Open AccessThe Effect of Mg 2+ on the Ca 2+ ‐Binding Properties and Ca 2+ ‐Induced Tyrosine‐Fluorescence Changes of Calmodulin Isolated from Rabbit Skeletal Muscle
Author(s) -
KOHSE Klaus P.,
HEILMEYER Ludwig M. G.
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb06366.x
Subject(s) - calmodulin , chemistry , divalent , egta , troponin c , calcium , phosphorylase kinase , binding site , ionic strength , fluorescence , chelation , biophysics , glycogen phosphorylase , biochemistry , enzyme , inorganic chemistry , troponin , biology , psychology , physics , organic chemistry , quantum mechanics , psychiatry , myocardial infarction , aqueous solution
Calmodulin from phosphoryiase kinase (the δ subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca 2+ and Mg 2+ was studied.1 Determination of the binding of Ca 2+ to calmodulin in a buffer of low ionic strength (0.001 M) showed that it contained six binding sites for this divalent cation. 2 Employment of a buffer of high ionic strength (0.18 M) allowed two Ca 2+ /Me 2+ ‐binding sites ( K Ca2+ d = 4.0 μM), which showed Ca 2+ –Mg 2+ competition ( K Ca2+ d = 0.75 mM), to be distinguished from two Ca 2+ ‐specific binding sites ( K Ca2+ d = 40 μM). The remaining two Ca 2+ ‐binding sites are not observed under these conditions and are probably Mg 2+ ‐specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca 2+ ‐binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250 , 4628–4633]. 3 The conformational states of calmodulin are defined by Ca 2+ , Mg 2+ and salt concentrations, which can be differentiated by their Ca 2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg 2+ induces a conformation which enhances the apparent affinity for Ca 2+ . Addition of Ca 2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca 2+ by chelation with EGTA. Only additional chelation of Mg 2+ with EDTA reduces the tyrosine fluorescence intensity. 4 Comparison of the Ca 2+ ‐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 Ca 2+ binding to the holoenzyme occurs by binding to the δ subunit exclusively. 5 Ca 2+ binding and Ca 2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca 2+ and Mg 2+ ‐induced conformational changes of calmodulin.