Submitted on May 2, 2007
Revised on September 10, 2007
Accepted on September 12, 2007
A rapid fluorescent assay for sphingosine-1-phosphate lyase enzyme activity
Padmavathi Bandhuvula, Henrik Fyrst, and Julie D. Saba
Research, Children's Hospital Oakland, Oakland, CA 94609
Corresponding Author: jsaba{at}chori.org
S1P lyase (SPL) catalyzes the conversion of sphingosine-1-phosphate (S1P) to ethanolamine phosphate and hexadecenal. The 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 employed for quantifying SPL activity is suboptimal. We have devised an assay utilizing a commercially available NBD omega-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following formation of NBD-aldehyde product, which is isolated from un-reacted 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 minutes and total protein concentrations of 20-200 mg/L. The sensitivity of the fluorescent assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 picomoles/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescent methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. The method is suitable for quantifying SPL activity in a variety of cell and tissue sources.
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