Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate.
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 omega-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 K(m) of 35 microM for BODIPY-sphingosine 1-phosphate.
Bandhuvula
Padmavathi
P
Cancer Biology Lab, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
Li
Zaiguo
Z
Bittman
Robert
R
Saba
Julie D
JD
D000446:Aldehyde-Lyases / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D001896:Boron Compounds / Q000737:chemistry*; D002384:Catalysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008246:Lysophospholipids / Q000737:chemistry*; D008722:Methods; D051379:Mice; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D013110:Sphingosine