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Updated: Jun 17, 2026
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Lipid Exchange Assay in Living Cells
Published on: March 21, 2025
Real-time assay method of lipid extraction activity
Toshihiko Sugiki1, Hideo Takahashi, Michiko Nagasu
1Japan Biological Informatics Consortium (JBiC), Aomi, Koto-ku, Tokyo 135-8073, Japan.
Analytical Biochemistry
|December 29, 2009
Summary
A new Surface Plasmon Resonance (SPR) assay quantifies ceramide transfer protein (CERT) activity. This method distinguishes lipid uptake from protein binding, advancing understanding of lipid metabolism disorders.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biophysics
Background:
- Intracellular lipid transfer is crucial for cellular lipid metabolism, mediated by lipid transfer proteins.
- Impaired lipid metabolism due to dysfunctional lipid transfer proteins can lead to severe health disorders.
- Current methods lack the resolution to observe key steps in protein-mediated lipid transfer, such as bilayer binding and lipid uptake.
Purpose of the Study:
- To develop a novel real-time assay for quantifying ceramide transfer protein (CERT) activity.
- To elucidate the molecular mechanisms underlying protein-mediated lipid transfer.
- To differentiate between lipid uptake and membrane binding events in lipid transfer proteins.
Main Methods:
- Utilized Surface Plasmon Resonance (SPR) to detect changes in response upon ceramide extraction by CERT.
- Developed a novel SPR-based assay for real-time, quantitative evaluation of CERT's ceramide extraction activity.
- Employed lipid bilayers with and without ceramide as ligands to specifically investigate ceramide uptake.
- Conducted mutagenesis experiments on CERT to determine the role of specific amino acid residues in lipid uptake versus bilayer binding.
Main Results:
- Demonstrated that ceramide extraction by CERT can be monitored via SPR signal changes.
- Established a novel SPR-based assay enabling quantitative assessment of CERT's ceramide extraction capabilities.
- Successfully differentiated ceramide uptake from other CERT-membrane binding events using specific lipid bilayer ligands.
- Mutagenesis studies elucidated the distinct roles of amino acid residues in either ceramide uptake or lipid bilayer interaction.
Conclusions:
- The developed SPR-based assay provides a powerful tool for studying lipid transfer proteins.
- This method allows for the separate evaluation of lipid extraction and membrane binding activities.
- Offers detailed insights into the molecular mechanisms of lipid transfer phenomena and their associated disorders.
