Identification and quantification of calcium-binding proteins in squid axoplasm (original) (raw)

1988, The Journal of Neuroscience

The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. %a-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (M. > 330 and 220 x 1 03) neurofilament proteins, an unidentified protein band that migrated around M, 55,000, and a diverse group of proteins that migrated together around M, 17,000. The low-molecularweight (M, 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 pmollkg axoplasm), 2 other M, 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium-binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca2+/kg axoplasm. i251-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca2+/CaM protein kinase II subunits. Calcium ions are generally recognized as being involved in a wide variety of biochemical and physiological processes in cells (Rubin, 1974; Rasmussen, 198 1; Evered and Whelan, 1986). These physiological actions of calcium usually occur when intracellular (cytosolic) free calcium rises from its resting level of around 0.1 PM to between 0.2 and 1.2 PM rarely, if ever, rising to over 10 FM (Rasmussen, 198 1). Higher intracellular concentrations are known to be deleterious to cell structure and function, e.g., in axons excessive calcium is well known to induce degeneration, in part, by activation of an intracellular calciumdependent neutral protease (Schlaepfer, 1974; Eagles et al., 1988). Hence, intracellular calcium is tightly regulated, often in a highly localized fashion within the cell (