Purification and characterization of human macrophage migration inhibitory factor: evidence for specific binding to glutathione and formation of subunit structure.
We cloned cDNA of human macrophage migration inhibitory factor (MIF) from a T-cell line encoding 116 amino acid residues and expressed it in E. coli. MIF specifically bound glutathione (dissociation constant = 600 microM), and the protein was effectively purified by an S-hexylglutathione Sepharose affinity column. MIF was homogeneous on SDS-PAGE and the molecular weight was calculated as 12.5 kDa. The molecular weight was in excellent agreement with that of the primary structure assumed from the cDNA. On the other hand, the molecular weight in the native form calculated by Sephadex G-100 gel chromatography was 24.3 kDa, which suggested that MIF formed a dimeric structure. To further confirm the native molecular weight, we carried out analytical ultracentrifugation. This precise hydrodynamic analysis revealed that the native molecular weight was 24.8 kDa, which confirmed that MIF existed as a homodimeric form.
Nishihira
J
J
Central Research Institute, Hokkaido University School of Medicine.
Kuriyama
T
T
Nishino
H
H
Ishibashi
T
T
Sakai
M
M
Nishi
S
S
D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000737:chemistry*; D014461:Ultracentrifugation
D018076:DNA, Complementary; D008263:Macrophage Migration-Inhibitory Factors; D005978:Glutathione