ATPase Assays

Tubulin (>5 mg/mL)

100 mM Mg·GTP

4 mM Taxol in DMSO

PM =	100 mM PIPES pH 6.8 2 mM EGTA 1 mM Mg2SO4

Motor protein (>95% purity; 15-20 µM)

Cuvettes (200 µL volume, 10 mm path length)

0.5 M Tris-OAc, pH 7.5

10 mM MgCl2

10 mM DTT

DDW

10 mM Mg·ATP

30 mM PEP

6 mM NADH (Make fresh in 10 mM Tris-OAc pH 7.5, 4.26 mg/mL = 6 mM)

PK/LDH (Sigma #P-0294, PK + LDH from Rabbit Muscle in 50% glycerol)

Procedure

1.	Assemble microtubules for assays.
	E.g.,	58 µL of 50 µM MTs = 50 µL 5.8 mg/mL tubulin + 0.5 µL 100 mM Mg·GTP, incubate for 30-60 min at 37°C, then add 7.5 µL 544 µM Taxol in PM (1.02 µL 4 mM Taxol + 6.5 µL PM) at 37°C.

2.	Determine protein concentration using the Bradford assay.

3.	Set up spectrophotometer to record OD340 at 10 second intervals for 300 seconds. A rate factor of 6.220 x 106 cm2 at 340 nm = molar extinction coefficient of NADH can be entered into the data collection program.

4.	Wash and invert 4 cuvettes to dry.

5.	Make Tris-MgCl2-DTT mix for all assays.






Tris-MgCl2-DTT mix = (n = # of assays + 1)

n x 20 µL 0.5 M Tris-OAc, pH 7.5 20 µL 10 mM MgCl2 20 µL 10 mM DTT 70 µL DDW




Add taxol to 10 µM (final concentration in assay = 6.5 µM).

6.

For each assay, add to cuvette in the following order: DDW = 19.5 µL – (volume of motor + MTs) 130 µL Tris-MgCl2-DTT 20 µL 10 mM Mg·ATP 20 µL 30 mM PEP in DDW 7 µL 6 mM NADH 3.5 µL PK/LDH Mix quickly with a plastic probe, then add the following: ___ µL motor ___ µL MTs (Note: the volume of motor and MTs used will vary with each assay. Click here for an example.) Mix quickly, put into spectrophometer, close chamber door and push “start” button.

7.	Collect data for 300 seconds, then remove cuvette and wash well with DDW. Invert to dry prior to next assay. Alternate use of 4 cuvettes.

8.

Determine the change in OD340 per minute after steady state has been reached, typically after the first 60 seconds. Convert to µM by dividing the change in OD340/min by 6.22 x 10-3/µM for NADH. Then divide by the motor concentration in µM. Divide by 60 s/min to convert to kcat (s-1). The control without motor or MTs is the background and should be subtracted from all other kcat values. The controls with MTs but without motor give the nucleotide hydrolysis by MTs and should be subtracted from corresponding values with motor and the same concentration of MTs. The kcat values (s-1) can be plotted vs MT concentration and the data points fit with the Michaelis-Menton equation using a program such as Kaleidagraph. The equation for the curve fit where y= kcat and x=[MTs] is: y = m1 * x / (m2 + x) m1 = Vmax and m2 = KM, MTs The initial values of m1 = 1, m2 = 1 can be entered to approximate the values of m1 and m2.

Notes