Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament.

Actin filament bundles isolated from Limulus sperm were used for quantitative electron microscope studies of F-actin assembly. The assembly rate constants were calculated. In addition, the critical concentrations (Cos) for both filament ends were directly determined. In 75 mM KCl and 1-5 mM Mg++, the Cos were 0.1 microM and 0.5 microM for the barbed and pointed ends, respectively. Substitution of Ca++ (20-200 microM) for Mg++ resulted in Cos of 0.4 microM for both filament ends. Consistent with these findings, filament growth occurred only from the barbed ends of Limulus bundles "seeded" into F-actin solutions in KCl and Mg++. Finally, filaments originally grown from the pointed filament ends of Limulus bundles were gradually lost as the actin solution reached steady state. These results demonstrate that actin filaments can "treadmill" under physiological conditions, albeit at very slow rates.