Partial purification of a high molecular weight hepatocyte growth stimulating factor from normal calf serum.
A heparin-binding protein, acting as a potent hepatocyte growth stimulating factor (HGSF) was extracted and partially purified from normal calf serum. HGSF stimulated DNA synthesis and proliferation in primary cultures of adult Balb/c mouse hepatocytes and in two liver-derived epithelial cell lines (C6 and C2.8) plated at low cell density in serum-free medium in the absence of epidermal growth factor. HGSF was non-dialyzable in M(r) 50,000 cutoff membranes, and was purified after chromatofocusing on PBE94 resin, (NH4)2SO4 precipitation (80% salt concentration) of the active fractions eluted at pH 5.7, flow chromatography and elution through Sephacryl S300 HR and HA-Ultrogel columns. The hepatotrophic activity was eluted with a protein fraction that was concentrated approximately 40,000 fold over the starting material. The effect was half maximal at approximately 50 ng/ml on adult hepatocytes in primary culture, HGSF had a molecular weight of 90,000-110,000 by gel filtration, was unstable on heat-treatment and was completely inhibited after trypsin digestion and after reduction with dithiothreitol. HGSF did not stimulate growth in Balb/c 3T3 fibroblasts. When injected into partially (40%) hepatectomized Balb/c mice, HGSF increased hepatic DNA synthesis 2 to 4-fold over the background stimulation, at 20 hours after the hepatectomy.
Barone
E
E
Mutagenic and Differentiation Institute, C.N.R., Pisa, Italy.
Van Boxel
T
T
Borney
F
F
Dal Monte
M
M
Chiavacci
L
L
D'Urso
M
M
Revoltella
R P
RP
D000818:Animals; D002417:Cattle / Q000097:blood*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D005260:Female; D017228:Hepatocyte Growth Factor / Q000097:blood / Q000302:isolation & purification* / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight
D017228:Hepatocyte Growth Factor; D004247:DNA