Revised Manuscript Received September 6, 1994® abstract: The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and themonocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF fromanterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MlF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dosedependent fashion, and both recombinant and native MIF exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-a and stimulated nitric oxide production by macrophages primed with interferon-y. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of/3-sheet and a-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establishthe biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.

The cytokine macrophage migrationinhibitory factor (MIF) 1 was described originally as a T-lymphocyte-associ-ated activity which inhibited the randommigration of guinea pig peritoneal macrophages (Bloom & Bennett, 1966; David, 1966). Over the years, MIF activity has been reported to be associated with delayed-type hypersensitivity (Bloom & Bennett, 1966; David, 1966), to be produced by lectinactivated T-cells (Weiser et al., 1981), and to enhance