The kinetics of the interaction between the actin‐binding domain of α‐actinin and F‐actin

Abstract

Measurement of the binding equilibrium for the interaction of α-actinin with F-actin is complicated by secondary reactions involving cross-linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin-binding domain (ABD) of chick smooth muscle α-actinin with F-actin. Stopped-flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F-actin yielding second order rate constants for association of 2 × 105, 1.8 × 106 and 4 × 106 M−1 · s−1 at 5°C, 15°C and 25°C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6 s−1, giving equilibrium constants of 0.83 and 2.4 μM, respectively. Optical changes on mixing intact α-actinin with F-actin were dominated by secondary bundling events