Identification of macrophage migration inhibitory factor isoforms in bovine brain

In the course of the study of the primary structures and molecular mechanis ms of action of immunologically active compounds of the nervous system we h ave isolated from the soluble fraction of total bovine brain two heat-stabl e proteins. The purification procedure was mainly based on DEAE-Servacel io n-exchange chromatography and reversed-phase HPLC. The proteins were identi fied by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences f or MIF1 and MIF2 were found to be identical. According to mass spectral ana lysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third pe ptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by t he oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this pro tein. A comparatively simple purification procedure is presented that may b e widely used for simultaneous isolation in one run of MIF isoforms.