Purification of the receptor for platelet-derived growth factor from porcine uterus

The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.     

Partial purification and characterization of porcine platelet-derived growth factor (PDGF)

Platelet derived growth factor (PDGF) has been partially purified from porcine platelets. Purification steps included heparin-agarose chromatography of the material released by thrombin-stimulated washed porcine platelets and Blue-Sepharose chromatography. Preparative isoelectric focusing showed that isoelectric point of porcine PDGF is at pH 10.0–11.0 and elution experiments from sodium dodecyl sulfate (SDS) polyacrymlam de gels indicated that its molecular weight is close to 30 kD. The immunoglobulin fraction prepared from anti-human PDGF serum inhibited the mitogenic activity of porcine PDGF. These experiments suggest a homology of porcine and human PDGF. Porcine platelet factor 4 and porcine platelet basic protein were devoid of significant mitogenic activity.     

Purification and characterisation of a growth factor from porcine bone

A growth factor was extracted from porcine bone matrix by demineralisation and purified by heat and acid treatment, hydroxyapatite chromatography and gel filtration under dissociative conditions and reverse-phase HPLC. Using the mitogenic response of osteoblast-progenitor cells from embryonic chicken, a mitogenic activity was purified 3000-fold. The mitogenic protein thus purified shows an apparent molecular mass of 13.5 kDa in both the nonreduced and reduced form on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The mitogenic activity is sensitive to proteinase K, dithiothreitol, and resistant to DNAse, RNase, heat (70 degrees C) and pH (3-10). The factor stimulates the proliferation of osteoblast-progenitor cells from embryonic chick at a concentration of 1 ng/ml. It is active on cells from skin, periosteum and sternum and has no or little activity on cells of the calvaria, intestine or kidney of embryonic chick or on mouse AKR-2B/Balb c/3T3 cell line.     

Purification and characterization of a platelet-derived growth factor and heavy metal-modulated nuclear protein

Platelet-derived growth factor (PDGF), fibroblast growth factor, and heavy metal salts such as sodium arsenite stimulated BALB/c-3T3 cells to synthesize a 31-kDa protein(s) (termed p31) in a concentration-dependent manner. p31 was also synthesized in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). V8 protease digestion of p31 purified from PDGF-, TPA-, and arsenite-treated cells showed identical fragmentation patterns, demonstrating that these agents modulate synthesis of the same (or a highly similar) protein. TPA-induced p31 synthesis was cell cycle-specific, occurring in density-arrested but not exponentially replicating cells. p31 was readily labeled with [35S]methionine but not with [35S]cysteine. Thus it is not a metallothionein. The protein associated with nuclei. It appears to be highly hydrophobic because solubilization required detergents or organic solvents. Reverse-phase high performance liquid chromatography (HPLC) provided further evidence of hydrophobicity. p31 has been purified to homogeneity using sodium dodecyl sulfate gels with electroelution and reverse-phase HPLC. It has an isoelectric point of 6.8. Its nuclear localization and amino acid analysis demonstrate that p31 is not heme oxygenase, a 32-kDa arsenite-induced microsomal protein. Stimulation of p31 synthesis by growth factors, PDGF and fibroblast growth factor; a tumor promoter, TPA; and heavy metal salts suggests that there is overlap in the pathways for mitogenic stimulation and heavy metal stress.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.     

The platelet-derived growth factor beta-receptor kinase insert confers specific signaling properties to a chimeric fibroblast growth factor receptor.

Signal transduction by tyrosine kinase growth factor receptors involves ligand-induced phosphorylation of substrates for the kinase, resulting in mediation of common or receptor-specific biological signals. We have compared signal transduction pathways for the fibroblast growth factor receptor-1 (FGFR-1), the platelet-derived growth factor beta-receptor (PDGFR-beta), and a chimeric FGFR-1 molecule, FGFRchim, in which the FGFR-1 kinase insert was replaced with that of the PDGFR-beta. The different receptors were characterized and found to be functional as ligand-stimulatable kinases, after expression of the respective human cDNAs in porcine aortic endothelial cells. Substrates for the receptors were analyzed by ligand stimulation of [32P]orthophosphate-labeled cells and immunoprecipitation with phosphotyrosine antiserum. A number of phosphoproteins were induced in all the different types of cells, but components specifically induced after stimulation of FGFR-1 and PDGFR-beta expressing cells could also be detected. Examination of receptor-associated substrates by in vitro kinase assays revealed phosphoproteins of 65 and 85 kDa, which were associated with PDGFR-beta and FGFRchim, but not with FGFR-1. The 85-kDa phosphoprotein could correspond to the regulatory subunit of phosphatidylinositol 3' kinase (PI3-K), since phosphatidylinositol 3' kinase activity was detected after ligand stimulation of FGFRchim- and PDGFR-beta- but not FGFR-1-expressing cells. In addition, ligand stimulation of FGFRchim- and PDGFR-beta-expressing cells, but not FGFR-1-expressing cells, led to induction of actin reorganization in the form of circular membrane ruffling. Thus, replacement of a discrete segment of the intracellular domain of the FGFR-1 with the corresponding stretch from the PDGFR-beta resulted in transfer of PDGFR-beta-specific signaling properties to the chimeric molecule.     

