Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor

Two mitogenic peptides in bovine liver extract were purified to apparent homogeneity by monitoring the purification steps with two in vitro bioassays; one based on stimulation of adult bovine aortic arch endothelial cell proliferation and the other incorporation of [3H]thymidine to mouse fibroblast 3T3 cells. The purification procedure involved cation-exchange chromatography followed by affinity chromatography on heparin-Sepharose and two steps of reversed-phase HPLC. The purified material showed the same biological activity as pituitary basic fibroblast growth factor (FGF). Amino acid analyses of the purified mitogen yielded a similar, but not identical composition to that of bovine pituitary basic FGF(1-146) reported previously. Gas-phase microsequencing identified two sequences in equal amounts in the purified preparation. Furthermore, the sequencing results are in accord with the theoretical data obtained when two truncated forms of basic FGF, corresponding to FGF(12-146) and (16-146), are being sequenced simultaneously. Basic FGF(12-146) is a novel truncated form of basic FGF which has not been isolated before although the (16-146) fragment has been found previously in kidney, corpus luteum, and adrenal. SDS-PAGE analysis could not separate the two forms and showed that both migrated as a protein of about 15,100 daltons, which is slightly smaller than intact basic FGF(1-146) (16,200 daltons). These results, taken together, indicate that at least some of the mitogenic activity in liver may be derived from basic FGF-related polypeptides.     

Isolation of an amino terminal extended form of basic fibroblast growth factor

Extraction of bovine pituitaries in the presence of enzyme inhibitors (2 mM PMSF, 2 mM sodium tetrathionate, 15 microM pepstatin A, and 1 mM EDTA) resulted in the isolation of two distinct forms of basic fibroblast growth factor. Partial characterization of both molecules showed one form to be identical to basic FGF(1-146) which has already been reported by our laboratory. The second form was estimated by SDS-PAGE to have a molecular weight of 17,000 Daltons which is slightly larger than that of basic FGF(1-146). Amino acid analysis shows the presence of 8 new residues more than basic FGF(1-146) which accounts for the difference in molecular weight. Gas-phase sequencing of this molecule indicated that it bears a blocked amino terminus. Furthermore, this higher molecular weight form of basic FGF did not show immunoreactivity with antibodies specific for the amino terminus of basic FGF(1-146) but cross reacted with antibodies generated against midportion fragments of basic FGF(1-146), indicating that the molecule is amino terminally extended. Like basic FGF(1-146), the molecule is a potent mitogenic factor for vascular endothelial cells. Taken together these results demonstrate the existence of a precursor form of basic FGF which is extended by 8 residues at the amino terminus with the first residue being blocked.     

Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis

Western blotting of low molecular weight (Mr) acidic and basic isoelectric point (pI) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, Mr 6045, pI 4.4), transforming growth factor type alpha (TGF alpha, Mr 5623, pI 6.8), insulin-like growth factor (IGF-I, Mr 7649, pI 8.3), and basic fibroblast growth factor (bFGF, Mr 15,000-17,000, pI 9.6) all transferred with high efficiency (74.1 +/- 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46-98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% (v/v) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGF alpha, and IGF-I were not detectable while 6.25-100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic pI proteins.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

Purification in high yield of brain fibroblast growth factor by preparative isoelectric focusing at pH 9.6

The purification of fibroblast growth factor (FGF) from bovine brain has been reported (Gospodarowicz, D., Bialecki, H., and Greenburg G. (1978) J. Biol. Chem. 253, 3736-3743). Further studies have shown that bovine brain fibroblast growth factor is composed of three fragments derived by limited proteolysis from myelin basic protein (Westall, F. C., Lennon, V. A., and Gospodarowicz, D. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4675-4678). Two of these fragments (FGF-1 and -2) had a high specific mitogenic activity. In the presence study, we have used isoelectric focusing to purify brain FGF to homogeneity. The principal active components of brain FGF preparations partially purified by gel filtration on Sephadex G-75 can be recovered in high yield from isoelectric focusing in sucrose. These components have a pI of 9.6 and 9.54, respectively. Their electrophoretic mobility, amino acid composition, and biological properties are identical with those previously reported for FGF-1 and -2.     

Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor

Monoclonal antibodies (McAbs) were developed that identify the complete (1-146 aa) and the NH2-terminal truncated (des 1-15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1-15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine-brain-derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2-terminal of complete bFGF. McAb 42 recognizes a "core" epitope found on both the complete and des 1-15 aa bFGFs. The McAbs are murine IgGs with affinity constants of 10(7)-10(8) liter/M for bovine-pituitary-derived bFGF. McAbs 8 and 42 have been used in a two-site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine-brain-derived aFGF. These McAbs should be useful in distinguishing the native and des 1-15 aa forms of bFGF from each other, and from aFGF and other growth factors.     

