescent protein; LCBP, long-chain base phosphate; NBD, {omega}(7-nitro-2-1,3-benzoxadiazol-4-yl)-D-erythro; S1P, sphingosine-1-phosphate; Sphk1, sphingosine kinase 1; SPL, sphingosine-1-phosphate lyase; TBAP, tetrabutylammonium-dihydrogen phosphate


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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.