Steady State ATPase Assays Coupled Enzyme System

互联网2013-09-05

1365

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 Mg₂ SO₄

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

Steady State ATPase Assays Coupled Enzyme System

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

For each assay, add to cuvette in the following order:

Mix quickly with a plastic probe, then add the following:

(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.

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 k_cat (s^-1 ). The control without motor or MTs is the background and should be subtracted from all other k_cat 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 k_cat 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= k_cat and x=[MTs] is:

The initial values of m1 = 1, m2 = 1 can be entered to approximate the values of m1 and m2.

1.	Assays are sensitive to some buffers and will not work, for example, in Tris-HCl. Avoid using phosphate buffers for ATPase assays.

The coupled enzyme ATPase assay is based on the conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase (PK) coupled to the conversion of pyruvate to lactate by lactate dehydrogenase (LDH). The latter step requires NADH which is oxidized to NAD⁺ . NADH absorbs strongly at 340 nm but NAD⁺ does not, enabling the ultilization of NADH to be followed by monitoring absorbance at 340 nm. The decrease in OD₃₄₀ can be converted into ATPase activity where 1 molecule of NADH oxidized to NAD⁺ corresponds to the production of 1 molecule of ADP by the motor ATPase.

Modified from Huang & Hackney (JBC 269, 16493-501 1994)