Figure 7
PAM interacts directly with actin but does not alter filament assembly/disassembly kinetics. (A) Filamentous actin (5 μM) and PAM-CD (10 μM) were incubated together or with buffer alone, and then sedimented. Supernatants and pellets were electrophoresed and stained with Coomassie blue; as the PAM-CD stains poorly with Coomassie blue, an immunoblot for this protein is also shown. In the absence of actin, nearly all the PAM-CD remained in the supernatant. However, in the presence of F-actin, approximately 50% of the PAM-CD was found in the pellet. In contrast, a control protein, GST/Furin-CD (also used at 10 μM), did not co-sediment with F-actin. The band at ~75 kDa in the GST/Furin-CD lanes is a contaminant that copurified with the fusion protein; the very minor ~110 kDa band present in the filamentous actin samples is α-actinin (AKL99 datasheet; Cytoskeleton Inc.). (B) Increasing concentrations of PAM-CD were incubated with 0.5 μM filamentous actin and the amount of PAM-CD present in the supernatants and pellets determined. The binding curve was fitted using a nonlinear regression single-phase exponential. The dissociation constant (kd) was 600 ± 150 nM (best-fit Scatchard analysis; n = 3). (C) The effect of PAM-CD (2.5 and 5 μM) on the rate of polymerization was determined using a fluorescent pyrene-actin assay with 5 μM G-actin. After an initial 30-minute incubation, polymerization was initiated by the addition of ATP; differences were not significant in a one-way ANOVA; P = 0.364. (D) PAM-CD (2.5 and 5 μM) was bound to pre-formed pyrene-actin filaments (5 μM), and the sample then placed under depolymerizing conditions; differences were not significant in a one-way ANOVA; P = 0.678.
