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1.
Pamela A. Maher 《Journal of cellular physiology》1993,154(2):350-358
Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibited of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled. © 1993 Wiley-Liss, Inc. 相似文献
2.
The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms. 相似文献
3.
Vibrational CD (VCD) and ir absorption data are reported for a series of films of Boc-(L -Ala)n-OMe homo-oligopeptides (n = 3–7) in the amide I and A regions. The data evidenced a sharp change between n = 3 and n = 4, which parallels the onset of β-structure formation, and another between n = 5 and n = 6, which parallels the full development of β-structure. This represents the first report of the application of VCD to oligopeptide conformation. The data resembled earlier reported film VCD studies of higher-molecular-weight polypeptides of known β-structure. 相似文献
4.
We have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross-linking experiments show that radio-labeled basic fibroblast growth factor-saporin (FGF-SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF-SAP. In a study of 4 different cell types, there is a decrease in the ED50 of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF-SAP which will result in cytotoxicity occurs very rapidly; 5 minutes of incubation of 10 nM basic FGF-SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that the basic FGF-SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti-FGF for therapeutic and research uses in vivo is discussed. 相似文献
5.
Vibrational CD (VCD) of amides A, I, and II vibrations of a variety of polypeptide films have been measured. VCD of films of α-helical and β-sheet structures are compared in the three regions. Reproducible spectra could only be obtained for thin films free of orientation dependence. The sign and band shape of the VCD of films are not always the same as that in solution. However, the magnitude of the observed VCD seems to correlate with the secondary structure such that α-helical molecules typically have much larger Δε/ε values than do β-sheet molecules. The possibility of interference by artifacts owing to light-scattering effects is discussed. 相似文献
6.
R K Dukor T A Keiderling V Gut 《International journal of peptide and protein research》1991,38(3):198-203
Vibrational Circular Dichroism (VCD) spectra of unblocked L-proline oligopeptides, (Pro)n n = 3 to 7, dissolved in D2O are reported. For these oligomers, the VCD spectra can be attributed to a conformational dominance of the trans amide conformation with subunits interrelated by a left-handed twist, particularly for the longer oligomers. As a function of oligomer length, formation of this conformation starts at n = 3; and by n = 5 a spectrum closely resembling that of the poly-L-proline II helix in shape and magnitude is seen. The VCD data are compared with previous (Pro)n results using IR, CD, Raman and NMR spectroscopies, and reasons for the variations in interpretation are discussed. 相似文献
7.
Thrombin inactivates acidic fibroblast growth factor but not basic fibroblast growth factor 总被引:7,自引:0,他引:7
R R Lobb 《Biochemistry》1988,27(7):2572-2578
Incubation of bovine brain derived acidic fibroblast growth factor (aFGF) with bovine or human thrombin, 0.5 NIH unit/mL, for 24 h at 37 degrees C results in cleavage of the mitogen, generating a 14-kilodalton fragment which has significantly reduced affinity for immobilized heparin as compared to aFGF, and is at least 50-fold less potent at stimulating mitogenesis. In addition, an 18 amino acid peptide, aFGF(123-140), is generated, identifying one of the thrombin cleavage sites as the Arg-122/Thr-123 bond. The peptide, aFGF(123-140), is neither mitogenic itself nor an inhibitor of the mitogenic activity of aFGF. The cleavage of aFGF by thrombin is inhibited by heparin (50 micrograms/mL) and is completely blocked by the irreversible thrombin inhibitors D-Phe-Pro-Arg chloromethyl ketone and hirudin. Incubation of aFGF with 50 units/mL thrombin at 37 degrees C results in rapid cleavage of the mitogen into several fragments. In contrast, incubation of bovine brain derived basic fibroblast growth factor with 1 unit/mL thrombin for 24 h, or 50 units/mL thrombin for 6 h, does not result in significant cleavage of mitogen. The results show that the C-terminal region of aFGF is of functional importance in both mitogenesis and heparin binding. Most importantly, a novel role for anionic heparin-binding growth factors and their fragments is indicated in physiologic and pathologic situations associated with thrombin generation. 相似文献
8.
Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin. 相似文献
9.
