Elastin isolation

It has been shown that vascular mineralization is associated with the elastin fibres334 of the components on the major arterial vessels. Elastin isolated from human aortas of varying ages undergoes mineralization in vitro at a faster rate when obtained from older tissues. This increase in the rate of mineralization may be seen in the reduction of what is called the lay period, in the uptake of calcium and phosphate during the in vitro calcification of elastin337. ... 

CONCENTRATIONS OF CROSSLINKS AND LYSINE RESIDUES IN ELASTIN ISOLATED FROM THE LIGAMENTUM NUCHAE OF CATTLE OF INCREASING AGEa... 

In different tissues the microscopic and submicroscopic organization of elastica takes different and highly characteristic forms and it is of interest to speculate if this differentiation springs from differences in the chemical constitution of the elastin molecule or if it is induced by environmental conditions at the site of growth. Further constitutional studies on purified elastins isolated from the different tissues may help to resolve these questions. 

Elastin shows a distinct increase with age (Blu-MENTHAL et al. 1964). Amino acid composition of elastins isolated from human pidmonary connective tissue by alkahne digestion demonstrated an increase in glutamic and aspartic acids (Fitzpatrick and Hospelhorn 1962). Within skin elastic fibres, starting of the fourth decade of hfe, electron dense materials accumidate in an age-dependent manner (Pasquali-Ronchetti and Baccarani-CoNTRi 1997). In very old subjects, these materials seem to have disappeared, leaving behind holes, which give to the fibre a cribriform appearance. 

Research into elastin, its properties, and the fiber formation was for a considerable period of time hindered due to its insolubihty. However, discovery of the soluble tropoelastin precursor made new investigations possible. The tropoelastin protein can be isolated from copper-deficient animals. However, this is a very animal-unfriendly and low yielding process [2]. Therefore, it is preferred to obtain tropoelastin from overexpression in microbial hosts such as Escherichia coli (E. coli). Most studies are thus based on tropoelastin obtained via bacterial production. 

Bailey AJ, Ranta MH, Nicholls AC, Partridge SM and Elsden DP (1977) Isolation of a -amino adipic acid from mature dermal collagen and elastin. Evidence for an oxidative pathway in the maturation of collagen and elastin. Biochem Biophys Res Comm 78, 1403-1410. 

Nakamura F, Yamazaki K and Suyama K (1992) Isolation and structural characterization of a new crosslinking amino acid, cyclopentenosine, from the acid hydrolysate of elastin. Biochem Biophys Res Comm 186, 1533-1538. 

The latter possibility appears the more likely of the two, especially in view of the results of the enzymatic digestion. These indicate that most of the material isolated from the human aortas was elastin, but some contamination with other substances did occur338). 

Calcium in this case would coordinate with acyl oxygens from the polypeptide backbone of the protein, because of its unique amino acid sequences and potential conformations348). Non-polar peptides have been isolated from elastin which consists almost entirely of the three non-polar amino acids glycine, valine, and proline349). Portions of porcine tropoelastin have been partially sequenced350). Repeating tetra-, penta-, and hexapeptides have been observed. The tetrapeptide contains the sequence —Gly—Gly—L—Val—L—Pro— the pentapeptide —L—Val—L—Pro-... 

Fibrillin microfibrils are widely distributed extracellular matrix assemblies that endow elastic and non elastic connective tissues with long-range elasticity. They direct tropoelastin deposition during elastic fibrillogenesis and form an outer mantle for mature elastic fibers. Microfibril arrays are also abundant in dynamic tissues that do not express elastin, such as the ciliary zonules of the eye. Mutations in fibrillin-1—the principal structural component of microfibrils—cause Marfan syndrome, a heritable disease with severe aortic, ocular, and skeletal defects. Isolated fibrillin-rich microfibrils have a complex 56 nm beads-on-a-string appearance the molecular basis of their assembly and... 

Hyaluronic acid is a component of the extracellular ground substance which surrounds the collagen and elastin fibres and cells of connective tissue [64], It is a member of the group of polysaccharides isolated from vertebrate connective tissues which were formerly called mucopolysaccharides and are now more commonly referred to as glycosaminoglycans [65,66], Glycosaminoglycans commonly occur in vivo as proteoglycans. 