Chemical and biological properties of a growth factor from human-cultured osteosarcoma cells: Resemblance with platelet-derived growth factor

A human osteosarcoma cell line, U-2 OS, cultured under serumfree conditions, was shown to produce a growth factor (osteosarcoma-derived growth factor, ODGF) for human-cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin-Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophoresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure. The biological activity of ODGF resided in a cationic, relatively heat-resistant, reduction-susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS-gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000-14,000 and 16,000-17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet-derived growth factor. ¹²⁵I-ODGF could be precipitated by an antibody to platelet-derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet-derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with ¹²⁵I-ODGF for binding to human-cultured glial cells.     

Crystal structure of human platelet-derived growth factor BB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.     

Purification and properties of stringent factor.

The stringent factor from Escherichia coli is the product of the relA locus. It is the enzyme that catalyzes the synthesis of pppGpp and ppGpp eliciting a pyrophosphate transfer from ATP to the 3'--OH of GTP (or GDP). This protein is responsible for the synthesis of pppGpp and ppGpp in stringent strains in response to an amino acid starvation. In vitro it catalyzes the synthesis of these guanosine compounds in either a ribosome-dependent reaction that requires a particular conformation of the ribosome i.e. the presence of an uncharged tRNA recognizing a codon in the acceptor (A) site of the ribosome or in a ribosome-independent reaction at temperatures under 30 degrees in the presence of only buffer, salts, and substrates. Here we report the purification of the stringent factor to near homogeneity. It is a monomeric protein with a molecular weight of 75,000. The properties of the ribosome-independent reaction are studied and it is shown that the presence of certain acidic proteins, such as the 50 S ribosomal proteins L7 and L12 or casein, or 20% methanol or both stimulates the reaction by creating an environment that together with the low temperature further stabilizes the stringent factor.     

Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling

Two homobifunctional cross-linking reagents have been used to cross-link 125I-platelet-derived growth factor (PDGF) to a cell surface component with an approximate Mr = 164,000 that has many characteristics of a specific PDGF receptor. Excess unlabeled PDGF competed for labeling of this component, while high concentrations of fibroblast growth factor, insulin, epidermal growth factor, low density lipoprotein or acetylated low density lipoprotein had no effect. Preincubation of cells with 125I-PDGF at 37 degrees C reduced specific 125I-PDGF binding (down regulation) and produced a parallel decrease in the amount of the 164,000-dalton receptor available for labeling. The 164,000-dalton component contains at least some protein that is accessible to trypsin in the extracellular medium. A complex of comparable Mr is seen on all PDGF-responsive cell types examined, but not on a nonresponsive cell type. 125I-PDGF does not become covalently cross-linked to this component in the absence of a cross-linking reagent.     

Purification of a growth factor related to platelet-derived growth factor and a type beta transforming growth factor secreted by mouse neuroblastoma cells. A general strategy for the purification of basic polypeptide growth factors.

A general strategy was developed for the purification of basic polypeptide growth factors. This method is a combination of gel filtration, weak-cation-exchange h.p.l.c. and reverse-phase h.p.l.c., separating the proteins according to size, charge and hydrophobicity respectively. All steps are carried out at low pH with exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by freeze-drying facilitate the detection of the growth factors by biological assays. By using this method, homogeneous preparations of two basic growth factors were purified in high yield from mouse-neuroblastoma-Neuro-2A-cell-conditioned medium. It is shown that these purified factors are biochemically and immunologically related to platelet-derived growth factor and type beta transforming growth factor from human platelets.     

Purification and properties of cathepsin D from porcine spleen.

Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species.     

Identification of a peptide antagonist for platelet-derived growth factor.

A series of peptides derived from the primary sequence of the B-chain of platelet-derived growth factor (PDGF) was analyzed for their ability to inhibit the binding of 125I-PDGF-AA and 125I-PDGF-BB to PDGF alpha-receptors and PDGF beta-receptors, respectively. A 13-amino acid peptide (ANFLVWEIVRKKP), corresponding to amino acids 116-121 and 157-163 in PDGF B-chain, was found to compete with binding to both alpha- and beta-receptors. Modification of this peptide on the tryptophan residue increased its receptor competing activity. The peptide was found to be a receptor antagonist, since it inhibited dimerization and autophosphorylation of PDGF receptors. When analyzed on intact cells, the peptide was found to have, in addition to the specific inhibitory effect at the receptor level, a nonspecific inhibitory effect on [3H]thymidine incorporation. Our study has identified two regions in PDGF that are of importance for receptor interaction.