Macrophage migration inhibitory factor (MIF) produced by a human T cell hybridoma clone.
A macrophage migration inhibitory factor (MIF) was purified to homogeneity from the serum-free culture supernatant of a human T cell hybridoma clone called F5. This clone was established by means of somatic fusion, using the emetine-actinomycin D selection method, and produced a large amount of MIF. The MIF activity in the culture supernatant of F5 cells was not due to contaminating interferon-gamma (IFN-gamma), which is known to possess MIF activity. Furthermore, other known cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF), were also revealed to have MIF activity, but our MIF was different from these known factors. F5 cells produced two species of MIF that could be separated on a phenyl-Sepharose column. MIF-1 (the more hydrophilic species of the two) was purified to homogeneity by sequential hydrophobic chromatography, ion-exchange chromatography, dye ligand affinity chromatography, and high-performance liquid chromatography (HPLC) on ion-exchange and reverse-phase columns. Finally, 4600-fold enrichment, as to specific activity, of MIF-1 was achieved. The purified MIF-1 was digested with endoproteinase Lys-C into some peptide fragments and the amino acid sequences of the peptides obtained were determined. No sequence identity between our MIF-1 and other proteins was observed. Then, antibodies were raised against a peptide synthesized according to the determined amino acid sequence. They specifically reacted with MIF-1 and reduced its migration inhibitory activity. Based on these results, we conclude that the determined amino acid sequence was certainly that of MIF-1.
Oki
S
S
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hirose
S
S
Higuchi
M
M
Osawa
T
T
D000595:Amino Acid Sequence; D006680:HLA Antigens / Q000032:analysis; D006801:Humans; D006825:Hybridomas / Q000378:metabolism*; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D008263:Macrophage Migration-Inhibitory Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D013601:T-Lymphocytes / Q000378:metabolism*
D006680:HLA Antigens; D008263:Macrophage Migration-Inhibitory Factors; D007371:Interferon-gamma