Basic fibroblast growth factor (bFGF) was modified by biotinylation via amino group substitution, using biotin-N-hydroxysuccinimide ester at molar reaction ratios of 20, 200, and 2000 per bFGF molecule (respectively named bio-bFGF.20, bio-bFGF.200, and bio-bFGF.2000). The biotinylated bFGF derivatives, bio-bFGF.20 and bio-bFGF.200, conserved the same affinity for heparin as native bFGF, in contrast to bio-FGF.2000 which lost this property. Bio-bFGF.20 and bio-bFGF.200 were as effective as native bFGF in their capacity to compete with 125I-bFGF for binding to bFGF receptor on bovine brain membranes. The biological activity of these bFGF derivatives was tested on CCL39 cells; bio-bFGF.20 and bio-bFGF.200 were as able as native bFGF to promote growth of CCL39. 相似文献
10.
L O Narhi T Arakawa M D McGinley M F Rohde K R Westcott 《International journal of peptide and protein research》1992,39(2):182-187
To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mM sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of alpha-helix, beta-sheet, and unordered structure. The addition of the alpha-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of alpha-helix, at the apparent expense of beta-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding. 相似文献
11.
12.
Dextran sulfate protected bFGF from heat and acid inactivation and from proteolytic degradation. The protective effect was stronger than that of heparin which is known as a stabilizer of bFGF. Dextran sulfate and bFGF formed a high molecular weight complex via ionic interaction when mixed together in aqueous solution. The complex was dissociated when the ionic strength was increased and the protective effect was completely abolished. Successive digestion of bFGF with Staphylococcus aureus V8 protease and pepsin followed by affinity chromatography on an immobilized dextran sulfate column and reversed-phase high performance liquid chromatography yielded three positively charged fragment peptides, Tyr24-Phe30, Tyr106-Trp114 and Tyr124-Leu138. These results suggest that dextran sulfate stabilizes bFGF by binding close to the putative heparin binding sites of the bFGF molecule. 相似文献
13.
In this article, we describe the mutual structural effect of the interaction between the model membranes and polylysine and poly-l-arginine. Vibrational circular dichroism (VCD), a method exceptionally sensitive to the polypeptide structure that has not been established in such studies before, was the primary method of this study. A complementary technique, electronic circular dichroism, was applied to verify the newly obtained results and as a bridge to the previous studies. We used micelles composed of sodium dodecyl sulfate (SDS) as a monolayer membrane model and large unilamellar vesicles composed of phospholipids as a bilayer membrane model. We describe the conformational changes of the polypeptides caused by the interaction with the model membranes. Among others, the presence of the liposomes in the solution generated special conditions for the formation of the α-helical structure of poly-l-arginine; the presence of SDS induced the formation of the β-structure of polylysine. From a methodological point of view, we emphasize the advantages of infrared spectroscopic techniques for the liposomic membrane studies as well as the preference of ultraviolet techniques for smaller micellar systems. 相似文献
14.
Vibrational circular dichroism (VCD) has been shown to be a useful technique for characterization of the qualitative secondary structure type for linear polypeptides and oligopeptides. A brief review of characteristic spectral responses and applications is given. Since VCD is dependent on relatively short range interactions, it detects residual structure in such oligomers even if long range order is lost. VCD studies presented here for Lys oligomers as well as Lys and Glu polymers as a function of length, salt added and temperature, confirm residual local order in these 'random coils'. Comparison to results with Pro oligomers, supports an interpretation that these extended structures have a left handed twist conformation. The 'coil' VCD is shown to be significantly reduced in intensity by temperature increase and by decrease in peptide length. By contrast, for partially alpha-helical Ac-(AAKAA)3GY-NH2 oligomers, the spectrum changes to the high temperature Lys(n) shape on heating, first losing then gaining intensity, indicating an equilibrium shift between structured states, from helix to coil (locally ordered) forms. VCD is shown to be a useful technique for monitoring local order in otherwise random coil structures. 相似文献
15.
Y D Sohn H J Lim K C Hwang J H Kwon H Y Park K H Chung S Y Cho Y Jang 《Biochemical and biophysical research communications》2001,284(4):931-936
Basic fibroblast growth factor (FGF-2) is a pleiotropic mitogen which plays an important role in cell growth, differentiation, migration, and survival in different cells and organ systems. Recently, several clinical applications for FGF-2 gene transfer are being evaluated in wound healing and collateral artery development to relieve myocardial and peripheral ischemia due to the ability of FGF-2 to regulate the growth and function of vascular cells. However, FGF-2 lacks a classical hydrophobic secretion signal peptide, the FGF-2 chimeras containing various signal sequences have been explored. In this study, a novel recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 (Met1 to Leu9) with eight amino acid residues of signal peptide of FGF-4 (Met1 to Ala8) to better increase the secretion level of FGF-2. When the recombinant FGF-2 gene, cloned into the expression vector with CMV promoter, was expressed in COS-7 cells, the recombinant 4sFGF-2 was highly secreted into the media. The secreted 4sFGF-2 showed the same biological activity as the native FGF-2 in the dose-response effects on DNA synthesis and cell growth of rat aortic smooth muscle cells (RASMCs) and NIH3T3 cells. The 4sFGF-2 also was able to activate MAPK as wild FGF-2 in RASMCs. These results indicate that a novel recombinant 4sFGF-2 may be useful as clinical applicability of angiogenic growth factor gene transfer. 相似文献
16.
