Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.
Article| August 01 1975
Preparation and purification of polymerized actin from sea urchin egg extracts.
R E Kane
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1975) 66 (2): 305–315.
R E Kane; Preparation and purification of polymerized actin from sea urchin egg extracts.. J Cell Biol 1 August 1975; 66 (2): 305–315. doi: https://doi.org/10.1083/jcb.66.2.305
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