Nucleotide-Binding Assay for aIF2 Protein

The nucleotide-binding assay was performed essentially as described (20 (link)). The purified aIF2 proteins (30 picomoles) were incubated 10 min at 65°C in a buffer containing 10 mM HEPES pH7.5, 200 mM NaCl, 5 mM MgCl₂, 10 mM 2-mercaptoethanol with increasing amounts (0–464 picomoles) of [³H]GDP (∼18 000 d.p.m/pmol). The final volume of the reaction was 50 μl. Aliquots (20 μl) were withdrawn and mixed with 1 ml of cold incubation buffer and then filtered through Millipore 0.22 μM nitrocellulose disks, which were washed with 2 ml of cold incubation buffer. The filters were then counted by liquid scintillation in a Beckman LS 6500 counter. Results were fitted with simple binding curves from which apparent dissociation constants and their associated standard errors were derived using the MC-Fit program (29 ).