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Related Experiment Video

Updated: Jan 27, 2026

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe
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New fluorogenic probes for neutral and alkaline ceramidases.

Mireia Casasampere1,2, Núria Bielsa1,2, Daniel Riba1

  • 1Spanish National Research Council (CSIC), Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Department of Biological Chemistry, Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.

Journal of Lipid Research
|March 31, 2019
PubMed
Summary

New fluorogenic probes were developed to study ceramidase activity. RBM14C24:1 efficiently detects neutral ceramidase, while RBM15C18:1 is a superior substrate for alkaline ceramidase (ACER)1 and ACER2.

Keywords:
ceramidessphingolipidssubstrateumbelliferone

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Area of Science:

  • Biochemistry
  • Enzymology
  • Chemical Biology

Background:

  • Ceramidases are crucial enzymes in sphingolipid metabolism, involved in cellular signaling and disease pathogenesis.
  • Development of selective and sensitive fluorogenic substrates is essential for studying ceramidase activity.
  • Previous probes, like RBM14C12, have limitations in specificity or efficiency for certain ceramidase types.

Purpose of the Study:

  • To design and synthesize novel fluorogenic substrates for various ceramidase enzymes.
  • To evaluate the efficiency and selectivity of new probes against acid, neutral, and alkaline ceramidases.
  • To identify optimal probes for high-throughput screening of ceramidase activity.

Main Methods:

  • Synthesis of new fluorogenic ceramidase substrates based on N-acyl modification of existing probes (RBM14 series).
  • Development of a second generation of substrates (RBM15 series) using a natural diol framework.
  • Assay development for measuring ceramidase activity in cell lysates and intact cells using the novel probes.

Main Results:

  • None of the new RBM14 derivatives surpassed RBM14C12 for acid ceramidase.
  • RBM14C24:1 demonstrated efficient and selective substrate activity for recombinant human neutral ceramidase.
  • RBM15C18:1 emerged as a highly effective fluorogenic probe for measuring alkaline ceramidase (ACER)1 and ACER2 activity in a 96-well format.

Conclusions:

  • Novel fluorogenic substrates targeting neutral and alkaline ceramidases have been successfully developed.
  • RBM14C24:1 is a valuable tool for studying neutral ceramidase.
  • RBM15C18:1 represents a significant advancement for the sensitive and high-throughput detection of ACER1 and ACER2 activity.