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A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction.

Rotem Tidhar1, Kacee Sims2, Eden Rosenfeld-Gur1

  • 1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Journal of Lipid Research
|November 5, 2014
PubMed
Summary
This summary is machine-generated.

A new assay quantifies ceramide synthase (CerS) activity using a fluorescent NBD-sphinganine substrate and solid-phase extraction. This rapid method aids in studying CerS roles in cell regulation and human diseases.

Keywords:
N-acylationceramideschromatographyenzymologyfatty acyl-CoAin vitro assaylong-chain basesphingolipids

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Mammalian ceramide synthases (CerSs) catalyze ceramide synthesis, a crucial lipid in cellular regulation and disease.
  • Each of the six CerSs exhibits specificity for fatty acyl-CoAs of distinct chain lengths.
  • Understanding CerS activity is vital due to their increasing association with human diseases.

Purpose of the Study:

  • To develop a rapid and reliable assay for measuring ceramide synthase (CerS) activity.
  • To provide a robust method for analyzing CerS function using minimal sample volumes.

Main Methods:

  • Utilized a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate for CerS activity detection.
  • Employed solid-phase extraction (SPE) C18 chromatography for efficient separation of NBD-labeled lipids.
  • Optimized the assay for minimal protein and reagent volumes, ensuring no degradation of NBD-sphinganine or NBD-ceramide.

Main Results:

  • Demonstrated a rapid and reliable method for quantifying CerS activity.
  • Showcased SPE C18 chromatography as a superior alternative to traditional TLC for lipid separation.
  • Confirmed the stability of the NBD-sphinganine substrate and NBD-ceramide product during the assay.

Conclusions:

  • The developed NBD-sphinganine-based assay provides a sensitive and efficient tool for CerS activity analysis.
  • This assay facilitates research into the roles of CerS in cellular processes and various human pathologies.
  • The method's optimization for small volumes and high reliability makes it valuable for diverse research applications.