Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure.
The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate processes, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength. The effect of 0.1 M KCl is to accelerate the slow process from 9.1 +/- 1.5 s-1 to 24 +/- 6.0 s-1 and to reduce the rate of the fast process from 650 s-1 to 240 +/- 50 s-1 at 25 degrees C. In the presence of 0.1 M KCl it was possible to determine activation parameters for the fast process: delta H# = 41 +/- 5 kJ mol-1 and delta S# = -63 +/- 17 J K-1 mol-1. These values are in good agreement with those obtained by 43Ca NMR. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the fast and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains. The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2+ dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the N-terminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.
D000634:Aminoquinolines; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000032:analysis*; D002147:Calmodulin / Q000032:analysis*; D002417:Cattle; D005456:Fluorescent Dyes; D007700:Kinetics; D008956:Models, Chemical; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000032:analysis; D011189:Potassium Chloride; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014268:Trifluoperazine; D014357:Trypsin
D000634:Aminoquinolines; D002147:Calmodulin; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D014268:Trifluoperazine; D011189:Potassium Chloride; D014357:Trypsin; C035739:Quin2; D002118:Calcium