Isolation of tropoelastin a from lathyritic chick aortae.
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ABSTRACT:
SUBMITTER: Rich CB
PROVIDER: S-EPMC1153251 | biostudies-other | 1984 Jan
REPOSITORIES: biostudies-other
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Project description:Chymotryptic fingerprint analyses of tropoelastin a and tropoelastin b demonstrated a very close relationship between these two polypeptides synthesized in a cell-free system under the direction of chick-embryo polyribosomal mRNA. A similar study on tropoelastin polypeptides extracted in their hydroxylated and under-hydroxylated forms from artery cells incubated with [3H]valine in the absence and presence of alpha alpha'-bipyridine or 3,4-dehydroproline confirmed this close relationship and suggested that tropoelastins a and b are likely to be the products of a single gene. Pulse-chase experiments in which the synthesis and secretion of tropoelastin by artery cells were monitored demonstrated that, after a pulse with [3H]proline, the polypeptides rapidly appeared in the medium and the half-time of tropoelastin secretion was approx. 30 min. Further pulse-chase studies, in which [3H]tropoelastin contents of subcellular fractions were determined, showed that rough and smooth microsomal fractions contained maximal amounts of tropoelastin at different times. The quantity of tropoelastin in the smooth-microsomal fraction was always only a small proportion of that in the rough-microsomal fraction, suggesting rapid translocation of the polypeptides to the plasma membrane. Incubation of the cells with 0.1 mM-colchicine did not markedly alter the rate of secretion or the distribution of tropoelastin between the subcellular fractions, whereas when 1 microM-monensin was included in the incubations the polypeptides were retained in the rough microsomal fraction. The results are consistent with the proposal that tropoelastin may follow a pathway of secretion from rough endoplasmic reticulum to the plasma membrane via secretory vesicles.
| S-EPMC1152605 | biostudies-other
Project description:The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.
| S-EPMC1146942 | biostudies-other
Project description:Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10--20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.
| S-EPMC1186406 | biostudies-other
Project description:Electrophoretic analyses of the products of cell-free translation of elastin mRNA isolated from 17-day chick-embryo thoracic arteries have demonstrated that the elastin mRNA codes for polypeptides that are slightly larger than the cellular tropoelastin polypeptides synthesized and secreted by matrix-free artery cells. Pulse-chase experiments with cells labelled with [3H]proline established that newly synthesized tropoelastin polypeptides were associated solely with membrane-bound particulate fractions. Cell-free translation of membrane-bound and free polyribosomes isolated from artery cells revealed that the tropoelastin mRNA was associated predominantly with the membrane-bound fraction. When rough-microsomal fractions, isolated from cells labelled with [3H]proline for 10 min, were treated with proteinases in the presence and in the absence of detergent, the nascent tropoelastin polypeptides were shown to be susceptible to proteolysis only when the integrity of the membranes was destroyed by detergent treatment. In similar experiments tropoelastin polypeptides synthesized by membrane-bound polyribosomes in the nuclease-treated reticulocyte lysate were also resistant to the proteolytic-enzyme treatment. The results suggest that tropoelastin polypeptides are synthesized on membrane-bound polyribosomes and discharged into the lumen of the endoplasmic reticulum with co-translational removal of a signal peptide.
| S-EPMC1144050 | biostudies-other
Project description:Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk-tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly-d-lysine. When patterned by drying on grooved polydimethylsiloxane (3.5 ?m groove width, 0.5 ?m groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk-tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs.
| S-EPMC4308504 | biostudies-literature
Project description:Tropoelastin, as the monomer unit of elastin, assembles into elastic fibers that impart strength and resilience to elastic tissues. Tropoelastin is also widely used to manufacture versatile materials with specific mechanical and biological properties. The assembly of tropoelastin into elastic fibers or biomaterials is crucially influenced by key submolecular regions and specific residues within these domains. In this work, we identify the functional contributions of two rarely occurring negatively charged residues, glutamate 345 in domain 19 and glutamate 414 in domain 21, in jointly maintaining the native conformation of the tropoelastin hinge, bridge and foot regions. Alanine substitution of E345 and/or E414 variably alters the positioning and interactive accessibility of these regions, as illustrated by nanostructural studies and detected by antibody and cell probes. These structural changes are associated with a lower propensity for monomer coacervation, cross-linking into morphologically and functionally atypical hydrogels, and markedly impaired and abnormal elastic fiber formation. Our work indicates the crucial significance of both E345 and E414 residues in modulating specific local structure and higher-order assembly of human tropoelastin.
| S-EPMC5686564 | biostudies-literature
Project description:A structural protein blend system based on silkworm silk fibroin and recombinant human tropoelastin is described. Silk fibroin, a semicrystalline fibrous protein with beta-sheet crystals provides mechanical strength and controllable biodegradation, while tropoelastin, a noncrystallizable elastic protein provides elasticity. Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) indicated that silk becomes miscible with tropoelastin at different blend ratios, without macrophase separation. Fourier transform infrared spectroscopy (FTIR) revealed secondary structural changes of the blend system (beta-sheet content) before and after methanol treatment. Atomic Force Microscopy (AFM) nano-indentation demonstrated that blending silk and tropoelastin at different ratios resulted in modification of mechanical features, with resilience from approximately 68%- approximately 97%, and elastic modulus between 2 and 9 Mpa, depending on the ratio of the two polymers. Some of these values are close to those of native aortic elastin or elastin-like polypeptides. Significantly, during blending and drying silk-tropoelastin form micro- and nano-scale porous morphologies which promote human mesenchymal stem cell attachment and proliferation. These blends offer a new protein biomaterial system for cell support and tailored biomaterial properties to match mechanical needs.
| S-EPMC2933948 | biostudies-literature
Project description:Antiserum was prepared in sheep against insoluble elastin isolated from embryonic-chick aortae. In an indirect immunoprecipitation test, the antiserum reacted quantitatively with small amounts of radioactively labelled purified tropoelastin prepared from embryonic-chick aortae. The antiserum did not cross-react with chick procollagen, and the antiserum uas used to identify radioactively labelled tropoelastin secreted by chick aorta cells in suspension culture.
| S-EPMC1163887 | biostudies-other
Project description:This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells.Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin.Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W.Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.
| S-EPMC5827807 | biostudies-literature
Project description:A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.
| S-EPMC1148163 | biostudies-other