An improved method to determine serine palmitoyltransferase activity
AUTOR(ES)
Rütti, Markus F.
FONTE
American Society for Biochemistry and Molecular Biology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2681407Documentos Relacionados
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