Primary structure of porcine tropoelastin.
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Biomedical subjects
Publications and source records attributed to L B Sandberg.
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Immunization of rabbits with either porcine tropoelastin, lung alpha-elastin, or aortic alpha-elastin resulted in the production of antibodies against the respective antigens. The assay of antibody activity with a radioimmunoassay indicated a high degree of cross reactivity between these three soluble elastin preparations. Through the use of competitive protein binding experiments it was possible to detect antigen-specific differences between tropoelastin and the two alpha-elastins. No antigenic differences were observed between lung or aortic alpha-elastin by any of the assay procedures uded in this investigation. Absorption of the various antisera with either insolubilized tropoelastin, lung elastin or aortic elastin allowed the preparation of non-crossreactive antibodies and provided further evidence for the antigenic differences between tropoelastin and the soluble alpha-elastins. The implications of the findings, as they relate to our current understanding of the molecular and structural arrangement of elastin and the elastin precursor, are discussed.
A highly sensitive method utilizing omicron-phthaldialdehyde for an automated fluorescent assay of proteins and peptides is described in this paper. As an example of the utility of omicron-phthaldialdehyde, the separation of bovine serum albumin monomer from bovine serum albumin oligomers is discussed.
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Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.
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1. Insoluble elastin has been prepared by several different methods from adult bovine and calf ligamentum nuchae. Highly purified tropoelastin has been prepared from copper-deficient porcine aorta. 2. Amino acid analyses indicated that all preparations, except that obtained from calf ligamentum nuchae by using an EDTA extraction followed by collagenase digestion (preparation E6), were typical of pure elastin having high concentrations of hydrophobic and low concentrations of hydrophilic amino acids. Preparation E6 was found to contain approx. 40% collagen. 3. The determination and composition of the carbohydrates associated with these preparations is reported. With the exception of preparation E6, the insoluble elastins contained only trace amounts of neutral sugars (0.13-0.35%, w/w) and amino sugars (0.01-0.06%, w/w). The porcine tropoelastin contained virtually no carbohydrate. 4. The results suggest that carbohydrate analyses can yield valuable information about the purity of elastin preparations.
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