FIGURE 4.

aSBDS inhibits the release of aIF6 induced by aEF2. (A) Density gradient fractionation of HSW ribosomes (70 pmol) or 50S subunits (50 pmol) incubated with aSBDS (140 and 100 pmol, respectively), and aEF-2 (140 and 100 pmol, respectively), at 65°C for 20 min. Each sample was incubated in presence of 1 mM GTP. The distribution of aIF6 was revealed by western blotting of the individual fractions with the anti-aIF6 antibodies. The distribution of endogenous and recombinant aSBDS was revealed by western blotting of the individual fractions with the anti-aSBDS antibodies. The distribution of 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. (B) GTPase activity of aEF-2 (40 pmol) was analyzed by incubating the recombinant protein in presence/absence of 70S HSW (20 pmol), aSBDS (40 pmol), and 1 mM GTP at 65°C for 20 min. At the end of the reaction, the inorganic phosphate released after GTP hydrolysis was detected, as described in the “Materials and Methods” section. Data are presented as mean value ± SD (n = 4).