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  The separation and characterization of two forms of Torpedo electric organ calelectrin.

Fritsche, U., von Kieckebusche, A., Potschka, M., Whittaker, V. P., & Witzemann, V. (1988). The separation and characterization of two forms of Torpedo electric organ calelectrin. Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology, 957(1), 122-130. doi:10.1016/0167-4838(88)90164-1.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-2F4D-1 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-2F4F-E
Genre: Journal Article

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Fritsche, U.1, Author              
von Kieckebusche, A., Author
Potschka, M.2, Author              
Whittaker, V. P.1, Author              
Witzemann, V., Author
Affiliations:
1Abteilung Neurochemie, MPI for biophysical chemistry, Max Planck Society, ou_578555              
2Department of Biochemistry and Cell Biology, MPI for biophysical chemistry, Max Planck Society, ou_578618              

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 Abstract: Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ ofTorpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7–8 mg · kg−1 wet weight of tissue, that is 4–5-times greater than the original method. The calelectrin so obtained could be resolved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% β-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32 000) than the H-form (34 000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34 000 and 32 000. The addition of 2 mM Ca2+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3–5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3 nm, corresponding to an apparent Mr of 44 000. It was unaffected by changes in ionic strength, pH or Ca2+ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent Mr of 36 000. Measurements of circular dichroism indicated that 78% of the molecule was in the α-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.

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Language(s): eng - English
 Dates: 1988-11-02
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/0167-4838(88)90164-1
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Title: Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology
Source Genre: Journal
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Pages: - Volume / Issue: 957 (1) Sequence Number: - Start / End Page: 122 - 130 Identifier: -