Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.
A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)
Watanabe
M
M
Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., Gunma, Japan.
Fukamachi
H
H
Uzumaki
H
H
Kabaya
K
K
Tsumura
H
H
Ishikawa
M
M
Matsuki
S
S
Kusaka
M
M
D000818:Animals; D001345:Autoradiography; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D009504:Neutrophils / Q000378:metabolism*; D011869:Radioligand Assay; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D007457:Iodine Radioisotopes; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; C070653:granulocyte colony-stimulating factor, Tyr(1)-Tyr(3)-; D016179:Granulocyte Colony-Stimulating Factor