Radiolabeled GM-CSF differentially partitions to the ternary GM-CSF receptor complex.

(A-C) A titration of purified sβc (0 to 7.03 μM) against a mixture of 3.2 μM sGMRα and 7.3 μM GM-CSF spiked with³²P-labeled SGMKIN. Reaction mixes were set up with 0 (dashed gray), 0.88 μM (dashed black), 1.76 μM (thin gray), 3.52 μM (thin black), 5.27 μM (thick gray), or 7.03 μM (thick black) sβc and incubated at 25°C for 1 hour before size-exclusion chromatography. Fractions were collected at 1-minute intervals. A control reaction was also prepared with 5.27 μM sβc and 3.2 μM sGMRα but no GM-CSF (dashed black). (A) Chromatogram of A₂₈₀ profiles for each sample with the location of the ternary complex (TC), binary complex (BC), and free ligand (GM) indicated. (B) Distribution of radioactivity among the ternary complex, binary complex, and free ligand for the reactions described in panel A. (C) Radioactive GM-CSF distributed into the ternary complex, expressed as a percentage of the total radioactive GM-CSF in ternary and binary complexes; comparing experimentally observed values for the reactions described in panel A (●) with a theoretical distribution based on 1GM:1α:2β (○) and 2GM:2α:2β (■) models. (D) Titration of GM-CSF (0 to 7 μM) spiked with³²P-labeled SGMKIN against a mixture of 3.5 μM sGMRα and 3.5 μM sβc. Reaction mixes were allowed to reach equilibrium at 25°C for at least 2 hours before being fractionated by size-exclusion chromatography. The distribution of radioactivity among ternary (●) and binary (○) complexes was determined and the radioactivity in each complex was expressed as a percentage of total bound counts where counts in TC plus counts in BC is 100%.