Retina- and eye-derived endothelial cell growth factors: partial molecular characterization and identity with acidic and basic fibroblast growth factors

Two retina-derived growth factors have been isolated on the basis of their ability to stimulate the proliferation of capillary endothelial cells in vitro. Gas-phase sequence analysis identified the amino-terminal sequence of the major form of the mitogen as being identical with residues 1-35 of bovine basic fibroblast growth factor (FGF). Amino-terminal sequence analysis of the second form identified 28 residues that are indistinguishable from those of brain acidic FGF (residues 1-28). The possibility that these retina-derived endothelial cell growth factors are related to, if not identical with, basic and acidic FGF is supported by observations that they have similar molecular weights (15000-16000), similar retention behavior on all steps of chromatography (ion-exchange, heparin-Sepharose), and similar amino acid compositions and that they cross-react with antibodies to basic and acidic FGF. The eye-derived growth factors, like FGF, are potent stimulators of capillary endothelial cell growth in vitro. The results identify the major retina-derived endothelial cell growth factor as indistinguishable from basic FGF and demonstrate the presence of an acidic FGF in the eye. They suggest that at least some of the mitogenic, angiogenic, and neovascularizing activities described as being present in the retina are due to the existence of FGF in this tissue. The implications of this finding on the etiology and pathophysiology of vasoproliferative diseases of the eye are discussed.     

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.     

Purification of bovine somatomedin.

A procedure for the purification of bovine somatomedin (SM⁴) is presented. The purification scheme utilizes ultrafiltration through membranes of nominal mol. wt. cutoffs, molecular sieve chromatography and finally iso-electric focusing. Two peaks of SM activity, measured by the $\text{in vitro}$ stimulation of ³⁵S-Na₂SO₄ and ³H-thymidine uptake by costal cartilage, were present after focusing; an acidic component having a pI of 6.0 – 6.7 and a basic component having a pI in the range of 7.8 – 8.3. The acidic component comprised 2% of the initial activity and was 120,000-fold purified: the basic component comprised 10% of the initial activity and was 350,000-fold purified relative to the starting material. These components are similar in molecular size and pI to SM-A and SM-C isolated from human plasma.     

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.     

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.     

Neuronal-derived factors regulating glial cell proliferation and maturation

We purified to homogeneity two active factors, named astroglial growth factors (AGFs: AGF2 and AGF1), from bovine brain after two and three chromatographic steps, respectively. We found that AGFs have a strong affinity for heparin. Therefore, heparin affinity chromatography was used to purify rapidly and efficiently these growth factors. The purified AGF1 is an acidic protein (pI: 5.5) with an apparent molecular weight of about 17,500 daltons; the AGF2 is a basic protein (pI: 9.5) of 18,500 daltons. The comparison of the physico-chemical properties, the aminoacid composition and the amino-terminal sequence of the AGFs with that of other growth factors isolated from the brain and affecting the proliferation of other cell types has indicated that AGF1 and AGF2 are identical to the acidic and basic fibroblast growth factor (FGF), respectively. Both factors stimulate the proliferation as well as the morphological and biochemical maturation of the astroglial cells. Both factors enhance also the multiplication of oligodendroglial cells. Polyclonal and monoclonal antibodies against AGFs have been prepared and used for immunocytochemical localization of these molecules in the rat brain and cerebellum. AGFs are found exclusively in neuronal cells.     

Fetuin: a serum component associated with rat Sertoli and spermatogenic cells in coculture

Cocultures of rat Sertoli-spermatogenic cells plated in a culture medium supplemented with 10% fetal bovine serum for 6-12 h and then maintained in serum free, hormone/growth factor-supplemented medium accumulated an acidic glycoprotein of molecular weight of 68,000 dalton (68 kD) and isoelectric point range of about 4.2-3.5. Anion exchange chromatography has allowed the partial purification of this protein, which consists of a major protein band of 68 kD and two minor, low molecular weight components. A rabbit antiserum raised against the 68 kD component also crossreacts with the two low molecular weight components, thus suggesting that these two minor components are antigenically related to the 68 kD protein. The 68 kD protein has been identified as fetuin, the major component of fetal bovine serum, based on similar molecular weight, isoelectric point, immunoreactivity and trypsin inhibitory activity. Labeling experiments with [14C]amino acid mixture show that 68 kD protein is not synthesized by cocultured rat Sertoli and spermatogenic cells. Immunocytochemistry and Western blot approaches carried out under various experimental conditions support the view that the fetuin-68 kD protein is taken up from serum by both Sertoli cells and pachytene spermatocytes. Because fetuin 1) behaves as a carrier protein for growth factors, 2) has protease inhibitory activity, 3) is preferentially internalized by Sertoli cells and pachytene spermatocytes and 4) fetal bovine serum-supplemented medium impairs spermatogenic cell viability, there is a need to further define appropriate conditions for optimizing long-term viability and differentiation of spermatogenic cells in vitro.     

