|Abstract||A method was improved to detect ('45)Ca('2+)-binding proteins in polyacrylamide gels. It was shorter, quantitative, highly reproducible, and resulted in a 40-fold enhanced sensitivity over the previous method. The technique could detect proteins in polyacrylamide gels with different modes of Ca('2+) binding and different dissociation constants. It could also detect EF hand containing proteins in polyacrylamide gels in the nanogram range. All major isoforms of actin were observed to bind ('45)Ca('2+). Sequence analysis revealed the presence of an highly conserved region in all actins that contained a variant EF hand. This variant EF hand was probably the calcium-binding site, possibly involved in inhibiting actin nucleation. Both (alpha) and (beta) tubulin were observed to bind ('45)Ca('2+). Sequence analysis revealed the presence of variant EF hands. Like the actins, this variant EF hand was probably the calcium-binding site, possibly involved in inhibition of tubulin nucleation. Proteins from each of the 5 classes of intermediate filaments bound ('45)Ca('2+). Sequence analysis revealed the presence of variant EF hands in the intermediate filament proteins of known sequence: vimentin, desmin, keratin, and glial fibrillary acidic protein. These variant EF hands were probably the calcium-binding sites, suggesting a regulatory function for calcium. Whole brain and synaptic subfractions from chick were examined for ('45)Ca('2+)-binding proteins. To ensure isolation of intact synaptosomes for ('45)Ca('2+) binding, an examination of the ultrastructure of chick synaptosomes was made. They were found to be resistant to lower osmotic pressure suggesting synaptosomal species differences. ('45)Ca('2+) analyses of whole brain subfractions (cerebra, cerebella, nuclei, microsomes, brain cytoplasm, myelin, extrasynaptosomal mitchondria) in polyacrylamide gels revealed several ('45)Ca('2+)-binding proteins. Many previously unreported proteins were observed. Synaptic subfractions (synaptosomes, synaptic vesicles, intrasynaptosomal mitochondria, presynaptic membranes, presynaptic cytoplasm) revealed several ('45)Ca('2+)-binding proteins. The majority of ('45)Ca('2+)-binding proteins were found in the presynaptic cytoplasm. Two-dimensional gel electrophoresis and ('45)Ca('2+) binding of this subfraction revealed 14 proteins other than calmodulin which could be triggers for neurotransmitter release.