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An Improved Isoform-Selective Assay for Sphingosine Kinase 1 Activity
Melissa R Pitman1, Lorena T Davies1, Stuart M Pitson2
1Molecular Signalling Laboratory, Centre for Cancer Biology, University of South Australia and SA Pathology, Frome Road, Adelaide, SA, 5000, Australia.
Methods in Molecular Biology (Clifton, N.J.)
|May 26, 2017
Summary
Researchers developed a new assay to selectively measure sphingosine kinase 1 (SK1) activity. This method uses CHAPS detergent to inhibit sphingosine kinase 2 (SK2), enabling precise SK1 analysis in various samples.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Sphingosine kinases (SK) produce sphingosine 1-phosphate (S1P), a crucial signaling lipid.
- S1P acts intracellularly and extracellularly through G-protein-coupled receptors.
- SK1 and SK2 enzymes have distinct and overlapping functions, regulated by localization and expression.
Purpose of the Study:
- To develop an improved method for selectively detecting SK1 activity.
- To enable precise analysis of SK1 in various biological samples.
Main Methods:
- Utilized the zwitterionic detergent CHAPS to selectively inhibit SK2 activity.
- Developed an assay measuring 32P-labeled S1P production from sphingosine and [γ-32P]ATP.
- Employed Bligh-Dyer extraction, thin-layer chromatography (TLC), and storage phosphor screen for quantification.
Main Results:
- The CHAPS-based assay effectively inhibits SK2, allowing selective SK1 activity measurement.
- The assay demonstrated sensitivity and reproducibility across different sample types.
- Successful detection of SK1 activity in tissue, cell, and recombinant protein samples.
Conclusions:
- A novel, selective, sensitive, and reproducible assay for SK1 activity has been established.
- This method facilitates the study of SK1 in diverse biological contexts.
- The assay provides a valuable tool for biochemical and cell biology research involving sphingolipid metabolism.
