Identification of a Calcium‐binding, Brain Specific Protein in the Axoplasm of Squid Giant Axons

Abstract

Abstract— An acidic protein has been isolated from the optic lobes of two cephalopods, Sepia officinalis and Loligo vulgaris. The protein has been obtained in pure form by fractionation with ammonium sulphate and chromatography on DEAE‐cellulose and Sephadex G 100. Its apparent molecular weight is 13,000–15,000. Glutamic and aspartic acids account for 35 per cent of the amino acid residues. The protein binds Ca2+ ions with an apparent dissociation constant of 2·5 × 10−5 M at physiological concentrations of KCI. Antibodies have been prepared against the protein purified from Sepia officinalis. By the micro‐complement fixation technique it has been shown that the protein is highly concentrated in the nervous system of cephalopods and that the amount in the axoplasm of squid giant axons is eight to nine‐fold higher than in the optic lobes of the same animal.

Calcium Binding Protein in Squid Brain: Biochemical Similarity to the 28,000‐Mr Vitamin D‐Dependent Calcium Binding Protein (Calbindin‐D28k)

“…low-molecular-weight calcium binding proteins in squid [ 13,500-M, (Alema et al, 1973) and 17,000-M, noncalmodulin protein (Head et al, 1983)] had previously been reported, using the chelex assay, we were unable to detect these other proteins. However, we were able to detect calmodulin using the more sensitive phosphodiesterase assay.…”

“…However, we were able to detect calmodulin using the more sensitive phosphodiesterase assay. It is possible that calmodulin and the low-molecular-weight calcium binding proteins reported by Alema et al (1973) and Head et al (1983) may be present in squid brain in lower concentrations than calbindin. It is also possible that, at the salt concentrations used in the chelex assay, the affinity of calcium for these other calcium binding proteins is too low to permit their detection by the chelex assay.…”

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).…”

Movement of axoplasmic organelles on actin filaments from skeletal muscle

“…In preparations of squid axoplasm treated with CaM, we found that the movement of tubular membranous organelles on actin filaments was much more frequent and the level of organelle motility in general increased. Squid axoplasm contains CaM which may constitute as much as 1.0-2.5% of total protein in the axon [Alema et al, 1973;Head and Kaminer, 1980;Krinks et al, 19881. The stimulatory effect of CaM on organelle motile activity in bulk axoplasm has also been observed (Rivera et al, manuscript in preparation).…”