Effect of Temperature on the Mechanism of Actin Polymerization

The rate of the Mg²⁺-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg²⁺, Ca²⁺, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513–6517] was found to fit the data from 10 to 35 °C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 °C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg²⁺-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg²⁺ binding becoming tighter, and Ca²⁺ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.