Figure 2.

Dependence of Arabidopsis SphK activity on time and protein amount. SphK activity in leaf lysates was measured with Sph (50 μm) added either as a BSA complex or as Triton X-100 mixed micelles. A, Leaf lysates (25 μg of protein) were incubated for the indicated times. B, The indicated amounts of leaf lysate protein were incubated for 30 min. When linearity and saturation were implied, a straight line and a saturation curve were fitted, respectively; R² = 0.9966 and 0.9912 for BSA and Triton X-100, respectively, in A; R² = 0.9986 and 0.9909 for BSA and Triton X-100, respectively, in B. Data are means ± se of three independent experiments. Note that, when error bars are not indicated, the se is less than the size of the symbols. Top images show representative TLCs demonstrating the formation of [³²P]S1P with increasing time (A) or amounts (B) of protein. The phosphorylated product of Sph produced by murine SphK1 was used as an authentic [³²P]S1P standard.