A rapid radioassay for sphingosine kinase.

Published

Journal Article

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.

Full Text

Cited Authors

  • Vessey, DA; Kelley, M; Karliner, JS

Published Date

  • February 2005

Published In

Volume / Issue

  • 337 / 1

Start / End Page

  • 136 - 142

PubMed ID

  • 15649386

Pubmed Central ID

  • 15649386

Electronic International Standard Serial Number (EISSN)

  • 1096-0309

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1016/j.ab.2004.10.032

Language

  • eng