FIGURE 3.

Kinetic analysis of SPTLC3 activity in HEK^Cnt and HEK^SPTLC3 for palmitoyl (palm)-CoA and myristoyl (myr)-CoA. A, for palmitoyl-CoA, both cell lines showed an identical kinetic behavior. The kinetics followed a Michaelis-Menten curve up to a substrate concentration of 0.1 mm (left panel). Above that concentration activity was reduced because of substrate inhibition. The Hanes-Woolf representation (right panel) revealed an inverse linear correlation between substrate concentration and reaction velocity. B, HEK^SPTLC3 cells showed significantly increased activity with myristoyl-CoA in comparison to control cells (HEK^Cnt). Also, with myristoyl-CoA we observed a Michaelis-Menten kinetic up to 0.1 mm and substrate inhibition at higher concentrations. Maximal activity was seen at myristoyl-CoA concentrations between 0.1 and 0.125 mm. The Hanes-Woolf plot for myristoyl-CoA also showed an inverse linear correlation between substrate concentration and reaction velocity. C, V_max and K_m values were deduced from the Hanes blot and confirmed by a hyperbolic regression analysis. With palmitoyl-CoA the V_max and K_m values were identical for both cell lines, whereas V_max for myristoyl-CoA was significantly lower in control cells (HEK^Cnt) compared with the SPTLC3-expressing cells (HEK^SPTLC3). In HEK^SPTLC3 cells, the K_m for myristoyl-CoA and palmitoyl-CoA were comparable, whereas V_max for myristoyl-CoA was about 50% lower than for palmitoyl (palm)-CoA. For HEK^Cnt cells the K_m and V_max of myristoyl (myr)-CoA could not be reliably determined (n.d.) because of low enzymatic activity with myristoyl-CoA in these cells.