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Characterization of Calelectrin, a Ca 2+ ‐Binding Protein Isolated from the Electric Organ of Torpedo marmorata
Author(s) -
Südhof Thomas C.,
Walker John H.,
Fritsche Ulrich
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb08757.x
Subject(s) - torpedo , calmodulin , egta , chemistry , affinity chromatography , membrane , fluorescence , electric organ , binding site , biochemistry , chromatography , biophysics , calcium , biology , enzyme , organic chemistry , physics , receptor , acetylcholine receptor , quantum mechanics
We report a fast (< 1 day) and efficient (2–3 mg protein/100 g tissue) isolation method for calelectrin, a protein of M r 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca 2+ . Purified protein was used to investigate the nature of its interaction with membranes and with Ca 2+ . Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca 2+ ‐dependent and ‐specific manner with half‐maximal binding between 3 and 7 μ M free Ca 2+ . This binding is totally inhibited by 1 m M mercaptoethanol. It is also shown that calelectrin directly binds Ca 2+ in solution by two techniques: at 1 and 10 μ M Ca 2+ it binds 45 Ca 2+ as measured by gel permeation chromatography, and it contains saturable Tb 3+ ‐binding sites that are Ca 2+ ‐displaceable. An investigation of the protein's endogenous fluorescence shows that although it contains both tryptophan and tyrosine, there is no change in the apparent quantum yield as a function of Ca 2+ . Ca 2+ ‐dependent hydrophobic affinity chromatography of the total soluble proteins from Torpedo electric organ shows that Torpedo calelectrin, like calmodulin and mammalian calelectrins, is specifically retained in the presence of Ca 2+ and eluted by EGTA. Calelectrin also contains high‐affinity sites for hydrophobic fluorescence probes such as N ‐phenyl‐1‐naphthylamine, 2‐CP‐toluidinylnaphthalene‐6‐sulfonic acid, and 1‐anilinonaphthalene‐8‐sulfonic acid, which again unlike calmodulin, show no changes as a function of Ca 2+ . We conclude that calelectrin is a Ca 2+ ‐binding protein whose binding to the lipid moieties of membranes is regulated by physiological changes in the Ca 2+ concentration. This binding must be due to specific mechanisms other than simple exposure of hydrophobic sites.