Figure 2.

Determination of the K_m (ATP), K_m (MT) for the minimal catalytic core of NOD (GST-NOD320): biochemical characterization of two NOD mutant proteins. (A) ATPase rate of 50 nM GST-NOD320 was measured in the presence of subsaturating levels of microtubules to determine the apparent K_m for ATP. These values were plotted by DeltaGraph and K_m values were obtained by curve fitting. The data was fit to the Michaelis-Menten kinetic model and the solid line was obtained from this curve-fitting procedure. The inset shows an SDS-PAGE gel showing molecular weight standards and the first two elutions of GST-NOD320. (B) ATPase rates of 50 nM GST-NOD320 were determined in the presence of various levels of polymerized tubulin and 0.5 mM ATP. The microtubule dimer concentration that led to half-maximum ATPase activation K_m (MT) and the k_cat were obtained by curve fitting with DeltaGraph. The solid line was obtained from the curve-fitting procedure (r = 0.97). (C) ATPase rates of various 50 nM GST-NOD fusions were measured and calculated as in B. Wild-type NOD-GST, NOD_DTW, and NOD_“DR2” are represented by filled circles, diamonds, and squares, respectively. K_m (MT) and the k_cat were obtained by curve fitting with DeltaGraph with the following r values: GST-NOD320, 0.97; NOD_DTW, 0.92; and NOD_“DR2”, 0.96.