Carbon-13 magnetic resonance spectra of aortic alpha-elastin in solution, coacervate and fibrous states.

Carbon-13 magnetic resonance spectra are reported for aortic α-elastin in solution, coacervate and fibrous states. The observation of spectra for the coacervate and fibrous states indicates retention of some chain mobility in these condensed phases and qualitative comparisons demonstrate selected and identifiable losses of resonance intensity on progressing from the solution state to the coacervate and fibrous states. Comparison of the carbonyl regions of α-elastin and the polypentapeptide, HCO-(Val₁-Pro₂-Gly₃-Val₄-Gly₅)_n-Val-OMe, spectra suggests that the polypentapeptide is a good model for the observable carbonyls of α-elastin. With the polypenta-peptide model, certain resonances of elastin in each of the three states can be assigned and related to previously defined features of sequence and of conformation. This approach will ultimately lead to substantial and detailed information on the structure and dynamics of fibrous elastin.     

Characteristic interaction of Ca2+ ions with elastin coacervate: Ion transport study across coacervate layers of α-elastin and elastin model polypeptide, (Val-Pro-Gly-Val-Gly)n

Ion transport characteristics across a macrocoacervate layer membrane composed of aqueous elastin model polypeptides with a specific repeating pentapeptide sequence, H-(Val-Pro-Gly-Val-Gly)_n-Val-OMe (n ≥ 40), were investigated. Transmembrane potential responses for NaCl, MgCl₂, and CaCl₂ concentration-cell systems were measured and examined systematically by comparing with those across a coacervate membrane composed of bovine neck ligamental α-elastin. In the case of the NaCl and MgCl₂ systems, potential responses across these protein liquid membranes were different noticeably from each other depending upon the molecular structure with and without charged peptide side chains, whereas in the CaCl₂ systems the transmembrane potential responses across the noncharged polypentapeptide coacervate membrane were comparable with those across the α-elastin coacervate membrane carrying both the positively and negatively charged amino acid residues as an amphoteric ion-exchange membrane. These results indicated that mechanisms of major Ca²⁺ ion transport are based on the specific and selective interactions with electrically neutral sites of elastin, such as the polypentapeptide backbone chain. © 1996 John Wiley & Sons, Inc.     

Solubilized elastin substrate for continuous fluorimetric assay of kinetics of elastases

Elastolysis is central to progression of emphysema and aortic aneurysms. Characterization of steady-state enzyme kinetics of elastolysis is fettered by the insolubility of mature elastin and the polydispersity of solubilized elastin. We prepared a fluor-tagged, 100-kDa fraction (fEln-100) from commercial α-elastin. It is soluble, less heterogeneous in mass, cross-linked like mature elastin, and likely to retain the capacity of α-elastin to self-assemble. fEln-100 has introduced the ability to compare quantitatively the apparentk_cat and K_m of elastases. For example, metalloelastase (MMP-12) displays higher apparent affinity for fEln-100, while MMP-2 displays faster catalytic turnover.     

DOES ELASTIN CONTRIBUTE TO THE PERSISTENCE OF CORPORA ALBICANTIA IN THE OVARY OF THE COMMON DOLPHIN (DELPHINUS DELPHIS)

The corpora albicantia (CAs) from the ovaries of 39 common dolphins ( Delphinus delphis ) caught in driving fisheries were used, and histologically and immunohistochemically examined. The area of each corpus albicans (CA) and the proportion of that area occupied by elastin were measured using NIH-image software. In all CAs, elastoid material (EM) was apparent although EM area varied in each CA. CAs increased in number with dolphin age. Smaller CAs contained a higher proportion of EM. EM was completely digested by elastase, but not by collagenase. Furthermore, EM was immunostained with anti-α-elastin antibody. These results demonstrated that EM was elastin. The present study is the first to describe the presence of elastin in cetacean CAs. The higher proportion of elastin in small-sized CAs of common dolphins is suggested as the likely cause of the persistence of CA.     

The salmonid elastic fibril. An investigation of some chemical and physical parameters.

Elastic fibrils were isolated, as electron microscopically homogeneous preparations, from salmon (Salmo salar) and trout (Salmo gairdneri) bulbus arteriosus by extraction of other tissue components with guanidinium hydrochloride. The preparations exhibited compositions widely at variance with that of bovine elastin, the differences including both the overall concentration and the relative proportions of the crosslinks. Absorption and fluorescence spectroscopy ruled out the presence of tyrosine-derived crosslinks. The wide-angle X-ray diffraction pattern of the salmonid preparations showed broad reflections corresponding to spacings of 9.8, 4.5, and 2.2 Å, similar to bovine elastin. The mechanical behavior of the salmon preparation was characterized by a linear response to stress, with minimal hysteresis, a Young's modulus of 5.5 × 10⁵ N m^?2, and a breaking strain of 1.5.     

Dielectric relaxation studies on bovine ligamentum nuchae

Dielectric relaxation studies of bovine ligamentum nuchae are reported over the frequency range of 1 MHz to 1 GHz and over the temperature range of 23–48°C. A temperature-dependent relaxation process was observed at low megahertz-frequency with the correlation time of around 40 ns. The result is quite similar to that of a synthetic polypentapeptide (VPGVG) and of α-elastin. The relaxation is proposed to arise in part from the peptide libration within the polypentapeptide of bovine ligamentum nuchae.     

Co-operation between plasmin and elastase in elastin degradation by intact murine macrophages.