Vibrational circular dichroism of polypeptides. VI. Polytyrosine alpha-helical and random-coil results 总被引:1,自引:0,他引:1
We have measured the VCD of polytyrosine in the amide I and II regions in dimethyl sulfoxide (DMSO) and in 80:20 mixtures of DMSO with trifluoroethanol, trifluoroacetic acid (TFA), and dichloroacetic acid and in 50:50 mixtures of DMSO and trimethyl phosphate (TMP). Additionally, VCD was obtained for deuterated polytyrosine in DMSO and DMSO:D2O, DMSO:TFA(d1), and DMSO:TMP mixtures as before. Amide A VCD was obtained in DMSO and DMSO:TMP mixtures. In the pure solvent, VCD of an opposite sign was seen as compared with that seen in the mixtures. The latter were characteristic in sign pattern and shape of right-handed α-helices for poly(L -tyrosine). The pure polytyrosine:DMSO results are similar to those of polylysine:D2O at neutral pH and poly(β-benzyl-aspartate) in DMSO and may be characteristic of random-coil VCD. 相似文献
17.
Site-directed PEGylation of human basic fibroblast growth factor 总被引:2,自引:0,他引:2
Through site-directed mutagenesis, three cysteines of human basic fibroblast growth factor (hbFGF) were replaced with serine residues, resulting in a hbFGF mutant named hbFGFSer25,69,92. The mutant with only one cysteine residue at the 87th position, whose mitogenic activity was comparable to that of wild-type hbFGF, was further coupled to polyethylene glycol with a molecular size of 5 kDa (PEG5K) via the cysteine residue to obtain another hbFGF derivative, PEG5K-hbFGFSer25,69,92. The optimal modification reaction was conducted at 4 degrees C for 4 h at a molar ratio of PEG5K to hbFGFSer25,69,92 of 20:1. The result of SDS-PAGE showed that the modification extent was up to 80%. The modified product was purified by ion exchange chromatography. Compared to the hbFGF mutant, the purified PEG5K-hbFGFSer25,69,92 still retained about 60% of the mitogenic activity of the former, which provided a good basis for further studying the bioactivity of the PEGylated protein in vivo. 相似文献
18.
Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. 总被引:29,自引:0,他引:29
J E Turnbull D G Fernig Y Ke M C Wilkinson J T Gallagher 《The Journal of biological chemistry》1992,267(15):10337-10341
The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF. 相似文献
19.
Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor 总被引:1,自引:0,他引:1
A Wellstein R Lupu G Zugmaier S L Flamm A L Cheville P Delli Bovi C Basilico M E Lippman F G Kern 《Cell growth & differentiation》1990,1(2):63-71
We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo. 相似文献
20.
S A Thompson 《The Journal of biological chemistry》1992,267(4):2269-2273
Basic fibroblast growth factor has 4 cysteine residues in its amino acid sequence, two of which are perfectly conserved within the fibroblast growth factor family of proteins suggesting a disulfide bond at this position. Furthermore, thiol titration of bovine pituitary basic fibroblast growth factor (bFGF) indicates the presence of two free thiols, which is consistent with an intramolecular disulfide. Direct analysis of natural and recombinant fibroblast growth factor proteins have not confirmed the existence of such a disulfide. Instead, the two nonconserved cysteines of bFGF purified from bovine pituitaries are S-thiolated with glutathione. Inclusion of 75 mM N-ethylmaleimide during the homogenization of the pituitaries effectively blocks the S-thiolation, demonstrating that this modification is an artifact of the purification procedure. Analysis of the N-ethylmaleimide purified bovine pituitary bFGF suggests that the natural protein is in the correct redox state when all 4 cysteines are in the reduced form. 相似文献