Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: cloning of its cDNA, purification and characterization of recombinant protein.
Two different forms of cDNA for F-TCF were isolated from cDNA library prepared with mRNA from human embryonic lung fibroblast, IMR-90 cells. One of them was completely identical to the cDNA for placenta type hepatocyte growth factor (HGF) and the other one was a variant cDNA for the HGF with a deletion of 15 base pairs in the coding region. The cDNAs were expressed in CHO cells and recombinant proteins were purified and characterized. The deleted form of recombinant F-TCF (rF-TCF) was slightly lower in heparin affinity than the intact form. Both rF-TCFs showed almost same dose-response curves for cytotoxicity on Sarcoma 180 or Meth A sarcoma cells. Dose-response curves for the stimulation of DNA synthesis in rat hepatocytes were also almost same before reaching maximal activity at 12.5 ng/ml but significantly different at higher concentrations. The deleted form of rF-TCF maintained maximal activity in the dose range of 12.5 to 100 ng/ml, although the intact form decreased the activity dose-dependently at more than 25 ng/ml. This suggests that the deletion of five amino acids results in a conformational change which alters heparin binding and hepatocyte growth stimulating activities.
Shima
N
N
Life Science Research Institute, Snow Brand Milk Products Co., Ltd., Tochigi, Japan.
Nagao
M
M
Ogaki
F
F
Tsuda
E
E
Murakami
A
A
Higashio
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004261:DNA Replication / Q000187:drug effects; D005260:Female; D005347:Fibroblasts; D015723:Gene Library; D014644:Genetic Variation; D006133:Growth Substances / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D017228:Hepatocyte Growth Factor; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008168:Lung; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010920:Placenta / Q000502:physiology; D011247:Pregnancy; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology
D006133:Growth Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D004247:DNA