Modified From: White, 1982 Meths. Enz. 85:701

ATPase Protocol

Make 1 Liter of Buffer and Stop solutions to use in all assays for a set of experiments, generally it will last for quite a while when experimenting. Store the buffer and stop solution at room temp. Make colorizing solution at the least once a week, preferably each day and store a 4°. The 100 mM ATP solution should be stored in a -20° freezer and thawed prior to experiments. The HMM and F-Actin solutions should be stored in a 4o refrigerator and never frozen.

Materials

1. Buffer Solution (pH 7.4)
   10 mM Imidazole (pH buffer)
   2 mM MgCl 
   1 mM DTT 
   0.1 mM K-EGTA
2. Stop solution (pH 7.4)
   13.3% SDS
   0.12 M Na-EDTA
3. Colorizing Solution
   0.5 % ammonium molybdate
   0.5 M H2SO4 
   0.5 % ferrous sulfate
4. 10 mM Stock phosphate solution (From H2PO4)
5. 100 mM NaATP
6. HMM (Heavy Meromyosin)
7. F-actin

Calibration of the Spectrophotometer

Absorption should be measured at 550 nm.

A. Phosphate Standard Curve

Using a 10 mM Na-H2PO4 stock solution, make the following concentrations of solution to a .25 ml volume.

These standards will be run in duplicate. Average the results.

1. Pipette the above aliquots of phosphate solution and buffer into 5 cuvettes.
2. Add 83 1/3 µl of stop solution.
3. Add 666 2/3 ml colorizing solution into each tube (The total measured volume in each tube should be 1 ml).
4. Spectrophotometer Blank:
   .083 ml Stop Solution
   .25 ml ATPase Buffer
   .667 ml Color Solution
5. Measure and record OD in spectrophotometer at 550 nm.
6. Plot absorbance vs. [Pi] for conversion of absorbance ([Pi]) in myosin ATPase measurements.

Calibration Curve

B. HMM ATPase assay

The HMM ATPase reaction is as follows:

You will be sampling the myosin ATP reaction at 5 time points – 0, 15, 30, 45, and 60 minutes. If the amount of Pi liberated is plotted against time the slope of the relationship is the myosin ATPase rate.

1. In a one to three glass tubes (Depending on how many sample at each time point are being taken) mix a solution of 1 uM HMM (Use C1V1 = C2V2), any peptides at their desired concentrations and buffer solution. Bring the volume to 2970 uL with buffer solution. (Total Volume will be 3000 ul = 3 ml). 
   1. Note: For each solution that contains a peptide in addition to HMM, run an identical reaction with an equal amount of buffer solution in place of the peptide.
2. To start the reaction, pipette 30 μl of 100 mM ATP stock (1 mM ATP in the tube) into the tube and start timer.
3. At this point make a solution to blank the spectrophotometer. The solution is composed of:
   1. .25 ml (250 ul) Buffer
   2. .083 ml (83 ul) Stop Solution
   3. .667 ml (667 ul) Color Solution
4. While the reaction proceeds, prepare duplicate tubes for each time point by pipetting 0.083 ml stop solution into each tube.
5. Upon reaching each time point, pipette 0.25 ml reaction solution into the proper cuvettes containing the stop solution.
6. Add .667 ml colorizing solution to the stopped reaction cuvettes in the same order that you pipetted the reaction solution. Check the solutions for precipitate and allow them to sit for 15 minutes.
7. Before each reading, blank the spectrophotometer with the blanking solution.
8. After 15 minutes measure and record absorbance @ 550 nm in spectrophotometer.
9. Plot [Pi] vs time. Convert absorbance to [Pi] using the calibration curve.
   1. Note: As a control, run a tube with buffer solution in place of HMM or one with no ATP.

HMM ATPase Measurements

Actomyosin Assay

You will be sampling the myosin ATP reaction at 5 time points – 0, 3, 6, 9 and 12 minutes. When the amount of Pi liberated is plotted against time the slope of the relationship is the actomyosin ATPase rate.

1. In a one to three glass tubes (Depending on how many sample at each time point are being taken) mix a solution of 0.1 to 1.0 µM HMM (Use C1V1 = C2V2), add any peptides at their desired concentrations and finally buffer solution. Bring the volume to 2970 uL with buffer solution. (Total Volume will be 3000 ul = 3 ml after the addition of ATP).
   1. Note: The concentration ratio of myosin to actin is generally 1:50. Using a concentration of 0.1-µM myosin and 5-µM actin is recommended.
   2. Note: For each solution that contains a peptide in addition to HMM, run an identical reaction with an equal amount of buffer solution in place of the peptide.
2. At this point make a solution to blank the spectrophotometer. The solution is composed of:
   1. .25 ml (250 ul) Buffer
   2. .083 ml (83 ul) Stop Solution
   3. .667 ml (667 ul) Color Solution
3. Before adding the ATP to the reaction tubes, pipet .25 ml of each reaction solution into the first time point cuvettes. Next add .083 ml of stop solution and .667 ml of color solution to the same cuvettes (total volume of 1.0 ml) and allow it to sit for 15 minutes. This will be your zero time point. (Due to the swift ATPase activity of the actomyosin complexes this measurement is taken to ensure a low initial phosphate level)
4. To start the reactions, pipette 30 μl of 100 mM ATP stock (Makes 1 mM ATP in the tube) into the tube and start timer.
   1. Note: Once a tube of ATP has been thawed and used, throw the remaining ATP solution out. Refreezing of ATP solutions makes errors in data more likely as reused ATP tends to perform at less than optimal levels.
5. While the reaction proceeds, prepare single to triplicate tubes for each time point by pipetting 0.083 ml stop solution into each tube.
6. Upon reaching each time point, pipette 0.25 ml reaction solution into each of the cuvettes containing the stop solution.
7. Add .667 ml colorizing solution to the stopped reaction cuvettes in the same order that you pipetted the reaction solution. Check the solutions for precipitate and allow them to sit for 15 minutes.
8. Before each reading, blank the spectrophotometer with the blanking solution.
9. After a cuvette has sat for 15 minutes measure and record the absorbance @ 550 nm using the spectrophotometer.
10. Plot [Pi] vs time. Convert absorbance to [Pi] using the calibration curve.

Note: As a control, run a tube with buffer solution in place of actin and myosin or one with no ATP. Also, a tube with just HMM and ATP may be run to determine the effect non-bound HMM is having on the Assay.

Actomyosin ATPase Measurements