Isolation of heparin-binding growth factors from bovine, porcine and canine hearts.
Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.
Quinkler
W
W
Max Planck Institute, Department of Experimental Cardiology, Bad Nauheim, Federal Republic of Germany.
Maasberg
M
M
Bernotat-Danielowski
S
S
Lüthe
N
N
Sharma
H S
HS
Schaper
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004285:Dogs; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006133:Growth Substances / Q000302:isolation & purification* / Q000494:pharmacology; D006321:Heart / Q000502:physiology*; D006493:Heparin / Q000302:isolation & purification* / Q000494:pharmacology; D007700:Kinetics; D008934:Mitogens / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013045:Species Specificity; D013552:Swine
D006133:Growth Substances; D008934:Mitogens; D006493:Heparin; D004247:DNA