Identification and quantification of calcium-binding proteins in squid axoplasm

Abstract

The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. 45Ca-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 (Mr greater than 330 and 220 X 10(3] neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 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. 125I-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.

Ca(2+)‐dependent inactivation of Ca2+ current in Aplysia neurons: kinetic studies using photolabile Ca2+ chelators.

“…ICa was represented as a uniform influx described by a Hodgkin-Huxley-type formula fitted to a typical record. Ca2" diffused inwardly with binding to the various forms of diazo-4 calculated from our model of photolysis (see Appendix) and to 0-44 mm of an immobile buffer of 4.4 fM affinity, similar to that reported in molluscs (Alema, Calissano, Rusca & Giuditta, 1973;Krinks, Klee, Pant & Gainer, 1988) and identical to singly photolysed diazo-4 to simplify calculations. Ca2" was extruded by uptake into organelles and a surface membrane pump adjusted to match experimental measurements (Smith & Zucker, 1980).…”

“…For a native buffer, we assumed an initial concentration of 1 mm, a binding constant of 108 M-l s-' and a dissociation rate of 2000 s-1, yielding a dissociation constant of 20 #M. The binding parameters match the quantity and buffering capacity of cytoplasmic buffer measured in molluscan neurons (Smith & Zucker, 1980;Krinks et al 1988); a fast binding rate is assumed by analogy with T-sites of Ca2+-binding protein in muscle (Robertson, Johnson & Potter, 1981), and because transients of unbuffered [Ca2+]i are not observed to brief Ca2+ influx, even on a millisecond time scale (Charlton, Smith & Zucker, 1982). A diffusion constant of 10-7 cm2 S-1 was assumed, appropriate for a large protein.…”

Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals

“…Comparative Southern blot analyses using all three AtCBL cDNAs as probes (Fig. 2) instead of EGTA (27). As shown in Fig.…”

“…Proteins were stained with Coomassie blue to monitor the mobility pattern of the proteins. For the 45 Ca 2ϩ -overlay assay, recombinant proteins were analyzed by SDS͞PAGE, transferred to nitrocellulose membrane, and blotted by 45 Ca 2ϩ as previously described (27).…”

Peptidyl-Prolyl Isomerase 1 Regulates Protein Phosphatase 2A-Mediated Topographic Phosphorylation of Neurofilament Proteins

“…We performed dose-dependent analysis of cyclosporine A to verify the neurofilament phosphorylation and observed no effect on perikaryal phosphorylation of NF. In previous studies using the squid giant axon system, we have shown that PP2B is abundant in the axons compared with cell bodies (Krinks et al, 1988). PP2B levels and activity are shown to be increased in AD brain (Liu et al, 2005).…”