Figure 4. Analysis of the activity of purified Mical^redoxCH protein.

Pyrene-labeled actin was used to monitor both the polymerization and depolymerization of actin, where the fluorescence intensity (a.u. [arbitrary units]) of the pyrene-labeled actin polymer is substantially higher than the pyrene-labeled actin monomer. As judged by the increase in fluorescence intensity over time which reveals actin polymerization, the addition of 600 nM of purified Mical^redoxCH protein alone to actin (black dots) does not alter the rate (r), extent (e), or steady-state level (ss) of actin polymerization (as compared to an actin only control incubated with the buffer used to store Mical^redoxCH; gray dots). In the presence of 100 μM of its NADPH coenzyme, however, purified Mical^redoxCH protein (Mical^redoxCH + NADPH; green dots) specifically alters the rate of actin polymerization (1) such that over time activated Mical^redoxCH induces a decrease in the extent of polymerization (2), the rapid depolymerization of F-actin (3), and the inability of actin to reinitiate polymer formation (4). These results are similar to what we observed with our previously purified active Mical^redoxCH enzyme [10-12].