Intact, thioglycollate-stimulated murine macrophages cultured on an insoluble [3H]-elastin substratum progressively hydrolysed the elastin. Cell lysates had little activity. We compared the effect of various proteinase inhibitors on elastinolysis by either live cells or cell-free, elastase-rich conditioned medium. Only known inhibitors of macrophage elastase blocked the activity of elastase-rich cell-conditioned medium whereas inhibitors of cathepsin B also suppressed intact cell activity. Serum proteinase inhibitors blocked cell-derived soluble elastase activity but not intact cell elastolytic activity. We also observed that plasminogen added to the cell cultures markedly increased elastinolysis by live macrophages or cell-free elastase-rich medium. Purified plasmin alone had no measurable effect on native elastin. Additional experiments indicated that the plasmin enhancement was due to elastin-dependent activation of latent macrophage elastase. These results indicate that live macrophage elastinolysis is a co-operative process involving multiple proteinases, especially a cysteine proteinase(s) and elastase. Plasmin may be a physiological activator of latent macrophage elastase.     

Effects of metal cations on coacervation of α-elastin

The quasi-elastic light scattering studies were carried out to investigate effects of metal cations such as Ca²⁺ and Na⁺ on the early stage of Coacervation process of α-elastin, a chemical fragmentation product originated from the biological elastomeric protein elastin, in aqueous solutions. In particular, our attention was focused on changes of two types of dynamical behaviors found in the earlier work, which are a remarkable increase and a monotonous decrease in the hydrodynamic radius R of molecules with temperature for critical and off-critical concentrations of α-elastin, respectively. For the critical α-elastin concentration, an addition of Ca²⁺ was found to exert little effects on the steep temperature profile of R observed in the absence of Ca²⁺. On the other hand, an addition of a slight amount of Na⁺ resulted in a monotonous decrease in R , but its further addition restored a remarkable increase in R similar to the critical behaviors in the salt-free system. In the case of off-critical sample, the addition of either Ca²⁺ or Na⁺ above a certain concentration induced a change in R from a monotonous decrease to a remarkable increase. For both critical and off-critical concentrations of α-elastin, Ca²⁺ and Na⁺ brought about an elevation and a lowering of the temperature at which the sample started to be turbid, respectively. © 1995 John Wiley & Sons, Inc.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.     

Comparative studies of the cross-linked regions of elastin from bovine ligamentum nuchae and bovine, porcine and human aorta.

1. The preparative Edman degradation of desmosine-containing peptides permitted the isolation of peptides C-terminal to the desmosine cross-links in bovine, porcine and human aortic elastin as well as bovine ligamentum nuchae elastin. This identifies the lysines in the tropoelastin which give rise to the desmosine cross-links. 2. The sequences from bovine aortic elastin were identical with those obtained from bovine ligamentum nuchae elastin but differed from those obtained from the other species. The most striking difference involves the occurrence of phenylalanine in bovine elastin and tyrosine in porcine and human elastin C-terminal to the desmosine cross-links. 3. The sequences of the C-terminal peptides were found to fall into two distinct classes, one starting with hydrophobic residues, the other starting with alanine. It is proposed that thehydrophobic residue prevents the enzymic oxidative deamination of the adjacent lysine e-amino group and this then contributes the nitrogen to the pyridinium ring of the cross-links.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.     

Amino acid sequences C-terminal to the cross-links in bovine elastin.

All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685--695].     

The application of collagenase, purified by affintiy chromatography, to the isolation of insoluble elastin from bovine aorta.

Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.16), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine aorta. The extremely low level of N-terminal residues (1.6 mol per 10(6) g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. The amino acid profile of the aortic elastin was found to be almost identical to that of insoluble elastin prepared from bovine ligamentum nuchae by the same method.     

Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of β-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii)

β-N-Acetylglucosaminidase (β-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that β-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of β-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for β-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of β-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of β-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that β-NAGase can play a significant role in the fertilization process in teleosteans.     

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin shows that there are considerable differences between these proteins. There is evidence that duplication of segments of DNA and gene rearrangement have occurred in the gene for chick elastin as compared with the gene for bovine elastin. The length of the polypeptide chain of elastin in both species is similar. Therefore, the duplications are compensated for by deletions in the gene for chick elastin.     

Conformational changes induced in α-elastin by cholesterol,taurocholate and unsaturated fatty acids

The ability of sodium taurocholate, cholesterol and oleic, linoleic and palmitoleic acids to induce conformational changes in α-elastin has been studied by circular dichroism. In addition, the influence of Ca²⁺ ions has been investigated. The formation of inelastic structure (α-helix, β-form) in the protein has been evidenced by spectral data. These results could be of interest in relation to aging and atherogenesis.     

Primary structures of bovine elastin a, b, and c deduced from the sequences of cDNA clones

Nucleotide sequence analysis of cDNA clones for bovine elastin revealed the occurrence of three mRNAs for elastin in fetal calf nuchal ligament, encoding three forms of elastins (a, b, and c, of 747, 733, and 713 amino acid residues, respectively). These forms arise as the result of the presence, at a single position, of 102 additional nucleotides in the mRNA for elastin a and of 60 of these nucleotides in the mRNA for elastin b as compared to the mRNA for elastin c. As expected, most lysines occur in pairs, separated by two or three small amino acid residues. However, at two places, lysines occur in groups of three. The occurrence of a group of three lysines followed by a hydrophobic residue (lysine 400, 404, and 407) offers an explanation for the formation of lysinonorleucine. The alignment of amino acid sequences of porcine tropoelastin tryptic peptides with the sequence for bovine elastin a results in the ordering of these tryptic peptides. The analysis of the complete primary structures of elastin a, b, and c provides further insight into the structure-function relations of elastin.