Chemical and biological characterization of a truncated form of acidic fibroblast growth factor from bovine brain

Abstract

Two forms of acidic fibroblast growth factor were isolated from bovine brain by a combination of ammonium sulfate precipitation, cation-exchange chromatography, heparin-Sepharose affinity chromatography, and reversephase high-performance liquid chromatography. Amino acid analysis, polyacrylamide gel electrophoresis and amino-terminal sequence analysis showed that one form corresponds to a protein with a molecular mass of 16 kDa and the amino-terminal sequence Phe-Asn-Leu-Pro-Leu-Gly-Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr-and thus represents the acidic fibroblast growth factor as previously characterized EMBO J. 5, 1951 -19561. The second mitogen form has a molecular mass of 15.5 kDa. The amino-terminal sequence was established as Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr-. This evidence indicates that the latter form represents an amino-terminally truncated acidic fibroblast growth factor, lacking the first six amino acid residues. Both forms of the protein are biologically active and equipotent with respect to stimulation of the proliferation in vitro of mesodermal cells such as vascular endothelial and adrenal cortex cells.Two potent growth factors for mesenchymal cells, basic fibroblast growth factor (bFGF) from bovine pituitary [l, 21 and acidic fibroblast growth factor (aFGF) from bovine brain [3 -61, have recently been isolated and their amino acid sequences determined. bFGF and aFGF are structurally distinct proteins but they share extensive sequence homology [6]. Both growth factors bind to the same cellular receptors [7]. In addition to being mitogens in vitro, the two proteins possess angiogenic activity in vivo [2, 5, 81. Although no physiological role has yet been established for those mitogens, they may represent physiological angiogenesis factors thought to be involved in the regulation of neovascularization [9]. Mitogens structurally identical or very closely related to bFGF also occur in bovine [lo]

Acidic fibroblast growth factor is present in nonneural tissue: isolation and chemical characterization from bovine kidney

“…It is well established (Bohlen et al, 1985a;Gautschi et al, 1985Gautschi et al, , 1986) that, with similarly prepared brain extracts, heparin-Sepharose fractions eluting at approximately 1-1.2 M NaCl contain aFGF. Therefore, the corresponding fractions from kidney (marked by a horizontal bar and designated "A" in Figure 1) were pooled and subjected to further analysis.…”

Microvascular and collateral adaptation in swine hearts following progressive coronary artery stenosis

“…We postulated (2) and favored the existence of biochemical transmitters because mitosis and DNA-synthesis occur (although at a lower rate) also in small veins accompanying growing collaterals where the distribution of physical forces differs, and we recently (26) isolated potent mitogenic peptides of the Fibroblast Growth Factor family (27,28,29) from bovine, porcine, and canine hearts. The role of these peptides in the process of vascular growth remains to be elucidated.…”

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

“…This aberrant chromatographic behavior is probably caused by aggregation of peptides in mixtures of truncated forms as described by McKeehan and Crabb [20]. The activity of proteolytic enzymes is a causative factor for the truncated forms of aFGF and has already been reported [7]. Additional extractions of bovine hearts with an inhibitor of proteolytic enzymes (pepstatin A) produced a peptide with a molecular mass of about 20 kDa which is mitogenic; when blotted on to nitrocellulose it reacted with the antibody against aFGF; it also has a blocked amino terminus.…”

“…aFGF and bFGF were isolated from the heart using the methods described for brain by Gautschi et al 1986 [7]. 3 kg of the left ventricle were homogenized in a Waring Blender with 6 10.1 5 M ammonium sulfate for 3 min.…”

“…These peptides were found to be identical to acidic and basic fibroblast growth factor by SDS/PAGE, by immunoblotting and by gas-phase amino acid microsequencing. The success of our procedures was aided by adopting gradient elution from the heparinSepharose column as described by Gautschi et al [7] which resulted in the pure fractions of aFGF and nearly pure fractions of bFGF (see Figs. 4 and 5).…”