Heterogeneity of free alpha-subunit in term placenta

Free alpha-subunit in normal term placenta was examined for molecular weight, electric charge and ability to combine with standard hCG-beta in comparison with extracellular free alpha-subunit and standard hCG-alpha dissociated from urinary hCG in vitro. The gel chromatography on Sephadex G-100 of the placental extract revealed three major immunoreactive hCG-alpha peaks, designated as P alpha-A (Kav = 0.32-0.46), P alpha-B (0.47-0.58) and P alpha-C (0.59-0.70), near the position of standard hCG-alpha. In the isoelectric focusing, while P alpha-A was mainly distributed over the acidic region, the major components of P alpha-B and P alpha-C were distributed over the basic region. Furthermore, in the combination study with standard hCG-beta, such a alpha-subunit with acidic pI scarcely showed any combining activity whereas alpha-subunit with basic pI revealed significant combining activity. These results suggest the following possibilities: that 1) the various size species of placental alpha-subunit may be responsible for the progressive glycosylation; 2) the small alpha-subunit with basic pI may combine with beta-subunit to form immunoreactive hCG; 3) the alpha-subunit, which has not associated with beta-subunit, may be converted to a large and incombinative form with acidic pI by further glycosylation, followed by secretion as a free alpha-subunit.     

Basic and acidic fibroblast growth factors interact with the same cell surface receptors

Despite quantitative differences, the activity of basic and acidic fibroblast growth factors (FGF) on a wide variety of normal diploid cells derived from neuroectoderm and mesoderm is intrinsically similar. This suggests that they bind to the same cell surface receptors. This was investigated using a baby hamster kidney cell line (BHK-21) as a model. BHK-21 cell membrane components that interact with basic and acidic FGF have been identified by covalent cross-linking to their respective 125I-labeled ligands. Under appropriate conditions, basic and acidic 125I-FGF were cross-linked, using disuccinimidyl suberate, to two receptor species with apparent molecular masses of 145,000 and 125,000 daltons, respectively. The labeling of those receptors is inhibited when either native basic or acidic FGF are present in excess during incubation of cells with either acidic or basic 125I-FGF. Competition of basic 125I-FGF with increasing concentrations of native acidic FGF results in a preferential decrease in the labeling of the 125,000-dalton species, whereas competition of acidic 125I-FGF with increasing concentrations of native basic FGF leads to a preferential decrease in the labeling of the 145,000-dalton species. The data suggest that qualitatively both mitogens interact with the same 145,000- and 125,000-dalton receptor species. The different affinities displayed by acidic and basic FGF toward their common receptor molecules could explain why acidic FGF, depending on the cell type considered, is 20-100-fold less potent than basic FGF.     

Purification and characterization of heparin-binding growth factors from porcine uterus.

Heparin-binding growth factors present in pig uterine tissue were purified by approx. 50,000-fold using a combination of ammonium sulphate precipitation, ion-exchange chromatography and heparin-affinity chromatography. Purification of the uterus-derived growth factors (UDGFs) was monitored by the stimulation of [3H]thymidine incorporation into Swiss 3T3 cells and by a radioreceptor assay using 125I-labelled epidermal growth factor (EGF) as the ligand. The latter was shown to be a novel, rapid and reliable assay for heparin-binding growth factors which utilizes their trans-modulation of EGF receptor affinity. UDGFs exhibit strong affinity for immobilized heparin and two forms, named alpha UDGF and beta UDGF, were distinguished by salt gradient elution from heparin-agarose affinity columns. beta UDGF activity was eluted from heparin-agarose between 1.5 M- and 1.8 M-NaCl, and was correlated with the elution of a protein doublet of 17.2 kDa and 17.7 kDa. Immunoblotting of heparin-purified beta UDGF indicated that the beta UDGF doublet is immunologically related to the 146-amino-acid form of bovine basic fibroblast growth factor (bFGF), and that the 17.2 kDa component is an N-terminally truncated form of the 17.7 kDa component. After purification by C4 reversed-phase h.p.l.c., this doublet was biologically active and greater than 95% pure as assessed by silver-stained SDS/PAGE. Amino acid composition and sequence analysis confirmed that these beta UDGF polypeptides were microheterogeneous forms of bFGF. Fractions containing alpha UDGF activity were eluted from heparin-agarose in 1.3 M-NaCl. These fractions contained a 16.5 kDa protein which co-migrated on SDS/polyacrylamide gels with recombinant human acidic FGF (aFGF) and which which cross-reacted with an antiserum raised against aFGF. The identification of heparin-binding growth factors in porcine uterus at the time of implantation raises the possibility that they function in the reproductive tract during early pregnancy.     

Bovine fibroblast growth factor: comparison of brain and pituitary preparations

Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl- 3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.     

The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei.

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.     

A growth factor from spinal cord.

Mitogenic activity is present at a variety of sites in the central nervous system. A growth factor was purified from neonatal bovine spinal cord. It has a pI of 9.5-9.8 and a molecular weight of about 11,000 daltons. Spinal cord growth factor is a basic polypeptide that is inactivated by extremely acid or basic conditions. Its mobility on SDS polyacrylamide gels suggests that this factor is different from pituitary FGF and brain FGF-1.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.