The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle.
Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology*; D009132:Muscles / Q000378:metabolism*; D009994:Osmolar Concentration; D010764:Phosphorylase Kinase / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D014443:Tyrosine; D010764:Phosphorylase Kinase; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium; D002118:Calcium