Facilitation of actin polymerization by interfilamentous factors

Abstract

The polymerization of actin in low ionic strength buffer at 0 degrees C in the presence of 0.25 mM Mg2+ was studied by viscometry, turbidity and absorbance at 232 nm. Under these conditions, significant polymerization occurred only in the viscometer and not in isotropic mixtures. The polymerization rate with 0.25 mM MgCl2, as judged from shear viscosity, was equal to or greater than that observed with 0.1 M KCl and 0.25 mM MgCl2 at 0 degrees C, and was characterized by a longer nucleation period. Measurements of the turbidity at 350 nm (detecting filament formation and aggregation) and the absorbance at 232 nm (detecting conformational changes of the G-F transition) showed no evidence for polymerization or nucleation in a bulk solution at 0 degrees C when Mg2+ was added to 0.25 mM and, furthermore, F-actin nuclei were ineffective as seeds under these conditions. However, nucleation and polymerization by these criteria could be induced by raising the temperature to 20 degrees C. These results demonstrate the existence of narrow conditions when elongation of F-actin is dissociated from nucleation of oligomeric acceptor nuclei, even if monitored on the sub-molecular level (absorbance at 232 nm). Under these conditions, elongation appears to require anisotropic F-actin, i.e. that filaments are ordered by laminar flow.

Regulation of endothelial nitric oxide synthase by the actin cytoskeleton

“…The actin was further purified by gel filtration on a 100-cm Sephacryl HR300 column (Amersham-Pharmacia, Piscataway, NJ). G-actin was polymerized into F-actin by incubation with 100 mM KCl at room temperature for 2 h, as reported by Cerven (7).…”

Cytoskeletal components enhance the autophosphorylation of retinal insulin receptor

“…G-actin was polymerized into F-actin by incubation with 100 mM KCl at room temperature for 2 h as reported [27]. Bovine rod outer segments (ROS) were prepared according to the method described earlier [8].…”