A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity.
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 approximately 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.
Bandhuvula
Padmavathi
P
Children's Hospital, Oakland Research Institute, Oakland, CA 94609, USA.
Fyrst
Henrik
H
Saba
Julie D
JD
D041721:3T3-L1 Cells; D000446:Aldehyde-Lyases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002396:Catechols / Q000737:chemistry; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009942:Organometallic Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002396:Catechols; D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; C042235:neodymium pyrocatechin disulfonate; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase)