Chick tropoelastin isolation and partial chemical characterization (original) (raw)
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Properties of chick tropoelastin
Biochimica et biophysica acta, 1973
I. Tropoelastin was purified from the aortas of copper-deficient chicks. The amino acid composition of this protein is very similiar to that previously described for porcine tropoelastin. The protein contains more than 4 ° residues of lysine per thousand total residues and no desmosine or isodesmosine. 2. Tropoelastin is easily solubilized by elastase. The rate of elastolysis was found to increase if tropoelastin was first preincubated in solutions containing sodium dodecyl sulfate, but not dipalmityllecithin or cholesterol. 3-Sodium dodecyl sulfate elicits a marked conformational change in tropoelastin. The circular dichroism spectrum of tropoelastin in the presence of sodium dodecyl sulfate was typical of proteins containing significant amounts of a-helix. 4. Most of the lysyl residues in tropoelastin appear to equilibrate with aldehyde functions in the formation of Schiff base products. The potential for such reactions is a prerequisite in the formations of elastin cross-links. Approx. 8o% of the lysyl groups in the protein appeared capable of forming Schiff base products.
Additional evidence for a proform to tropoelastin from chick aorta
Biochemical Journal, 1979
Evidence is presented for the presence of a precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100000 daltons in size. It is suggested to be a precursor to tropoelastin (72000 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 10-20min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (<I p.p.m. to inhibit crosslinking) containing e-aminohexanoic acid (0.2 %) to retard proteolysis and then injected with [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 pH7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100000 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 100000-dalton protein subunit.
Biochemical Journal, 1986
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...
Connective Tissue Research, 1991
Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten-to 30-fold more sensitive than the existing competitive ELISA assays.
Comparison of aortic and ear cartilage tropoelastins isolated from lathyritic pigs
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1980
Tropoelastin was isolated from aortae and auricular cartilage obtained from lathyritic piglets. The two tissue-specific tropoelastins were judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on a high-pressure liquid chromatograph. Comparative studies of the tropoelastins were performed. Amino acid analysis revealed that the aortic and cartilage tropoelastins were very similar, if not identical, with the only exception that the cartilage tropoelastin contained more hydroxyproline and less lysine residues, both of which can be attributable to post-translational modifications. Both tropoelastins possess an apparent molecular weight of 70 000 and exhibit similar peptide fragments with limited trypsin cleavage. Antiserum raised to the aortic tropoelastin was used to show immunological identity between the two tissue tropoelastins.
Connective Tissue Research, 1999
Tropoelastin, which is secreted from the cell in a soluble form, contains specific alanine rich repeat domains that are destined to form covalent desmosine and isodesmosine crosslinks in mature insoluble elastin. We raised a monospecific polyclonal antibody to a AKAAAKAAAKA synthetic peptide (AKA) which represents this alanine rich region of tropoelastin. The antibody was reactive with the original peptide antigen and purified tropoelastin, but not with mature crosslinked elastin isolated from several animal species. Conditioned media from chick aorta smooth muscle cells in culture reacted in an ELISA with the AKA antibody, but only in the presence of BAPN to block the conversion of the e-amino groups to aldehydes. lmmunofluorescence demonstrated that the AKA antibody decorated newly deposited tropoelastin assembled in fine fibrils in matrix produced by cultured human skin fibroblasts. EM-immunogold specifically localized this antibody to the immature elastic fibers present in fetal sheep ductus arteriosus. Moreover, immunohistochemistry demonstrated that the antibody recognized nonpolymerized tropoelastin assembled on the periphery of elastic fibers in the aorta of chicks raised on copper deficient and BAPN containing diets. These studies demonstrate that this new anti-tropoelastin antibody can be used as a useful tool to investigate elastin metabolism where it is important to distinguish between tropoelastin and mature crosslinked elastin.
A high molecular weight species of soluble elastin
Biochemical and Biophysical Research Communications, 1976
The isolation of a high molecular weight species (130–140,000 daltons) of soluble elastin from the aortas of lathyritic chicks is described. In comparison to chick tropoelastin, the higher molecular weight material possesses an increased amount of acidic and hydroxyl amino acids and in contrast to tropoelastin, contains histidine, methionine and cystine residues. This molecular weight species of soluble elastin is susceptible to proteolytic degradation and can be shown to readily breakdown to lower molecular weight components including tropoelastin.