A rapid radioassay for sphingosine kinase.
A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.
Vessey
Donald A
DA
Liver Study Unit, Department of Veterans Affairs Medical Center, San Francisco, CA 94121, USA. donald.vessey@med.va.gov
Kelley
M
M
Karliner
Joel S
JS
D000818:Animals; D007700:Kinetics; D008246:Lysophospholipids / Q000032:analysis; D009206:Myocardium / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D012588:Scintillation Counting; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D014316:Tritium; D000099157:Sphingosine Kinase
D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D014316:Tritium; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate