Frederick Esch

Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues.

Publisher Summary This chapter provides an overview of molecular characterization and the distribution and biological activities of fibroblast growth factor (FGF) in various tissues. FGF in the pituitary plays a nonmitogenic role in the maintenance of prolactin and thyrotropin responsiveness, while the same molecule in macrophages may promote wound healing and in the corpus luteum angiogenesis. It is based on the diverse biological activities of somatostatin in brain, hypothalamus, stomach, and pancreas and the wide distribution of numerous neuropeptides in both the central nervous system and the gastrointestinal tract. The concept is equally applicable to FGF and perhaps other growth factors that show a wide distribution in many tissues. The results presented in the chapter suggest that heretofore unidentified biologic activities may be related if not identical to acidic and basic FGFs. These would include ovarian growth factor, macrophage-derived growth factor, cartilage growth factor, tumor angiogenic factor, eye-derived growth factor, retina-derived growth factor, corpus luteum angiogenic peptide, follicular angiogenic factor, adrenal vascularizing factor, and endothelial cell growth factors.

Primary structure of bovine brain acidic fibroblast growth factor (FGF)

The major anionic mitogenic polypeptide for endothelial cells, acidic fibroblast growth factor (FGF), has been purified to homogeneity from bovine brain and its complete primary structure established by gas-phase sequence analysis. The 140 amino acid (Mr 16,000) protein has been previously shown to be a potent growth factor for many diverse cell types of mesodermal origin, in vitro, and an angiogenic agent, in vivo. The amino acid sequence of bovine brain acidic FGF has a 53% absolute homology with that of bovine pituitary basic FGF suggesting that these endothelial cell mitogens are derived from a single ancestral gene.

Acidic fibroblast growth factor (FGF) from bovine brain: amino-terminal sequence and comparison with basic FGF.

Acidic fibroblast growth factor (FGF) from bovine brain has been isolated by a combination of salt precipitation, ion‐exchange chromatography, heparin‐Sepharose affinity chromatography and reverse phase h.p.l.c. The amino acid composition of the mitogen is indistinguishable from that of acidic FGF previously purified. The amino‐terminal sequence of acidic FGF was established as Phe‐Asn‐Leu‐ Pro‐Gly‐Asn‐Tyr‐Lys‐Pro‐Lys‐Leu‐X‐Tyr‐X‐Ser‐Asn‐Gly‐X‐Tyr‐Phe‐Leu‐Arg‐Il e‐Leu‐Pro‐Asp‐Gly. Acidic FGF is structurally different from basic FGF as judged by mol. wt., amino acid composition and sequence. In vitro biological comparison of the two growth factors indicates that acidic and basic FGFs possess the same intrinsic activities to stimulate the proliferation of aorta, vein or capillary endothelial cells and adrenal cortex cells, but acidic FGF is 30‐100 times less potent, depending on the cell type.

Somatocrinin (growth hormone releasing factor) in vitro bioactivity; Ca++ involvement, cAMP mediated action and additivity of effect with PGE2.

Abstract The release of GH induced by purified hypothalamic GRF or native or synthetic tumor-derived GRF is antagonized by the presence of CoCl2; it is simulated by 8Br .cAMP, IBMX, cholera toxin, forskolin, with identical maximal effects (Emax). Somatocrinin (GRF) stimulates the efflux of cAMP by the pituitary cells in parallel to the release of GH. Addition of either 8Br .cAMP, IBMX, cholera toxin or forskolin to a maximally stimulating dose of GRF does not increase the response which remains GRF-Emax. In contradistinction with these results PGE2 releases GH with a dose-response curve different from that of GRF, and the combination of PGE2 + GRF produces an Emax far greater than that due to either agonist alone; showing a true additivity. The name somatocrinin is proposed to replace the acronym GRF.

