PMC

Determintation of sphingosine kinase activity of cell/tissue extracts using the FlashPlate method. Total sphingosine kinase activity of U937 cells (filled square) and mouse kidney (open circle) lysates was measured as a function of sphingosine. Michaelis-Menten constants (K_m) was calculated using non-linear regression. Experimental values are averages of three independent experiments.

Comparison between the current method (“FlashPlate”) and the classical method (“TLC”) in terms of their sensitivities of S1P detection. The amounts of S1P produced in SphK1- and SphK2-catalyzed reactions were measured using both methods. Reactions were carried out for 20 min using 10 μM sphingosine and 250 μM [γ-³³P]ATP as substrates. Values are means of triplicate measurements ± SD. Black and gray bars represent TLC- and FlashPlate-based assays, respectively.

Kinetic analysis of SphK1 and SphK2 using the FlashPlate method. Activity as a function of sphingosine (panel A) or ATP (panel B) was measured with the FlashPlate method and the Michaelis-Menten constants (K_m) were calculated for each substrate using non-linear regression. In both cases, purified SphK1 (filled squares) and SphK2 (open circles) were used. SphK2 activity was measured also as a function of selective SphK2 substrate, FTY720 (panel C). Experimental values are means ± SD of three independent measurements. Fitted values are shown as a continuous line. Calculated K_m values are shown next to the corresponding plots.

Time and amount of enzyme dependence of S1P production assessed with the FlashPlate method. Values are means of triplicate measurements ± SD for SphK1 (filled squares) and SphK2 (open circles). (A) Time dependence: assays were carried out in the presence of 10 μM of sphingosine, 250 μM of ATP and SphK1 or SphK2 (2 μg of cell lysate protein) for the indicated times. (B) Amount of enzyme dependence: reactions were carried out as described above for 30 min in the presence of varying amounts of cell lysates as indicated in the figure.