Characterization of calelectrin, a Ca2+-binding protein isolated from the electric organ of Torpedo marmorata.
We report a fast (less than 1 day) and efficient (2-3 mg protein/100 g tissue) isolation method for calelectrin, a protein of Mr 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca2+. Purified protein was used to investigate the nature of its interaction with membranes and with Ca2+. Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca2+-dependent and -specific manner with half-maximal binding between 3 and 7 microM free Ca2+. This binding is totally inhibited by 1 mM mercaptoethanol. It is also shown that calelectrin directly binds Ca2+ in solution by two techniques: at 1 and 10 microM Ca2+ it binds 45Ca2+ as measured by gel permeation chromatography, and it contains saturable Tb3+-binding sites that are Ca2+-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 Ca2+. Ca2+-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 Ca2+ 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 Ca2+. We conclude that calelectrin is a Ca2+-binding protein whose binding to the lipid moieties of membranes is regulated by physiological change in the Ca2+ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Südhof
T C
TC
Walker
J H
JH
Fritsche
U
U
D000818:Animals; D017302:Annexins; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000032:analysis*; D005456:Fluorescent Dyes; D008081:Liposomes / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D013050:Spectrometry, Fluorescence; D014101:Torpedo
D017302:Annexins; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D008081:Liposomes; D009419:Nerve Tissue Proteins; D008623:Mercaptoethanol; D002118:Calcium