THE KINETICS OF THE INTERACTION BETWEEN THE ACTIN-BINDING DOMAIN OF ALPHA-ACTININ AND F-ACTIN

Measurement of the binding equilibrium for the interaction of alpha-ac tinin with F-actin is complicated by secondary reactions involving cro ss-linking and/or bundling of the actin filaments. To quantitate the i nitial binding event, we studied the interaction of the bacterially ex pressed actin-binding domain (ABD) of chick smooth muscle alpha-actini n 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 x 1 0(5), 1.8 x 10(6) and 4 x 10(6) M(-1) s(-1) at 5 degrees C, 15 degrees C and 25 degrees 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 mu uM, respectively. Optical changes on mix ing intact alpha-actinin with F-actin were dominated by secondary bund ling events.