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Journal article

An improved method to determine serine palmitoyltransferase activity.

  • 2009-02-03
Published in:
  • Journal of lipid research. - 2009
Carbon Radioisotopes
Cell Line
Chromatography, High Pressure Liquid
Dithiothreitol
Humans
Kinetics
Palmitoyl Coenzyme A
Palmitoyl-CoA Hydrolase
Recombinant Proteins
Reproducibility of Results
Sensitivity and Specificity
Serine C-Palmitoyltransferase
Sphingomonas
Substrate Specificity
English Serine palmitoyltransferase (SPT) catalyzes the condensation of l-serine and palmitoyl-CoA, which is the rate-limiting step in the de novo synthesis of sphingolipids. SPT activity is commonly measured by monitoring the incorporation of radiolabeled l-serine into 3-ketodihydrosphingosine. In this article, we introduce several adaptations of the established protocol to improve sensitivity, reproducibility, and practicability of the assay. A significant improvement of this new protocol is the possibility to measure SPT activity in total cell lysate instead of microsomes. The assay is furthermore extended by the introduction of a nonradioactive, HPLC-based detection protocol. The suggested HPLC method offers several advantages, most importantly, a 20-fold lower detection limit compared with the radioactive assay and the possibility to use an internal standard to correct for variation in the extraction.
Language
  • English
Open access status
bronze
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Persistent URL
https://sonar.rero.ch/global/documents/280206
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