Figure 3. Microtubule stimulated ATPase activity is inhibited by a phosphomimetic mutation.

a) Measurement of basal ATPase activity of 250 nM p60 and p60^S131E with increasing ATP concentration. b) Measurement of ATPase activity of 250 nM p60 and p60^S131E in response to increasing microtubule concentration and 1mM ATP, corrected for basal activity. ATPase activity was determined using an enzyme-coupled assay as previously described except that reactions were in 20 mM HEPES pH 7.7, 25 mM potassium glutamate, 10% glycerol, 0.02% Triton-X100, 10 mM MgCl₂²⁸. Data was fit to the Michaelis-Menton equation and estimates of the error in fitted parameters was determined by monte carlo sampling of data within the experimental error and fitting each resulting dataset²⁷. The maximum likelihood mean±SD was then determined from 200 individual fits within the experimental error. c) ATPase activity of p60^S131E relative to wild-type at 1mM ATP and 5 μM microtubules with propogated error. Data for basal and 250nM are the same as in part a and b, respectively. All data is from at least 3 independent experiments with two separate preparations of each protein.