ATPase assays using32P-ATP
References: Pollard, T.D. (1982) Methods in Cell Biology 24, 333-371
V -32P-ATP, 50,000 dpm/assay 1 uCi = 2.2 x 106 dpm
100 mg/ml Norit A in 1 N HCl, 10 ml
Beckman microfuge or equivalent @ 4 C
F-actin, 4-5 mg/ml in F-buffer:
0.1 M CaCl2
0.1 M MgCl2
20 mM EDTA
0.5 mM DTT
Actin Activated ATPase Buffer:
50 mM KCl
5 mM MgCl2
20 mM Imidazole pH 7.2
High Salt ATPase Buffer:
0.714 M KCl (25 ml 2 M)
14.3 mM Imidazole (1 ml 1 M)
dd H2O (44 ml)
I. Assay Design:
1. Two different types of assays are used to determine myosin's ATPase activity. First, the ATPase activity of myosin in high salt is measured in the presence of EDTA, calcium, or magnesium. Myosin's ATPase is highest in EDTA, and lowest in magnesium. Second, the ATPase activity of myosin in low salt +/- F-actin is measured. If BB myosin is phosphorylated, it should have a higher ATPase activity in the presence of actin. However, phosphorylation has no effect upon the high salt ATPase activities of BB myosin.
2. High salt ATPase assays:
Total volume will be 50 uL.
Mix the following:
35 uL High Salt Atpase buffer
5 uL EDTA, Ca, or Mg
5 uL myosin
5 uL 10 mM ATP, 50,000 dpm/assay
Order of mixing is shown above; all assays should be mixed first with buffer, EDTA,Ca, or Mg, & myosin. Then, start the assays at timed intervals by adding ATP and incubating at 35o C. Prepare one assay which is stopped immediately by adding 50 uL of Norit A to determine background.
3. Stop the assay at 30 minutes by adding 50 uL of 100 mg/ml Norit A in 0.1 N HCl. Vortex 2 seconds and place on ice for 15 minutes. Vortex two more times @ 5 & 10 minutes.
4. Spin at 10,000g in the microfuge for 10 minutes @ 4oC.
5. Remove 45 uL of supernatant: mix with 0.2 ml of ddH2O in a scintillation vial: add 3 ml of scintillation cocktail. Pipet 5 uL of stock ATP in one vial to determine total counts. Count & determine % conversion. Calculate nmoles Pi released/mg/min.
6. Total conversion should be <10%. If greater hydrolysis occurs, dilute the myosin stock and repeat the assay.
II.Actin Activated ATPase Assays:
1. Skeletal muscle actin is polymerized by the addition of 50 mM KCl, 2 mM MgCl2, and ATP in 0.2 mM CaCl2, 0.2 mM mercaptoethanol and 2 mM Tris pH 8.0. Since this ionic composition is near that of the assay, no attempt is made to change buffers.
2. Myosin must be concentrated, and is assayed at a final concentration of 0.1-0.2 mg/ml. As long as myosin is >2 mg/ml, the addition of the high salt buffer in the myosin is ignored. Dilution of any assay components should be in the Actin Activated ATPase buffer.
3. Assays are performed at 25o or 35o C.
Prepare these stocks: Add volume:
a: 5 mM ATP stock/ 50,000 dpm/assay 10 uL/assay
b: 1.25 mg/ml F-actin in assay buffer 40 uL/assay
c: 1.25 mg/ml F-actin +.125 mg/ml myosin 40 uL/assay
d: 0.125 mg/ml myosin 40 uL/assay
Final volume: 50 uL/assay
4. Pipet 40 uL of F-actin, F-actin + myosin, or myosin in duplicate assays into eppendorf tubes. Start assays at defined time points by adding ATP, vortex,and place in the water bath. One assay should be performed in triplicate, and one sample stopped immediately after addition of ATP by adding 50 uL of Norit A/HCl to determine the background Pi. Since actin has a low ATPase activity, its contribution to the total Pi release should be measured and subtracted from that of myosin.
5. Incubate 30 minutes; stop assays by adding 50 uL of Norit A and proceed as described above.