Protein synthesis in rabbit reticulocytes. A study of the mechanism of interreaction of fluorescently labeled co-eIF-2A with eIF-2 using fluorescence polarization.

5-Dimethylaminonaphthalene-1-sulfonyl (dansyl)-Co-eIF-2A was prepared using homogeneous Co-eIF-2A. Dansyl-Co-eIF-2A was as active as untreated Co-eIF-2A when assayed for stimulation of ternary complex formation and also for protection of the ternary complex from dissociation by aurintricarboxylic acid. The mechanism of interaction of dansyl-Co-eIF-2A with eIF-2 was studied by measuring changes in fluorescence polarization. These studies indicate that dansyl-Co-eIF-2A interacts specifically with the ternary complex and does not interact with free eIF-2 or with two other high molecular weight protein complexes, Co-eIF-2B and Co-eIF-2C. Mg2+ inhibits ternary complex formation by eIF-2 and Co-eIF-2C relieves this Mg2+ inhibition of ternary complex formation. In both cases, the changes in fluorescence polarization of dansyl-Co-eIF-2A correlate well with the extent of ternary complex formed.