Table I.
Kinetic parameters of Arabidopsis SphK
Phosphorylation of Sph presented in Triton X-100 micelles was measured as a function of the substrate's bulk concentration at set surface concentrations of Sph in leaf lysates. Kinetic parameters were calculated from Figure 4, C and D, according to Equation 2 of the surface dilution kinetic model depicted in Figure 3A. Apparent KsA (mm) is the dissociation constant describing the interaction of the enzyme with the mixed micelles. Apparent Vmax (picomoles per minute per milligram) is the true Vmax at an infinite mole fraction and an infinite bulk concentration of Sph. Apparent KmB (mole fraction) is the interfacial Km. Phosphorylation of Sph presented as a BSA complex was measured at various substrate concentrations in lysate, cytosolic, and membrane fractions from leaves. Apparent Vmax (picomoles per minute per milligram) and Km (μm) values were determined from the data depicted in Figures 5A and 6, A and B, using GraphPad Prism by a weighted nonlinear regression fit to the equation V = Vmax A/(Km + A), where V is the initial rate and A is the substrate concentration.
| Fraction | Vmax(app) | Km(app) | Ks(app)A | Km(app)B | |
|---|---|---|---|---|---|
| Triton X-100 | Lysate | 8.3 | - | 1.2 | 0.0052 |
| BSA | Lysate | 17.3 | 0.18 | - | - |
| BSA | Cytosol | (2.4)a | (3)a | - | - |
| BSA | Membrane | 49.6 | 0.94 | - | - |
Values in parentheses are estimates because saturation of the enzyme with Sph was not reached.