DIFFERENTIAL PURIFICATION OF AUTOCRINE MOTILITY FACTOR DERIVED FROM AMURINE PROTEIN-FREE FIBROSARCOMA

We have previously shown that a protein-independent growing fibrosarco ma, Gc-4 PF has a high motile response to its cultured medium, which i s associated with an increase in expression of gp78, a cell-surface re ceptor for autocrine motility factor (AMF). Here we show that the cult ured medium contains two motile activities, acidic and basic AMFs with regard to binding features on ion exchange chromatography. These two AMFs were purified by sequential DEAE anion exchange, CM cation exchan ge, and gel filtration chromatographies. However, both acidic and basi c AMFs have a similar size of 55 kDa and 65 kDa under non-reducing and reducing conditions, respectively, with the same pl of 6.5. The stimu lated motility of both AMFs was inhibited by the pertussis toxin (PT), but not by Streptomyces hyaluronidase. These two AMFs significantly s timulated the lung colonizing properties of the self-producing cells b y 1.5-fold. These results suggest that both acidic and basic AMFs may correspond to the previously reported AMF and confirm directly that th e AMF-gp78 signaling pathway is involved in cell motility associated w ith metastatic property.