Determination of sphingosine kinase activity for cellular signaling studies.
Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The KM of sphingosine kinase for its fluorescently labeled substrate was 38 +/- 18 microM with a Vmax of 0.4 +/- 0.2 microM/min and a kcat of 3900 s-1. Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.
Lee
Katherine J
KJ
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Mwongela
Simon M
SM
Kottegoda
Sumith
S
Borland
Laura
L
Nelson
Allison R
AR
Sims
Christopher E
CE
Allbritton
Nancy L
NL
D000818:Animals; D045744:Cell Line, Tumor; D019793:Fluorescein / Q000737:chemistry; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D013997:Time Factors; D000099157:Sphingosine Kinase
D019793:Fluorescein; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate