Characterization of the Ca2+-binding properties of calgizzarin (S100C) isolated from chicken gizzard smooth muscle

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Abstract
Calgizzarin is a Ca2+-binding protein of the S100 family that has been implicated in the regulation of cytoskeletal function through its Ca2+-dependent interaction with annexin I. The Ca2+-binding properties of calgizzarin (S100C) have not previously been thoroughly characterized. Calgizzarin, therefore, was purified from chicken gizzard smooth muscle by exploiting its Ca2+-dependent interaction with the hydrophobic matrix phenyl-Sepharose and is shown by 45Ca2+ overlay to bind Ca2+ more weakly than does calmodulin. Gel filtration in the absence and presence of Ca2+ suggested a dimeric structure of calgizzarin and indicated a more compact structure in the presence of Ca2+. Flow dialysis experiments indicated that, at physiological ionic strength, calgizzarin binds two Ca2+ ions per monomer (four per native dimer), as predicted from the deduced amino acid sequence which contains two putative EF-hands, with [Ca2+]0.5 of 0.52 mM and nH of 1.4 in the absence of Mg2+ and [Ca2+]0.5 of 0.3 mM and nH of 1.2 in the presence of 10 mM MgCl2. The hydrophobic fluorescent probe 2-p-toluidinylnaphthalene-6-sulphonate was used to demonstrate Ca2+-dependent exposure of a hydrophobic site(s) in calgizzarin. This approach also indicated the ability of calgizzarin to bind Zn2+. Interestingly, the affinity of calgizzarin for Ca2+ was enhanced approximately 10-fold in the presence of the hydrophobic probe, possibly reflecting an increased affinity for Ca2+ when calgizzarin binds to a target protein. Finally, the distribution of calgizzarin among chicken tissues was examined by immunoblotting: calgizzarin was expressed at its highest levels in lung tissue, followed by smooth muscle tissues (oesophagus, large intestine, trachea, and gizzard), kidney, liver, brain, and heart; it was not detected in small intestine or skeletal muscle.Key words: calcium-binding protein, calgizzarin, S100C, smooth muscle, zinc.