Back to Search
Start Over
An Improved Isoform-Selective Assay for Sphingosine Kinase 1 Activity.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2018; Vol. 1697, pp. 9-20. - Publication Year :
- 2018
-
Abstract
- Sphingosine kinases (SK) are the sole enzymes responsible for the production of sphingosine 1-phosphate (S1P). S1P is a signaling molecule with a plethora of targets, acting as both a second messenger intracellularly and extracellularly via a family of cell surface G-protein-coupled S1P receptors. The two sphingosine kinases, SK1 and SK2, arise from different genes and have some distinct and overlapping cellular functions that are regulated in part by differential cellular localization, developmental expression, and catalytic properties. Here, we describe an improved method for selectively detecting SK1 activity in vitro and cell lysates via the use of the zwitterionic detergent CHAPS, which effectively inhibits SK2 activity and thus allows selective analysis of SK1 activity in a range of cell samples. The assay measures the production of <superscript>32</superscript> P-labeled S1P following the addition of exogenous sphingosine and [γ <superscript>32</superscript> P]ATP. The S1P product can be purified by Bligh-Dyer solvent extraction, separated by thin layer chromatography (TLC), and the radiolabeled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 activity in tissue, cell, and recombinant protein samples.
- Subjects :
- Animals
Cholic Acids chemistry
Chromatography, Thin Layer
Detergents chemistry
Humans
Signal Transduction
Sphingosine metabolism
Lysophospholipids metabolism
Phosphotransferases (Alcohol Group Acceptor) isolation & purification
Phosphotransferases (Alcohol Group Acceptor) metabolism
Sphingosine analogs & derivatives
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 1697
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 28540558
- Full Text :
- https://doi.org/10.1007/7651_2017_41