Calcium release from calmodulin and its C‐terminal or N‐terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped‐flow fluorescence with Quin 2 and intrinsic tyrosine

Abstract

Calcium dissociation from the C‐terminal and N‐terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped‐flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C‐terminal half of calmodulin, which contains only the two high‐affinity calcium‐binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s⁻¹ and 12.8 s⁻¹, respectively, in the C‐terminal half and 10.5 s⁻¹ and 10.8 s⁻¹, respectively, in intact calmodulin (pH 7.0, 25°C, 100 mM KCl). Alkylation of the C‐terminal half resulted in a biphasic calcium dissociation (Quin 2: k_obs 1.90 s⁻¹ and 0.73 s⁻¹ respectively; tyrosine: k_obs 1.65 s⁻¹ and 0.61 s⁻¹ respectively). Alkylation of intact calmodulin resulted in a fourphase calcium dissociation measured with Quin 2 (K_obs 85.3 s⁻¹, 11.1 s⁻¹, 1.92 s⁻¹ and 0.59 s⁻¹); the latter two phases are assumed to represent calcium release from high‐affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (k_obs 2.04 s⁻¹ and 0.53 s⁻¹ respectively) and the rate parameters determined in the C‐terminal half. Evidently perturbation of the calcium‐binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high‐affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N‐terminal half resulted in biphasic calcium release with rates (k_obs 153 s⁻¹ and 10.9 s⁻¹ respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin‐melittin or fragment‐melittin complexes, measured with Quin 2, were slower and monophasic in the C‐terminal half (k_obs 1.12 s⁻¹), biphasic in the N‐terminal half (K_obs 140 s⁻¹ and 26.8 s⁻¹ respectively) and triphasic in intact calmodulin (k_obs 126 s⁻¹, 12.1 s⁻¹ and 1.38 s⁻¹). Calmodulin antagonists thus increase the apparent calcium affinity of high and low‐affinity sites mainly due to a reduced calcium ‘off rate’, presumably because of conformation restrictions. Phenoxybenzamine‐alkylated calmodulin and calmodulin‐melittin complex inhibit the endogenous calcium/calmodulin‐dependent protein kinase of cardiac sarcoplasmic reticulum. The alkylated calmodulin is actually an activator with lower affinity than the unmodified calmodulin suggesting a perturbation of both the calciumbinding domains as well as the enzyme‐binding domain(s).