Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling.
The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-125I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.
Chiba
S
S
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Shibuya
K
K
Miyazono
K
K
Tojo
A
A
Oka
Y
Y
Miyagawa
K
K
Takaku
F
F
D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D001710:Biotin; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008970:Molecular Weight; D010777:Photochemistry; D010920:Placenta / Q000737:chemistry; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; C029534:hydroxysuccinimidyl-4-azidobenzoate; D001710:Biotin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor