Salt-soluble elastin from lathyritic chicks

The isolation of a salt-soluble homogeneous elastin from the aortas of lathyritic chicks by chromatography on DEAE-cellulose and salt precipitation is described. These new techniques, as well as some previously published by other workers, were evaluated with the help of antiserum raised in sheep against insoluble chick elastin. The purified elastin was very basic and behaved in a predictable manner in coacervation studies. The protein migrated in sodium dodecyl sulphate-polyacrylamide gels as a single band moving slightly faster than pyruvate kinase (mol.wt. 57000).     

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

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.     

Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies.

Insoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37 degrees C. Small amounts of B3H4 (11 mg--2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin. A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)...) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin cross-links. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.     

Isolation of elastin from bovine auricular cartilage.

Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.24.3), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine auricular cartilage. The low level of N-terminal residues (2.8 mol per 10⁶g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. The amino acid composition of the preparation showed an overall two-fold increase in polar residues, and a 20% reduction in valine, when compared to those of aortic and ligamentum nuchae elastin, while the concentration of cross-links was almost identical in the three preparations. Analysis of peptides, isolated by gel-exclusion chromatography after digestion of auricular elastin with elastase (pancreatopeptidase E, EC 3.4.21.11) in the presence of sodium dodecyl sulfate, revealed elevated levels of polar residues in all fractions examined, with no correlation between the concentration of these amino acids and that of the lysine-derived cross-links. Comparison of auricular and ligamentum nuchae elastin by fingerprint analysis of their elastase digests also suggested that the two proteins were compositionally distinct. Finally, treatment of auricular elastin with either trypsin or chymotrypsin produced no significant reduction in the level of polar residues. It is concluded that elastin exhibits tissue-related compositional variability.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Isolation and translation of elastin mRNA from chick aorta.

mRNA was isolated from 15-day chick embryo aortae by digestion of the tissue at 40° with Proteinase K in 1% sodium dodecyl sulfate followed by chromatography on oligo(dT)-cellulose columns. The mRNA was translated using a reticulocyte lysate system and the tropoelastin immunoprecipitated using a specific antiserum prepared against insoluble elastin. The precipitated product was characterized by polyacrylamide gel electrophoresis and a major peak corresponding to a molecular weight of approximately 70,000 daltons was observed. No higher molecular weight product was observed.     

Putative forms of soluble elastin and their relationship to the synthesis of fibrous elastin

Radiochemical labeling experiments suggest that there may be forms of soluble elastin which behave as proforms to tropoelastin, a known precursor to fibrous elastin. These protein subunits behave as 135,000 and 100,000 dalton subunits in SDS-polyacrylamide gels. However, they are only observed after chemical modification, specific for sulfydryl groups.     

Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.     

Additional evidence for a proform to tropoelastin from chick aorta.

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.