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 t...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2007-12-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S002222752042927X |
| _version_ | 1818731270177292288 |
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| author | Padmavathi Bandhuvula Henrik Fyrst Julie D. Saba |
| author_facet | Padmavathi Bandhuvula Henrik Fyrst Julie D. Saba |
| author_sort | Padmavathi Bandhuvula |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T23:15:00Z |
| format | Article |
| id | doaj.art-86e3719c83c54762af0097985abd62fa |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-17T23:15:00Z |
| publishDate | 2007-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-86e3719c83c54762af0097985abd62fa2022-12-21T21:29:01ZengElsevierJournal of Lipid Research0022-22752007-12-01481227692778A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activityPadmavathi Bandhuvula0Henrik Fyrst1Julie D. Saba2Children's Hospital, Oakland Research Institute, Oakland, CA 94609Children's Hospital, Oakland Research Institute, Oakland, CA 94609Children's Hospital, Oakland Research Institute, Oakland, CA 94609Sphingosine-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.http://www.sciencedirect.com/science/article/pii/S002222752042927Xhigh-performance liquid chromatographyω(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro-sphingosinesphingosine-1-phosphatedihydrosphingosine-1-phosphate |
| spellingShingle | Padmavathi Bandhuvula Henrik Fyrst Julie D. Saba A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity Journal of Lipid Research high-performance liquid chromatography ω(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro-sphingosine sphingosine-1-phosphate dihydrosphingosine-1-phosphate |
| title | A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity |
| title_full | A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity |
| title_fullStr | A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity |
| title_full_unstemmed | A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity |
| title_short | A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity |
| title_sort | rapid fluorescence assay for sphingosine 1 phosphate lyase enzyme activity |
| topic | high-performance liquid chromatography ω(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro-sphingosine sphingosine-1-phosphate dihydrosphingosine-1-phosphate |
| url | http://www.sciencedirect.com/science/article/pii/S002222752042927X |
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