Effect of different CaM species on the kinetics of PDE1 determined by isothermal titration calorimetry.

<p>(<i>A</i>) The plot presents the PDE1 activity (normalized for 0.04 units of enzyme) at increasing concentrations of cAMP in the absence (<i>filled triangles</i>) and presence of wild type CaM (<i>open circles</i>) or CaM(Y99D/Y138D) (<i>filled circles</i>) (3.8 μM) in a buffer containing 50 mM imidazol-HCl (pH 7.5), 200 mM NaCl, 5 mM MgCl₂, 0.4 mM EGTA, and 0.5 mM CaCl₂ using ITC as described in Materials and Methods setting the micro-calorimeter chamber 37°C. (<i>B</i>) The trace corresponds to a typical experiment showing the rate of heat produced by PDE1 over time after injections of different pulses of cAMP from which the activity of the enzyme can be derived as described in Materials and Methods. This particular trace corresponds to an experiment performed in the absence of CaM using 0.04 units of PDE1. Similar traces were obtained in other conditions tested.