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.
Highlights
Serine palmitoyltransferase (SPT) catalyzes the condensation of L-serine and palmitoyl-CoA, which is the ratelimiting step in the de novo synthesis of sphingolipids
Other data indicate that sphingolipids are involved in the pathological aggregation of amyloid-b fragments, which is a key process in the development of Alzheimers disease [9]
The addition of DTT is considered to have a beneficial effect on SPT activity by protecting active site Cys residues from oxidation
Summary
Serine palmitoyltransferase (SPT) catalyzes the condensation of L-serine and palmitoyl-CoA, which is the ratelimiting step in the de novo synthesis of sphingolipids. We introduce several adaptations of the established protocol to improve sensitivity, reproducibility, and practicability of the assay. An improved method to determine serine palmitoyltransferase activity. Other data indicate that sphingolipids are involved in the pathological aggregation of amyloid-b fragments, which is a key process in the development of Alzheimers disease [9]. To address these issues on a biochemical level, a robust, sensitive, and reproducible SPT activity assay would be desirable. We provide a protocol for a standardized SPT activity assay to improve reproducibility and interassay comparability
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