Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

  • Bandhuvula P
  • Li Z
  • Bittman R
 et al. 
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Abstract

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an ω-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a Kmof 35 μM for BODIPY-sphingosine 1-phosphate. © 2009 Elsevier Inc. All rights reserved.

Author-supplied keywords

  • Assay
  • BODIPY
  • Fluorescent
  • NBD
  • Sphingolipid
  • Sphingosine 1-phosphate
  • Sphingosine 1-phosphate lyase

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Authors

  • Padmavathi Bandhuvula

  • Zaiguo Li

  • Robert Bittman

  • Julie D. Saba

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