Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

J Biol Chem. 1990 Nov 15;265(32):19777-81.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels / metabolism*
  • Azides / metabolism*
  • Biotin
  • Cell Membrane / chemistry
  • Chromatography, Affinity
  • Cross-Linking Reagents
  • Electrophoresis, Gel, Two-Dimensional
  • Female
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Humans
  • Iodine Radioisotopes
  • Isoelectric Point
  • Macromolecular Substances
  • Molecular Weight
  • Photochemistry
  • Placenta / chemistry
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / chemistry
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / isolation & purification*
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / metabolism

Substances

  • Affinity Labels
  • Azides
  • Cross-Linking Reagents
  • Iodine Radioisotopes
  • Macromolecular Substances
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor
  • hydroxysuccinimidyl-4-azidobenzoate
  • Biotin
  • Granulocyte-Macrophage Colony-Stimulating Factor