Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Enzymatic Assays 343
2.8. CaM Kinase II Assay
1. A scintillation counter, water bath, P81 paper and purified CaM as in Subheading
2.7.
2. Caldesmon substrate copurified with CaM kinase II (1–2 mg/mL). Stable for
years at –80°C.
3. 50 mM Tris-HCl (pH 7.5), 20 mM MgCl 2, 0.4 mM CaCl 2, 0.2% (v/v) Tween-80.
4. [γ- 32 P]ATP stock solution of 2 mM with a specific activity of approx 300 cpm/pmol.
3. Methods
3.1. Phosphodiesterase Assay
We have previously shown that CaM-dependent PDE will hydrolyze
2'-methylanthraniloyl-cyclic GMP (Mant-c-GMP) to 3'-Mant-GMP resulting
in a twofold decrease in its fluorescence (15). If Mant-c-GMP fluorescence is
followed as a function of time, it provides a continuous assay for PDE activity.
Figure 1 shows the CaM- dependent activation of PDE as monitored by the
decrease in Mant-c-GMP fluorescence.
To conduct this assay:
1. Add 8 µM Mant-c-GMP to 11 mL of 10 mM MOPS, 200 µM EGTA, 90 mM KCl,
0.5 mM CaCl 2, 5 mM MgCl 2 (pH 7.0) in a plastic tube. This is enough for 10
assays, using a different [CaM] in each 1-mL assay.
2. Mix the solution by inverting 4–5 times and let it equilibrate to the desired assay
temperature.
3. Place 1 mL of the solution in a quartz cuvet and place it in the fluorimeter.
4. Set the excitation wavelength to 280 nm and the emission wavelength to 450 nm.
5. Adjust the sensitivity so that the Mant-c-GMP emission is reading approx
80–90% of full scale.
6. Record the fluorescence intensity continuously for 1–2 min, being sure the fluorescence
intensity is stable (as in Fig. 1).
7. Add approx 15 nM of purified PDE and mix rapidly (see Note 1). Record the
fluorescence intensity with time for approx 2 min. The fluorescence should begin
to decrease slowly because of basal PDE activity (as shown in Fig. 1).
8. Add CaM or CaM mutant at the desired concentration. Mix rapidly and continue
following the time-dependent decrease in Mant-c-GMP fluorescence. Figure 1
shows the rate of decrease in Mant-c-GMP fluorescence when 0, 5, 7.5, 10, 15, or
50 nM CaM were added to 1 mL of the buffer + PDE solution aforementioned. As
[CaM] increases, the reaction rate increases until saturation occurs.
9. The rate of decrease in Mant-c-GMP fluorescence, at any [CaM], gives the extent
of PDE activation. This CaM-dependent activation of PDE can be expressed
as fold activation by dividing the rate of the fluorescence decrease in the presence
of CaM by the rate in the absence of CaM. In Fig. 1, 50 nM CaM produces
a 50-fold activation of PDE. Alternatively, the rate in the presence of maximal
[CaM] can be defined as 100% activation and the rate in the absence of CaM as