Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.

摘要: Three photoactive derivatives of the 7-methylguanosine-containing cap eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive specific labeling was observed with previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe synthesized that an azidophenyltyrosine derivative m7GTP [( 125I]APTM), monoanhydride m7GDP [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This allowed rapid quantitative introduction radioactivity in last rather than first step placed radioactive label on protein-proximal side weak P-N bond. dissociation constant 6.9 microM determined for [125I]APTM, which is comparable published values m7GTP. APTM equally effective as competitive inhibitors [125I]APTM. Like [32P]BPM, [125I]APTM labeled both full-length (25 kDa) polypeptide a 16-kDa degradation product, designated eIF-4E*, occurring proportion amounts each present. third azidophenylglycine 32P]APGM), [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, also shown specifically. Unlike [32P]BPM however, [32P]APGM eIF-4E* approximately 4-fold more readily intact eIF-4E. Tryptic CNBr cleavage suggested consists protease-resistant core retains cap-binding site residues 47-182.