Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides: Identification of GTP- and ATP-binding domains

Abstract

Abstract We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.