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A fluorescent assay for ceramide synthase activity.

Hyun Joon Kim¹, Qiao Qiao, Hamish D Toop

¹Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, 2052, NSW, Australia.

Journal of Lipid Research

|June 5, 2012

Summary

Researchers developed a new fluorescent assay to measure ceramide synthase (CerS) activity. This method is more accessible and cheaper than existing radioactive or LC-MS/MS assays, aiding sphingolipid research.

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Area of Science:

Biochemistry
Cell Biology
Lipid Metabolism

Background:

Sphingolipids, including ceramide, are crucial for cellular functions.
Ceramide is synthesized by six ceramide synthases (CerS1-6).
Current CerS assays rely on radioactive substrates or LC-MS/MS.

Purpose of the Study:

To develop a novel, accessible, and cost-effective fluorescent assay for CerS activity.
To validate the use of NBD-labeled sphinganine as a substrate for CerS.
To investigate the inhibitory effects of FTY720 on CerS activity.

Main Methods:

Development of a fluorescent assay using NBD-labeled sphinganine.
Quantification of endogenous CerS activity in cell and tissue homogenates.
Analysis of FTY720's inhibition mechanism on CerS.

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Main Results:

The fluorescent assay detects CerS activity with lower protein concentrations than radioactive assays.
NBD-sphinganine exhibits similar Michaelis-Menten kinetics to unlabeled sphinganine, confirming its suitability as a substrate.
FTY720 competitively inhibits CerS activity, dependent on its lipid tail and amine headgroup.

Conclusions:

A novel fluorescent assay for CerS activity offers an alternative to radioactive and LC-MS/MS methods.
This assay is sensitive, cost-effective, and suitable for studying CerS function and inhibition.
Findings provide insights into the mechanism of FTY720 as a CerS inhibitor.