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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D900001-JLR200 on January 29, 2009

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Journal of Lipid Research, Vol. 50, 1237-1244, June 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

An improved method to determine serine palmitoyltransferase activity

Markus F. Rütti, Stéphane Richard, Anke Penno, Arnold von Eckardstein and Thorsten Hornemann1

Institute for Clinical Chemistry, University Hospital Zurich, CH-8091 Zurich, Switzerland

This work was supported by the Hartmann Müller Foundation, the Herzog-Egli Foundation, the EMDO Foundation, the Foundation for Scientific Research (University of Zurich), and the 6th Frame-work Programme of the European Commission (LSHM-CT-2006-037631).

Published, JLR Papers in Press, January 29, 2009.

1 To whom correspondence should be addressed. e-mail: thorsten.hornemann{at}usz.ch

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.

Supplementary key words sphingolipids • high-performance liquid chromatography • acyl-CoA thioesterases

Abbreviations: 3KDS, 3-ketodihydrosphingosine; LOD, limit of detection; OPA, ortho-phthalaldehyde; SML, sucrose monolaurate; SPT, serine palmitoyltransferase


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