Affinity Labeling of Eukaryotic Initiation Factor 2 and Elongation Factor 1 Alpha Beta Gamma with GTP Analogs

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Archives of Biochemistry and Biophysics

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As part of an attempt to understand the specific function and role of each subunit in multisubunit protein synthesis factors, we have attempted to identify the nucleotide binding peptides of eukaryotic initiation factor 2 (eIF-2). To ensure that the interactions were of a specific nature, two general controls were used: first, other protein factors with characterized GTP binding activity were tested; second, all affinity labeling was checked for nucleotide specificity by protection with the authentic nucleotide at a 10-fold molar excess over the affinity reagent. Results with a number of GTP modifying reagents ([α-32P]GTP, [α-32P]GDP, oxidized [α-32P]GTP, 3′-p-azidobenzoyl-[α-32P]GTP, 3′-p-azidobenzoyl-[α-32P]GDP, and 5′-p-[8-3H]fluorosulfonylbenzoyl guanosine) indicate that appropriate conditions for both nucleotide and subunit specific labeling have been achieved. Under these conditions all reagents modified the β subunit of eIF-2. Complementary studies with subunit-deficient forms of eIF-2 also suggest that the β subunit of eIF-2 is involved with GTP binding. Coupled with other data suggesting that the γ subunit of eIF-2 might be involved in GTP binding and amino acid sequence data of eIF-2-γ from which a part of a GTP binding consensus sequence can be localized, support is provided for the concept of alternate GTP binding domains or a GTP binding domain shared between different subunits of eIF-2.