IDENTIFICATION OF AN ADENINE-NUCLEOTIDE-BINDING SITE ON INTERFERON ALPHA-2

Using P-32-labeled 2-azidoadenosine 5'-triphosphate (2N(3)ATP) and 8-a zidoadenosine 5'-triphosphate (8N(3)ATP), we have identified a site on human interferon alpha 2 (IFN-alpha 2) that binds adenine nucleotides , The results from saturation and competition experiments demonstrated the specificity of the nucleotide interaction, Half-maximal saturatio n of IFN-alpha 2 was observed at 10 mu M 2N(3)ATP or 35 mu M 8N(3)ATP. ATP effectively decreased photoinsertion of both photoaffinity analog s of ATP. Photoinsertion of 8N(3)ATP was enhanced by MgCl2, independen t of the ionic strength, and exhibited an optimum pH between 7.0 and 7 .5. Immobilized-Al3+ affinity chromatography and HPLC were used to pur ify the modified peptides from IFN-alpha 2 that had been photolabeled with 8N(3)ATP and digested with trypsin or chymotrypsin, Overlapping-s equence analysis localized the sites of photoinsertion to the region c orresponding to Lys121-Tyr135 in the amino acid sequence of IFN-alpha 2, which almost perfectly overlaps a nuclear-localization signal (R120 KYFQRITLYLKEKKY135).