Figure 2.

Activity of recombinant eEF-2 kinase in vitro. A large-scale (0.5 ml) reaction using a mixture of Cefk-1 and Cefk-2 plasmids was run as in Fig. 1, with the omission of labeled methionine. In the control experiment, the reaction was run with a plasmid containing a luciferase gene. (A) The reaction mixtures were separated by chromatography on a Mono Q column as described. (B) eEF-2 kinase activity in fractions was measured as the ability to phosphorylate purified rabbit eEF-2 in the presence of [γ-³²P]ATP. Purified rabbit reticulocyte eEF-2 kinase was used in the (+) control experiments. (C) Ca²⁺/calmodulin-dependency of recombinant C. elegans eEF-2 kinase. Mono Q fraction 25 was assayed in a standard eEF-2 kinase assay in the presence and absence of Ca²⁺ and calmodulin and 20 μM trifluoperazine (TFP) or N-(6-aminohexyl)-5-chloro-1-napthalene-sulfonamide (W7). (D) Ca²⁺/calmodulin-dependency of recombinant human eEF-2 kinase. Human eEF-2 kinase cDNA was expressed in a coupled transcription/translation system as described above and eEF-2 kinase activity was assayed without further purification.