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

Journal Article · Thu Nov 15 04:00:00 EST 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6324444

Chiba, S; Shibuya, K; Miyazono, K; Tojo, A; Oka, Y; Miyagawa, K; Takaku, F ^[1]

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1. Univ. of Tokyo (Japan)

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 {sup 125}I-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-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

• OSTI ID: 6324444
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:32; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CELL MEMBRANES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
FETAL MEMBRANES
FRACTIONATION
GROWTH FACTORS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MAN
MEMBRANE PROTEINS
MEMBRANES
MITOGENS
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLACENTA
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SEPARATION PROCESSES
TRACER TECHNIQUES
TWO-DIMENSIONAL ELECTROPHORESIS
VERTEBRATES