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.
Publication types
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Comparative Study
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Carbon Radioisotopes
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Cell Line
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Chromatography, High Pressure Liquid / methods*
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Chromatography, High Pressure Liquid / standards
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Chromatography, High Pressure Liquid / statistics & numerical data
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Dithiothreitol / pharmacology
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Humans
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Kinetics
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Palmitoyl Coenzyme A / metabolism
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Palmitoyl-CoA Hydrolase / metabolism
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Recombinant Proteins / analysis
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Reproducibility of Results
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Sensitivity and Specificity
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Serine C-Palmitoyltransferase / analysis*
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Serine C-Palmitoyltransferase / genetics
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Serine C-Palmitoyltransferase / metabolism
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Sphingomonas / enzymology
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Sphingomonas / genetics
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Substrate Specificity
Substances
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Carbon Radioisotopes
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Recombinant Proteins
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Palmitoyl Coenzyme A
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Serine C-Palmitoyltransferase
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Palmitoyl-CoA Hydrolase
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Dithiothreitol