Purification and characterization of human hepatic stimulator substance

Human hepatic stimulator substance (hHSS) was purified from fetal liver with 6,000-fold decrease in protein content and 840-fold increase in specific growth stimulative activity. Purification procedures included the heating of a homogenate in 35% (W/V) Tris-HCL at 95 degrees C for 20 min, high and ultra speed centrifugation, passage over Sephadex G100 gel filtration, DEAE-cellulose ion exchange, TSK G3000 SWG high performance liquid chromatography (HPLC) and YWG C-18 reverse phase HPLC techniques. The most purified material (HP-HSS) revealed cell-specific and dose dependent increase in 3H-TdR incorporation into cellular DNA. As little as 38 ng of the HP-HSS per ml of culture medium produced a 2.5-fold increase in DNA synthesis. Further studies indicate that HP-HSS in combination with insulin and epidermal growth factor stimulate DNA synthesis 16-fold compared with serum and hormone free controls and nearly 3-fold over hepatoma growth with HP-HSS alone. Sodium dedecyl sulfate polyacrylamide gel electrophoresis with silver stain and ultrascan XL laser densitrometer quantitative scanning revealed only one band at 12,800.