We have investigated the nature of endothelial cell growth factors in 14‐day embryonic and adult chick brain extracts. Mitogenic activity was isolated by a combination of cation‐exchange, heparin‐Sepharose affinity, and reverse‐phase HPLC. Two major mitogenic fractions eluted from heparin‐Sepharose at 0.8‐1.3 M and 1.5‐2 M. Biologically active proteins eluting at 0.8‐1.3 M NaCl, after purification to homogeneity from embryonic and adult brain, were found to possess the same amino‐terminal sequence as human acidic fibroblast growth factor (aFGF). The notion that the isolated mitogens represent chick aFGF is further supported by the findings that their affinity for heparin and their retention behavior in highly resolutive HPLC are indistinguishable from those of genuine aFGF. Mitogenic activities eluting at 1.5‐2 M NaCl were also present in embryonic and adult brain, but in quantities insufficient for preliminary characterization. The high specific mitogenic activity for endothelial cells, high affinity for heparin and cross‐reactivity with antibodies against bovine basic FGF (bFGF) suggest a relationship of those materials with basic FGF. Our data also suggest that the sequence of aFGF is highly conserved among vertebrates. While angiogenesis occurs predominantly in the embryonic brain, the absence of notable differences in the contents of the potent angiogenic factors aFGF and bFGF in embryonic versus adult chick brain is interesting.

A heparin-binding protein with neurotrophic activity for perinatal rat neurons, termed HBNF, was purified to homogeneity from bovine brain utilizing pH 4.5 extraction, ammonium sulfate precipitation, cation exchange and heparin-Sepharose affinity chromatographies, and reverse phase HPLC. In the presence of protease inhibitors during extraction, a protein with an apparent molecular weight of 18 kDa was obtained in a yield of approximately 0.5 mg/kg brain tissue. The amino acid sequence of the first 114 residues of HBNF was determined and found to highly homologous to the cDNA-derived amino acid sequence of human HBNF, a 136-residue protein. Bovine and human HBNFs have identical molecular weights as judged by SDS gel electrophoresis and very similar amino acid compositions. This and overall sequence conservation suggest that bovine HBNF is also a 136 amino acid protein with a calculated molecular weight of approximately 15.5 kDa. The apparent discrepancy between calculated and observed molecular weights of bovine HBNF (and of human HBNF of which the complete sequence is known) is most likely a result of the highly basic nature of HBNF. If protease inhibitors were omitted during tissue extraction, two additional proteins with lower apparent molecular weights and identical N-terminal sequences were isolated, with the smallest forms being the major product. Amino acid analysis showed that the smaller forms correspond to C-terminally truncated HBNFs with calculated molecular weights of 13.6 and 12.4 kDa, lacking approximately 14 and 22 residues. Comparison of the HBNF protein sequence with sequences stored in the Protein Identification Resource/Genbank databases reveals high homology to the translation product of the MK-1 gene, which is retinoic acid-inducible in embryonic carcinoma cells and developmentally expressed during gestation in mice.

Endothelial cell growth factor activity purified from bovine kidney by heparin-Sepharose affinity chromatography was previously identified as basic fibroblast growth factor [Baird, A., Esch, F., Böhlen, P., Ling, N., & Gospodarowicz, D. (1985) Regul. Pept. 12, 202-213]. We now show that a major mitogenic fraction, isolated from heparin-Sepharose-purified material by Mono-S cation-exchange chromatography and reverse-phase high-performance liquid chromatography, is related to acidic fibroblast growth factor (aFGF). Sequence analysis showed the amino-terminal sequence to be Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr-X-Ser-Asn-Gly-Gly-Tyr-Phe-Leu-Arg-Ile-Le u-Pro- Asp-Gly-Thr-Val-Asp-. The molecular mass of the protein, as determined by polyacrylamide gel electrophoresis, was 15.5 kDa. In combination, those data strongly suggest that this mitogen is amino terminally truncated acidic fibroblast growth factor. So far, aFGF has only been found in neural tissues, i.e., in the brain and retina. Our results strongly suggest that this mitogen also occurs in extraneural tissue.

Recently, the partial structural characterization of a novel bovine brain protein was reported (1). Because of its mitogenic activity for vascular endothelial cells and its ability to strongly bind heparin it was termed heparin-binding brain mitogen (HBBM). Although HBBM shares these properties with members of the fibroblast growth factor (FGF) family of growth factors, its amino-terminal sequence is not homologous to that of the FGFs. Now, we report the isolation and partial structural characterization of HBBMs from human, rat and chick brain. Proteins were isolated by tissue extraction at pH 4.5, ammonium sulfate precipitation, cation exchange chromatography, heparin-Sepharose affinity chromatography and reverse-phase HPLC. The amino-terminal sequences of the HBBMs from human, bovine and rat brain are identical, whereas that of chick HBBM reveals a single amino acid substitution. The high sequence homology among the HBBMs from different species suggests an important biological role of the protein.

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