Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

344 Walsh et al.
Fig. 1. CaM-dependent activation of PDE-catalyzed hydrolysis of Mant-c-GMP.
Additions of PDE and CaM were made as indicated and the experimental conditions
are described in Subheading 3. The rate of PDE-induced decrease in Mant-c-GMP
fluorescence is shown as a function of increasing concentrations of CaM (0, 5, 7.5, 10,
15, and 50 nM). A plot of% PDE activation as a function of [CaM] is also shown.
100% activation occurred at approx 20 nM CaM and represented a 50-fold increase in
the rate of hydrolysis relative to the basal rate.
0% activation. This allows one to plot the% PDE activation as a function of
increasing [CaM] as shown in Fig. 1.
10. Using this assay, the K act and V max of any CaM mutant or isoform for PDE activation
can be quickly and accurately determined (see Notes 2 and 3). In addition,
the effect of CaM inhibitors can be readily tested by determining their ability to
inhibit CaM stimulation of PDE (see Note 2).
3.2. Calcineurin Assay
When the protein phosphatase CaN is activated by CaM, it dephosphorylates
4-methylumbelliferyl phosphate (MUF) producing a large time-dependent
increase in fluorescence. Anthony et al. (16) have used MUF to develop a
continuous assay for CaN. Figure 2 shows an example of this assay and CaM
stimulation of CaN's dephosphorylation of MUF.
To conduct this assay:
1. Add 100 µM MUF to 11 mL of 50 mM Tris-HCl, 200 µM EGTA, 0.5 mM CaCl 2,
5 mM MgCl 2 (pH 7.4) in a plastic tube. This is enough for 10 assays, using different
[CaM] in each 1-mL assay.