Isolation of heparin‐binding growth factors from bovine, porcine and canine hearts

Abstract

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 γ‐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. Copyright © 1989, Wiley Blackwell. All rights reserved