Identification of an adenine-nucleotide-binding site on interferon alpha2.
Using 32P-labeled 2-azidoadenosine 5'-triphosphate (2N3ATP) and 8-azidoadenosine 5'-triphosphate (8N3ATP), we have identified a site on human interferon alpha2 (IFN-alpha2) that binds adenine nucleotides. The results from saturation and competition experiments demonstrated the specificity of the nucleotide interaction. Half-maximal saturation of IFN-alpha2 was observed at 10 microM 2N3ATP or 35 microM 8N3ATP. ATP effectively decreased photoinsertion of both photoaffinity analogs of ATP. Photoinsertion of 8N3ATP was enhanced by MgCl2, independent of the ionic strength, and exhibited an optimum pH between 7.0 and 7.5. Immobilized-Al3+ affinity chromatography and HPLC were used to purify the modified peptides from IFN-alpha2 that had been photolabeled with 8N3ATP and digested with trypsin or chymotrypsin. Overlapping-sequence analysis localized the sites of photoinsertion to the region corresponding to Lys121-Tyr135 in the amino acid sequence of IFN-alpha2, which almost perfectly overlaps a nuclear-localization signal (R120KYFQRITLYLKEKKY135).
Olcott
M C
MC
College of Pharmacy and the Lucille P. Markey Cancer Center, University of Kentucky, Lexington 40536-0082, USA.
Haley
B E
BE
D000227:Adenine Nucleotides / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010777:Photochemistry; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins
D000227:Adenine Nucleotides; D000345:Affinity Labels; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D021382:Protein Sorting Signals; D011994:Recombinant Proteins