Primary structure of PDC-109, a major protein constituent of bovine seminal plasma

The two major protein components of bovine seminal plasma, PDC-109 and BSP I, have been purified by gel filtration, partition chromatography and reverse-phase high performance liquid chromatography from an 86% ethanol precipitate of bovine seminal plasma ejaculate. The complete 109-residue amino acid sequence of PDC-109 has been established by automated Edman degradation of the intact peptide as well as its proteolytic digestion and cyanogen bromide cleavage fragments. The 12,774 dalton structure has two structurally similar domains of 38 and 41 amino acids, each containing two disulfide bonds.

High biological activity of the synthetic replicates of somatostatin-28 and somatostatin-25

We have isolated form extracts of ovine hypothalami two molecules characterized as somatostatin-28 and somatostatin-4-28 (referred to as somatostatin-25). They were reproduced by solid hase synthesis. In equimolar ratio and depending upon the experimental conditions, synthetic somatostatin-28 ans somatostatin-25 are 3-14 times more potent than somatostatin-14 to inhibit the basal in vitro secretion of growth hormone or as stimulated by prostaglandin (PGE2). In early studies in vivo, somatostatin-28 and somatostatin-25 are also more potent than somatostatin-14 in inhibiting the secretion of growth hormone acutely stimulated in the rat by injection of morphine; somatostatin-28 is also longer-acting than somatostatin-14. These results suggest that somatostatin-14, as originally isolated, is a biologically active fragment of a larger molecule of greater specific activity; it should be considered as another form of somatostatin with high biological activity present in some tissues and likely secreted y the tissues along with somatostatin-14 and possibly other somatostatin-peptides of diverse sizes.

Isolation and characterization of the bovine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 500 bovine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analysis. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala -Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln -Gly-Ala-Lys-Val-Arg-Leu-NH2 using approximately 2 nmol of material.

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH2 using approximately 6 nmol of material.

Isolation and partial characterization of basic fibroblast growth factor from bovine testis

A basic fibroblast growth factor (FGF) has been purified to homogeneity from bovine testis, using ammonium sulfate precipitation of the crude extract followed by three chromatographic steps, involving cation-exchange, heparin-Sepharose, and reversed-phase HPLC. Gas-phase sequence analysis showed the amino-terminal amino acid sequence of the isolated polypeptide as His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-, which is identical to the amino-terminal of the (16-146) fragment of basic FGF previously characterized from corpus luteum, adrenal, and kidney. The purified FGF was shown to have the same biological activity as that of basic FGF (1-146). This finding suggests that basic FGF is present in testis and may act as a local regulator of testicular function. In addition, testicular FGF might play an important role in spermatogenesis and/or the development of testis.

Isolation and partial characterization of an endothelial cell growth factor from the bovine kidney: homology with basic fibroblast growth factor

Two kidney-derived mitogens have been isolated by ion exchange, heparin-Sepharose and reverse-phase high-performance liquid chromatography on the basis of their capacity to stimulate the proliferation of bovine vascular endothelial cells in vitro. Gas phase sequence analysis identified the amino terminal sequences His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-Phe-Phe-Leu and His-Phe-Lys-Asp-Pro-Lys-Arg-Leu, respectively. The sequences are identical to residues 16-32 and 16-23 of bovine basic pituitary Fibroblast Growth Factor (FGF). The possibility that these kidney-derived mitogens are related, if not identical, to pituitary basic FGF is supported by the observations that they have similar molecular weights (15-16 kDa), similar retention behavior on all steps of chromatography and similar amino acid compositions, and they share at least some structural homology. Moreover, the kidney-derived growth factors, like basic FGF, are potent stimulators of capillary endothelial cells, granulosa cells, adrenocortical cells and vascular smooth-muscle cells (ED50 = 50 pg). The results demonstrate the existence of a kidney-derived FGF and suggest that at least some of the mitogenic, angiogenic and neovascularising activities described to be present in the kidney are due to the presence of an FGF-like molecule in this tissue.