An improved method to determine serine palmitoyltransferase activity.
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.
Rütti
Markus F
MF
Institute for Clinical Chemistry, University Hospital Zurich, CH-8091 Zurich, Switzerland.
Richard
Stéphane
S
Penno
Anke
A
von Eckardstein
Arnold
A
Hornemann
Thorsten
T
D002250:Carbon Radioisotopes; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010170:Palmitoyl-CoA Hydrolase / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D051102:Serine C-Palmitoyltransferase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D020578:Sphingomonas / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D011994:Recombinant Proteins; D010171:Palmitoyl Coenzyme A; D051102:Serine C-Palmitoyltransferase; D010170:Palmitoyl-CoA Hydrolase; D004229:Dithiothreitol