Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Enzymatic Assays 349
10. Wash each column with three consecutive 1-mL aliquots of water and collect the
flowthrough in 15-mL scintiverse cocktails.
11. Count the cocktails in a scintillation counter (see Note 10).
12. Determine enzyme activity from the specific activity of the radioactive substrate.
3.7. MLCK Assay
MLCK activity is absolutely dependent on Ca2+ and CaM. This assay measures
the incorporation of 32P from [γ-32P]ATP into LC20 at a fixed time following
addition of radiolabeled ATP to start the reaction. Figure 4 shows the
concentration dependence of CaM activation of MLCK.
To conduct this assay:
1. To 15 µL of buffer in a plastic 1.5-mL microfuge tube (without cap) on ice, add
LC 20 (10 µM), CaM (0–1 µM), and MLCK (0.05 µg/mL).
2. Add distilled, deionized H 2O to give a total volume of 29 µL.
3. Mix the solution well by vortexing and let it equilibrate to 30°C in the water bath.
4. Start the reaction by adding 1 µL of stock [γ- 32 P]ATP.
5. Incubate the reaction mixture at 30°C for 10 min.
6. With a pipetman, transfer 20 µL of the reaction mixture to a square (1 × 1 cm) of
P81 paper and immerse immediately in a glass 600-mL beaker containing
500 mL of 0.5% (v/v) H 3PO 4 and a stainless steel wire mesh basket. This stops
the reaction.
7. Wash the paper squares three times for 5 min each with stirring in 500 mL of
0.5% (v/v) H 3PO 4 and once for 2 min with acetone.
8. Remove the wire basket from the beaker, place on a paper towel and dry the P81
papers with a hair dryer.
9. Transfer the dried paper squares to plastic scintillation vials and quantify 32 P by
ˆ
Cerenkov counting (no scintillant or other liquid) in a scintillation counter using
3 H window settings.
10. Determine enzyme activity from the specific activity of the radiolabeled ATP.
3.8. CaM Kinase II Assay
This assay measures the incorporation of 32P from [γ-32P]ATP into
caldesmon at a fixed time following addition of radiolabeled ATP to start the
reaction. Figure 5 shows the characterization of the CaM kinase II/caldesmon
preparation: caldesmon phosphorylation is observed only in the presence of
both Ca2+ and CaM, and is completely inhibited by [Ala9]autocamtide 2, a
CaM kinase II inhibitor. Figure 6 shows the concentration dependence of CaM
activation of CaM kinase II.
To conduct this assay:
1. To 15 µL of buffer in a plastic 1.5-mL microfuge tube (without cap) on ice, add
CaM (0–10 µM) and caldesmon containing CaM kinase II (0.2 mg/mL).
2. Add distilled, deionized H 2O to give a total volume of 27 µL.