PURIFICATION AND CHARACTERIZATION OF HEPATOCYTE GROWTH-FACTOR (HGF) FROM HUMAN SEMINAL PLASMA

Naturally occurring forms of hepatocyte growth factor/scatter factor ( HGF/SF) have been purified by heparin-Sepharose chromatography, follow ed by cation exchange chromatography fi-om a pool of human seminal pla sma. Using an enzyme-linked immunosorbant assay, MDCK scatter assay, a nd Western blot analysis, it was found that, after heparin-Sepharose c hromatography, human HGF/SF present in seminal plasma eluted in two di fferent fractions, between 0.72 and 0.85 M NaCl (fraction I) and betwe en 0.95 and 1.10 M NaCl (fraction II). Further purification of fractio n I by cation exchange chromatography resulted again in two fractions which eluted at 0.2-0.3 and at 0.6-0.8 M NaCl. The fraction which elut ed at 0.2-0.3 M NaCl consisted of two biologically less active heavy c hains of the heterodimeric form of HGF/SF (107.1 U/ng immunoreactive H GF), with approximate molecular weights of 65 and 62 kDa under reducin g conditions. The second fraction, which eluted at 0.6-0.8 M NaCl, rev ealed three bands with molecular weights of 87, 65 and 62 kDa, respect ively. The 87 kDa form is thought to be a single chain precursor of HG F/SF devoid of biological activity. After subjecting fraction II to ca tion ex-change chromatography, only one major peak eluted between 0.9 and 1.0 M NaCl, and consisted of two biologically active heavy chains of the heterodimeric form of HGF/SF (708.3 U/ng immunoreactive HGF), w ith approximate molecular weights of 65 and 62 kDa under reducing cond itions. Nonreducing conditions for both fraction I and fraction II rev ealed only one band with a molecular weight of 68 kDa. The ratio of pr o-HGF/SF and less biologically active HGF/SF (fraction I) over mature heterodimeric HGF/SF (fraction II) was approximate to 1:3, in seminal plasma from sperm donors. In seminal plasma, pro-HGF/SF represents an 87 kDa glycoprotein which, apparently, is converted by limited proteol ysis into several bands with molecular weights of 65 and 62 kDa. This is the first report showing the presence of pro-HGF/SF and heterodimer ic mature HGF/SF, as well as less biologically active forms of HGF/SF in human seminal plasma.