|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Cdc8 requires N-terminal acetylation to associate with actin filaments. (A) 10 µM actin incubated with increasing concentrations of heterologous Cdc8 (1-20 µM in experiment shown) at 20°C for 30 minutes in 20 mM MOPS, 30 mM KCl, 5 mM MgCl2, pH 7.0. The actin was pelleted at 100,000 g and the equivalent samples of the pellet (upper panel) and supernatant (lower panel) were run on an SDS-PAGE gel, which was stained with Coomassie Blue. (B) Binding constants (K50%) were measured as the free Tm dimer concentration at which the actin filament is half saturated by Cdc8 dimer, and was determined as the ratio of density of actin against the free concentrations of Cdc8 (circles), Cdc8-AS (+) or endogenous Cdc8 (
) dimers, as measured by quantitative analysis of supernatant and pellet co-sedimentation gels (A), and calculated using the Hill equation (see Table 1). (C) Supernatant and pellet co-sedimentation gels of unacetylated and endogenous Cdc8. The faster migrating endogenous Cdc8 band associated with actin in the pellet, while the slower migrating band was the prominent form in the supernatant. (D) Endogenous Cdc8 purified from actin co-sedimentation pellets from C migrated more slowly than unacetylated Cdc8 purified from E. coli.
|
