An improved method to determine serine palmitoyltransferase activity |
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Authors: | Markus F R??tti St??phane Richard Anke Penno Arnold von Eckardstein and Thorsten Hornemann |
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Institution: | Institute for Clinical Chemistry, University Hospital Zurich, CH-8091 Zurich, Switzerland |
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Abstract: | 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. |
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Keywords: | sphingolipids high-performance liquid chromatography acyl-CoA thioesterases |
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