PURIFICATION AND CHARACTERIZATION OF BIOLOGICALLY-ACTIVE SCATTER FACTOR FROM RAS-TRANSFORMED NIH 3T3 CONDITIONED MEDIUM

Scatter factor (SF), a glycoprotein produced by cultured fibroblasts, acts in vitro on epithelial cells causing separation and increased local motility. In this study, the polypeptide was purified to apparent homogeneity in high yields with conserved biological activity from medium conditioned by ras-transformed NIH 3T3 cells, by a three-step procedure involving ammonium sulphate fractionation, cation-exchange and hydroxyapatite chromatography. After purification, SF specific activity increased from approximately 0.3 units/mu-g in unprocessed conditioned medium to approximately 5 units/ng, and cumulative recovery of biological activity was approximately 38%. Treatment of pure SF with N-glycanase resulted in a decreased M(r), but no concomitant effect was observed on biological activity. Proteolytic activity was absent from samples of both partially purified and pure SF. Our biochemical studies showed that SF, which is highly aggregated in low-ionic-strength media, is not aggregated in 0.4 m-salt. Under non-reducing conditions, pure SF migrated as a single stained band at M(r) 67000 on SDS/PAGE, and biological activity was eluted from unstained gels with an identical M(r). SF was electrofocused sharply at pI 8.5 with no degradation of activity. From ultracentrifugation studies (under non-aggregating conditions), the sedimentation coefficient of active SF was 3.7 S and fp.l.c. molecular sieve chromatography indicated a Stokes' radius of 2.95 nm. The calculated M(r) from these data was 61400. The appearance of three stained polypeptides of M(r) 82000, 57000 and 32000 derived from the M(r)-67000 constituent after reduction with mercaptoethanol suggests that SF may be a heterodimer of M(r)-57000 and -32000 subunits. Data from protein sequence analysis of the hydroxyapatite-purified protein confirms that SF has sequence identity with both rat hepatocyte growth factor and human fibroblast tumour cytotoxic factor.