With respect to one of these precursors, the term, tropoelastin, has been used as a designation for a non-cross linked elastin precursor of approximately 70,000 daltons (1). Since it is currently the best characterized of the non-crosslinked elastins and is used extensively by those familiar with elastin, this term will be retained. Elastin will be used to designate the protein in its crosslinked form. This term, however, is at best operational, since elastin is only isolated from tissues or cell culture by procedures that would be offensive to most protein chemists. As a component of extracellular matrices, elastin is extremely insoluble and in close association with many other extracellular components (2). In order to remove these components, harsh treatments such as autoclaving, extraction with alkali or... 

The exact form in which non-crosslinked elastin is secreted from smooth muscle cells is yet to be clearly defined. Foster et al. (36) have suggested that a non-cross linked elastin (pro-elastin) is secreted from smooth muscle cells in a form that is approximately 120,000 to 140,000 daltons. They have suggested that proelastin is cleaved to smaller molecular weight forms of non-crosslinked elastin. It should be noted, however, that this view is not entirely supported by data from other laboratories. There are two reports on the use of isolated mRNA from chick aorta suggesting only a 70,000 dalton non-cross linked elastin is the major product of translation (37,38). There is also a recent report suggesting that aortic mRMA translates a 200,000 dalton putative elastin product (39). We have recently isolated a non-crosslinked elastin from the aortas of copper deficient chicks that appears to be 100,000 daltons (27). Its amino acid composition is similar to that for tropoelastin (Table III). A major problem in resolving these points is that the trypsin-like proteinase associated with elastin is not easily denatured or separated from the non-crosslinked forms of elastin. The proteinase is also not readily inhibited by commonly used inhibitors for trypsin-like proteinases (26). 

For example, there are several reports that indicate many of the previously reported changes in elastin composition during atherosclerosis are related to the methods used in the initial isolation of the elastins (52,53). Although in certain forms of emphysema there appears to be evidence that elastic fibers are destroyed, the data related to compositional changes and alter-... 

The most ready source of elastin of high purity is the ligamentum nuchae of the larger ruminants, and from this source a protein of constant composition can be isolated by a variety of methods depending on the solubilization and removal of other less inert tissue components. Because of the ease with which it may be isolated the elastin of bovine ligamentum nuchae has come to be regarded as the type standard, but it is by no means certain that elastins from other mammals or other tissues in the same mammal are of identical constitution. 

Similar conclusions were reached by Kao et al. (1961) who injected female Wistar rats, 5 weeks, 8 months, and 2 years old with uniformly labeled Ci -lysine. The animals were sacrificed at intervals up to 40 days after injection. Elastin and collagen were isolated from aortas, tendon, uterus, and skin and examined for radioactivity. The results showed that with the exception of the uterus, insoluble collagen and elastin were synthesized at a significantly higher rate by 5-weeks-old rats than at 8 months or 2 years, but at all ages the turnover rate relative to other proteins was low. In agreement with the results of Slack, the elastin of aorta did not decay in activity in any age group above 5 weeks old. 

In an attempt to separate the normal effects of age from those due to visible atheromatosis, Lansing (1954) collected human aortas which possessed an over-all minimum of atheromatous plaques. Little difficulty was experienced in selecting suitable tissue from young subjects but considerable selection was necessary for the older specimens. From this material atheroma-free sections were cut out and the adventitia and intima were stripped off. Elastin was isolated from the separated tissue and its... 

Just as collagen fibers may suffer changes in staining properties so may elastin if it is damaged by the action of enzymes or by too severe isolation procedures. Rupture of a peptide bond results in the appearance of a new a-amino and a new a-carboxyl group and these added to the very few acidic and basic groups in elastin result in a much increased affinity for acidic and basic dyes. Concurrently the affinity for phenolic dyes may be reduced, particularly in acid or alkaline solutions, due to the repulsive forces set up by the increased charge. 

Little recent work has been reported on the water relations and sw elling properties either of native elastin or elastin that has been isolated by various procedures or treated with enzymes or hydrolytic reagents. This is to be deplored since the study of the hydration of a protein fiber is often a sensitive means of revealing the pattern of lateral bonds which lends structural stability. A comparison of the hydration of elastins derived from different animals, or different tissues of the same animal, would be particularly valuable since the methods are relatively insensitive to the preseruic... 

IV. Isolation and Analytical Characterization op Elastin A. Purification Procedures... 

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