Identification and characterization of a nucleotide binding site on recombinant murine granulocyte/macrophage-colony stimulating factor.
Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a regulatory cytokine important in the proliferative and functional activation of hematopoietic cells. It belongs to a family of 20 kDa or less acidic glycoprotein molecules found in a broad range of cellular sources. On the basis of the previously reported nucleotide-binding properties of interleukin-2 (IL-2), atrial natriuretic factor (ANF), and glucagon, the interaction of GM-CSF with nucleotides was investigated. Using radiolabeled 8-azidoadenosine-containing photoprobes of ATP ([gamma-32P]-8N3ATP) and Ap4A, the putative biological alarmone ([beta'-32P]-8N3Ap4A), we have identified a nucleotide binding site on recombinant murine GM-CSF (rmGM-CSF). Specificity of binding was demonstrated by saturation and competition experiments. Saturation of photoinsertion by [gamma-32P]-8N3ATP and [beta'-32P]-8N3Ap4A occurs with apparent Kd's of 10 and 0.7 microM, respectively. Using an immobilized Fe3+ affinity chromatography technique, developed specifically for the isolation of photolabeled peptides, a single radiolabeled peptide was isolated. It was identified as amino acids 5-14 near the N-terminus of GM-CSF. This peptide region has been shown in previous studies to be critical for biological activity. Also consistent with this observation is our finding that the photolabeled GM-CSF has lost most, if not all, of its biological activity, as determined by a cellular proliferation assay.
Doukas
M A
MA
College of Medicine, Lucille P. Markey Cancer Center, University of Kentucky, Lexington.
Chavan
A J
AJ
Gass
C
C
Boone
T
T
Haley
B E
BE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D002846:Chromatography, Affinity; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D009711:Nucleotides; D011994:Recombinant Proteins; C017040:8-azidoadenosine 5'-triphosphate; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000255:Adenosine Triphosphate