FIGURE 2.

aEF-2-induced release of aIF6 from ribosomes. (A) The GTPase activity of recombinant aEF-2 protein was analyzed by incubating 20 or 40 pmol of the protein with 20 pmol of 70S HSW and 1 mM GTP at 65°C for 20 min. At the end of the reaction, the inorganic phosphate released after GTP hydrolysis was revealed as described in the “Materials and Methods” section. Data are presented as mean value ± SD (n = 4). (B) Density gradient fractionation of 70S HSW (70 pmol) or 50S (50 pmol) incubated at 65°C for 20 min in the presence of aEF-2 (70 or 50 pmol) and 1 mM GTP or GMP-PNP. The distribution of aIF6 and recombinant aEF2 was revealed by western blotting of the individual fractions with anti-aIF6 and 6(His) antibodies, respectively. The distribution of the ribosomal subunits was identified by the optical scans at OD254 nm of the gradients. A representative image of at least three independent sucrose density experiments is shown for each analysis.