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 approximately 15,500 daltons that is synthesized and secreted after activation by antigen or mitogen. By using the analogs 8-azidoadenosine 5'-[gamma-32P]triphosphate [( gamma-32P]8N3ATP) and nicotinamide 2-azidoadenine [adenylate-32P]dinucleotide [( alpha-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 microM [gamma-32P]8N3ATP; a half-maximal decrease of its photoinsertion at 10 microM was obtained with 22 microM ATP. Saturation of photoinsertion with [alpha-32P]2N3NAD+ was observed at 180 microM; a half-maximal decrease of its photoinsertion at 10 microM was effected by 10 microM NAD+ and by 5 microM 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 [gamma-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 [gamma-32P]8N3ATP as well as nonphotoactive nuceotide. The biological significance of these interactions is unknown, but their specificity suggests that nucleotide binding may be involved in the bioactivity of IL-2.
Campbell
S
S
Department of Biochemistry, University of Kentucky, Lexington 40536.
Kim
H
H
Doukas
M
M
Haley
B
B
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism* / Q000031:analogs & derivatives; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D002413:Cations, Divalent; D007376:Interleukin-2; D011994:Recombinant Proteins; D012265:Ribonucleotides; D009243:NAD; C062879:nicotinamide 2-azidoadenine dinucleotide; D004533:Egtazic Acid; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid