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Published on: March 29, 2020
An improved method to determine serine palmitoyltransferase activity
Markus F Rütti1, Stéphane Richard, Anke Penno
1Institute for Clinical Chemistry, University Hospital Zurich, CH-8091 Zurich, Switzerland.
Journal of Lipid Research
|February 3, 2009
Summary
We enhanced the serine palmitoyltransferase (SPT) assay for sphingolipid synthesis. The improved method offers higher sensitivity and can be performed on cell lysates, not just microsomes.
Area of Science:
- Biochemistry
- Cell Biology
- Metabolomics
Background:
- Sphingolipids are crucial for cellular function.
- Serine palmitoyltransferase (SPT) controls sphingolipid de novo synthesis.
- Current SPT assays have limitations in sensitivity and sample requirements.
Purpose of the Study:
- To develop a more sensitive, reproducible, and practical assay for SPT activity.
- To adapt the assay for use with total cell lysates.
- To introduce a non-radioactive detection method.
Main Methods:
- Adaptation of the established radioactive L-serine incorporation assay.
- Development of a non-radioactive, High-Performance Liquid Chromatography (HPLC)-based detection protocol.
- Application of the assay to total cell lysates.
Main Results:
- The enhanced assay demonstrates improved sensitivity and reproducibility.
- SPT activity can be accurately measured in total cell lysates.
- The HPLC method achieves a 20-fold lower detection limit than the radioactive assay.
- Introduction of an internal standard enhances accuracy by correcting for extraction variability.
Conclusions:
- The optimized SPT assay provides a more robust and sensitive method for studying sphingolipid metabolism.
- The non-radioactive HPLC protocol simplifies the assay and expands its applicability.
- This improved assay facilitates research into diseases associated with sphingolipid metabolism.
