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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-' and 12.8 s-', respectively, in the C-terminal half and 10.5 s-' and 10.8 s-', respectively, in intact calmodulin (pH 7.0, 25"C, 100 mM KCI).Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-' and 0.73 s-' respectively; tyrosine: k& 1.65 s-' and 0.61 s-' respectively). Alkylation of intact calmodulin resulted in a fourphase calcium dissociation measured with Quin 2 (k& 85.3 s-', 11.1 s-', 1.92 s-l and 0.59 s-'); 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&s 2.04 s-' and 0.53 s-' 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&s 153 s-' and 10.9 s-' respectively) similar to those observed in intact alkylated calmoddin. The rates of calcium dissociation from calmodulinmelittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (/cobs 1.12 s -I ) , biphasic in the N-terminal half (kobs 140 s-' and 26.8 s-' respectively) and triphasic in intact calmodulin (kobs 126 s-', 12.1 s-' and 1.38 s-'). 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).The calcium-binding protein calmodulin activates a vari-calmodulin-dependent activation of enzymes in vitro by reety of enzymes by facilitating calcium-dependent activation versible calcium-dependent binding to hydrophobic domains on calcium binding t...

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Calcium-independent calcium efflux from heavy sarcoplasmic reticulum (HSR) of skeletal muscle was found to be biphasic, with half-times of 2 -6 s and 200 -400 s for the first and second phase, respectively. Calcium-, AMP-and caffeine-induced calcium efflux was triphasic, with half-times of 0.05 -0.2 s, 1 -5 s and 100-400 s for the first, second and third phases, respectively. This very fast first phase is certainly due to calcium efflux via the calcium-release channel of HSR vesicles. Both ruthenium red and neomycin inhibited the first phase of the calcium-independent calcium efflux and the first phase of the calcium-, AMP-or caffeine-induced calcium efflux completely, whilst the second phase was fully inhibited by ruthenium red only and partially inhibited by neomycin at high concentrations, indicating that the second phase of calcium release also occurs via the calcium-release channel.Various models for calcium efflux through the release channel have been tested by simulation. Activation and inhibition of the channel-mediated calcium efflux from HSR cannot be explained by two states of the calciumrelease channel (open or closed), but requires the existence of at least three states. A channel with one open state and two closed states, resulting in a rapid inactivation, is the most simple model compatible with the experimental data. According to this model, activation is assumed to reduce inactivation of the channel, whilst inhibition assumes an acceleration of channel inactivation. This mechanism most likely applies to neomycin. An additional open-blocked state has to be assumed for inhibition by ruthenium red.Skeletal muscle contraction is initiated by a release of calcium from the terminal cisternae of sarcoplasmic reticulum [I -61. The calcium release occurs via the calcium-gated calcium channel of skeletal-muscle sarcoplasmic reticulum which is activated by calcium, adenine nucleotides and caffeine, and inhibited by magnesium, ruthenium red, several drugs such as local anaesthetics, amino-glycoside antibiotics and calmodulin [7 -171.The ryanodine-receptor -calcium-release channel complex has recently been purified from the terminal cisternae and is a homotetramer of four subunits with an apparent molecular mass of 0.4 MDa [18 -201. Reconstitution into planar lipid bilayers revealed a high-conductance channel as in native sarcoplasmic reticulum vesicles [19 -231. In the present study we have investigated the effect of activators (calcium, caffeine and AMP) and inhibitors (ruthenium red and neomycin) on calcium-channel-mediated calcium efflux from passively calcium loaded HSR vesicles using a rapid-filtration apparatus [24] in order to measure the fast calcium efflux in the millisecond range. Ruthenium red and neomycin were chosen as inhibitors since they are frequently used, and it was found that both drugs inhibit calciumdependent and calcium-independent calcium efflux from HSR. Several models for channel-mediated calcium efflux have been considered to explain the experimental data. It is shown that the ...