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An improved method to determine serine palmitoyltransferase activity


Rütti, M F; Richard, S; Penno, A; von Eckardstein, A; Hornemann, T (2009). An improved method to determine serine palmitoyltransferase activity. Journal of Lipid Research, 50(6):1237-1244.

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.

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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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Clinical Chemistry
Dewey Decimal Classification:610 Medicine & health
540 Chemistry
Language:English
Date:2009
Deposited On:30 Nov 2009 13:34
Last Modified:09 Aug 2017 06:39
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0022-2275
Additional Information:This research was originally published in Rütti, M F; Richard, S; Penno, A; von Eckardstein, A; Hornemann, T (2009). An improved method to determine serine palmitoyltransferase activity. Journal of Lipid Research, 50(6):1237-1244. © the American Society for Biochemistry and Molecular Biology.
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1194/jlr.D900001-JLR200
PubMed ID:19181628

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