Highly purified α actinin can be made by using the low ionic strength extraction procedure and then subjecting the crude α actinin fraction obtained with this extraction procedure to successive chromatography on DEAE cellulose and hydroxyapatite. Hydroxyapatite chromatography specifically removes a protein having a subunit molecular weight of 42,000 on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Hydroxyapatite purified α actinin sediments entirely as a 6.21 S boundary in the analytical ultracentrifuge with no trace of the small 9 to 10 S boundary seen in earlier α actinin preparations purified by DEAE cellulose chromatography. In 100 mM KCl, 20 mM Tris.acetate, pH 7.5, hydroxyapatite purified α actinin has a diffusion coefficient (Do 20,w) of 2.71 x 10 -7 cm 2.s -1, an intrinsic viscosity of 20.6 ml.g -1, a molecular weight of 201,000 ± 4,300 (plus or minus least squares standard error) as determined by sedimentation equilibrium, and a molecular weight of 210,000 as determined by sedimentation diffusion. In 6 M guanidine HCl, hydroxyapatite purified α actinin has a molecular weight of 106,000 ± 6,300 as determined by sedimentation equilibrium and a molecular weight of 100,000 as determined by a calibrated 4% agarose gel permeation column. SDS polyacrylamide gel electrophoresis gives a molecular weight of 96,000 to 100,000 for hydroxyapatite purified α actinin. Rod shaped particles 44 x 390 to 400 Å are seen in electron micrographs of negatively stained α actinin. By assuming 45% hydration and a molecular weight of 206,000, dimensions of approximately 40 x 500Å can be calculated for the α actinin molecule by using either So 20,w, Do 20,w, intrinsic viscosity, or a calibrated 6% agarose gel permeation column. Hydroxyapatite purified α actinin has an α helical content of 74% as measured by circular dichroism at 208 nm.
|Original language||English (US)|
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|State||Published - 1976|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology