The separation and characterization of two forms of Torpedo electric organ calelectrin.
Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ of Torpedo 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 obtain could be resoloved 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% beta-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32,000) that 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 3nm, 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 alpha-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.
Fritsche
U
U
Abteilung Neurochemie, Max-Planck-Institut fur biophysikalische Chemie, Göttingen, FRG
von Kieckebusch
A
A
Potschka
M
M
Whittaker
V P
VP
Witzemann
V
V
D000818:Animals; D017302:Annexins; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004557:Electric Organ / Q000032:analysis*; D004586:Electrophoresis; D006860:Hydrogen Bonding; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D014101:Torpedo; D014461:Ultracentrifugation
D017302:Annexins; D002135:Calcium-Binding Proteins