Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor (GEF), Met-tRNA(f), GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2.GEF for GDP concentrations from 0.01 to 20 μM. This is in disagreement with the model: eIF-2.GDP + GEF of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2.GEF. We propose an asymmetrical model of ternary complex (eIF-2.GTP.Met-tRNA(f)) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the α subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(αP) nor from greatly enhanced binding of GEF. The inhibition results from the requirement of very high levels of GTP (100 μM) to dissociate the eIF-2(αP).GEF complex.
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - Aug 23 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology