Studies on the Isolation and Molecular Properties of Homogeneous Globular Actin: EVIDENCE FOR A SINGLE POLYPEPTIDE CHAIN STRUCTURE

Abstract

Solutions of globular actin have been isolated from rabbit skeletal muscle and purified by repetitive cycles of the reversible globular ⇄ fibrous transformation. Electrophoretic, ultracentrifugal, and gel filtration studies of these preparations revealed the presence of multiple macromolecular components. On the other hand, solutions of G-actin isolated by molecular exclusion chromatography exhibited a high degree of particle homogeneity. An appreciable, yet variable, fraction (15 to 75%) of the total protein isolated from acetone powders by reversible polymerization procedures is not globular actin. The predominant impurity is devoid of both ADP and ATP, and it is not transformed to fibrous actin in the presence of dilute aqueous KCl. Gel filtration chromatography (Sephadex G-200) eliminates this material. In agreement with earlier studies of Adelstein, Godfrey, and Kielley (4), this procedure represents a valuable preparative feature in the isolation of chemically homogeneous, monomeric actin. In the presence of dilute aqueous solutions of KCl, the chromatographically isolated protein is quantitatively transformed to F-actin. Molecular weight studies indicate that the mass of electrophoretically homogeneous G-actin is close to 46,000 g per mole (Mn0 = 46,500 and Mw0 = 45,800). When the reduced S-carboxymethylated protein is transferred to concentrated solutions of guanidine-HCl, the particle weight remains essentially unchanged (Mn0 = 47,600 and Mw0 = 47,800). This information implies that globular actin is constructed from a single, covalently linked poly-peptide chain.