Pollard, T.D. (1982) Methods in Cell Biology 24, 333-371
Needed Items:
V -32P-ATP, 50,000 dpm/assay 1 uCi = 2.2 x 106 dpm
cold ATP
100 mg/ml Norit A in 1 N HCl, 10 ml
Beckman microfuge or equivalent @ 4 C
ice
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)
pH 7.6
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.
