FIG. 7.

Time courses of in vitro reactivation of suicidally inactivated holoEAL (A) and in vitro activation of the EAL-CN-Cbl complex (B) by purified EutA. Suicidally inactivated holoEAL was formed by incubation of purified apoEAL (0.11 U; 5.9 μg) at 37°C for 45 min with 20 μM AdoCbl in 40 mM potassium phosphate buffer (pH 8.0) containing 0.4 M ethanolamine and 0.4% Brij 35 in a total volume of 25 μl. The EAL-CN-Cbl complex was formed by incubation of apoEAL (0.11 U; 5.9 μg) at 37°C for 15 min with 4 μM CN-Cbl in 40 mM potassium phosphate buffer (pH 8.0) containing 0.4 M ethanolamine and 0.4% Brij 35 in a total volume of 25 μl. In vitro reactivation and activation were assayed as described in the text with (circles) or without (triangles) 33 μg of EutA in the presence (solid symbols) and absence (open symbols) of ATP plus MgCl₂ (20 mM each). AdoCbl was added to a final concentration of 10 μM. Noninactivated EAL was treated similarly to controls to estimate the extent of (re)activation. Error bars, average ± SD for the results of three experiments.