PURIFICATION, BIOACTIVITY, AND SECONDARY STRUCTURE-ANALYSIS OF MOUSE AND HUMAN MACROPHAGE-MIGRATION INHIBITORY FACTOR (MIF)

The cytokine macrophage migration inhibitory factor (MIF) has been ide ntified to be secreted by the pituitary gland and the monocyte/macroph age and to play an important role in endotoxic shock. Despite the rece nt molecular cloning of a human T-cell MIF, characterization of the bi ochemical and biological properties of this protein has remained incom plete because substantial quantities of purified, recombinant, or nati ve MIF have not been available. We describe the cloning of mouse MIF f rom anterior pituitary cells (AtT-20) and the purification of native M IF from mouse liver by sequential ion exchange and reverse-phase chrom atography. For comparison purposes, human MIF was cloned from the Jurk at 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 milligra m quantities by a simple two-step procedure. The molecular weight of n ative mouse MIF (12.5 kDa monomer) was identical with that of recombin ant mouse MIF as assessed by gel electrophoresis and mass spectroscopy . No significant post-translational modifications were detected despit e the presence of two potential N-linked glycosylation sites. Recombin ant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF exhibited comparable biological activi ties. MIF induced the secretion of tumor necrosis factor-alpha and sti mulated nitric oxide production by macrophages primed with inteferon-g amma. Circular dichroism spectroscopy revealed that bioactive mouse an d human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated tha t MIF is of low to moderate thermodynamic stability. These studies est ablish the biochemical identity of native and recombinant MIF and prov ide a first insight into the three-dimensional structural properties o f this critical inflammatory mediator.