PHOTOAFFINITY-LABELING OF ATP AND NAD+ BINDING-SITES ON RECOMBINANT HUMAN INTERLEUKIN-2

Interleukin 2 (IL-2) is a T-cell-derived lymphokine critical in the activation and proliferation of T cells, B cells, and lymphokine-activated killer cells. It is a glycoprotein of ≈15,500 dal tons that is synthesized and secreted after activation by antigen or mitogen. By using the analogs 8-azidoadenosine 5′-[γ-32P]triphosphate ([γ-32P]8N3ATP) and nicotinamide 2-azidoadenine [adenylate-32P]dinucleotide ([α-32P]2N3NAD+) as photoaffinity probes, we have detected specific, metal ion-requiring nucleotide binding sites on recombinant human IL-2 (rhIL-2). The specificity of these nucleotide interactions with rhIL-2 was demonstrated by saturation effects and by competition by the parent nucleotides at physiologically relevant concentrations. Saturation of photoinsertion into rhIL-2 occurred at 50 μM [γ-32P]8N3ATP; a half-maximal decrease of its photoinsertion at 10 μM was obtained with 22 μM ATP. Saturation of photoinsertion with [α-32P]2N3NAD+ was observed at 180 μM; a half-maximal decrease of its photoinsertion at 10 μM was effected by 10 μM NAD+ and by 5 μM 3-aminobenzamide. The extent of photoinsertion of both photoprobes into rhIL-2 varied with the presence of different divalent metal ions. rhIL-2 photolabeling with [γ-32P]8N3ATP appeared to be dependent on the presence of metal ion. It was effectively labeled in the presence of Mg2+ and photoinsertion was increased with the addition of Zn2+ at micromolar concentrations. Also, rhIL-2 underwent slow autophosphorylation by an intramolecular mechanism using [γ-32P]8N3ATP as well as nonphotoactive nucleotide. The biological significance of these interactions is unknown, but their specificity suggests that nucleotide binding may be involved in the bioactivity of IL-2.