Identification of a soluble intermediate during synthesis of elastin by embryonic chick aortae

Murphy, Lois; Harsch, Margaret; Mori, Tomohiko; Rosenbloom, Joel

doi:10.1016/0014-5793(72)80116-9

Cited by 50 publications

(12 citation statements)

References 12 publications

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“…present in glycoproteins but largely absent from elastin and collagen (8,16)]. Proline was chosen as a label for all components, and valine is 10 times more prevalent in elastin than in collagen (7,17). Because crosslinked elastin is not hydrolyzed by trypsin, but elastase digests proteins other than elastin (18), the sequence of enzyme treatment was always trypsin followed by elastase.…”

Section: Resultsmentioning

confidence: 99%

Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells.

Jones¹,

Scott‐Burden²,

Gevers³

1979

Proc. Natl. Acad. Sci. U.S.A.

150

107

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Smooth muscle cells from rat heart secreted extracellular matrix components at high rates for many generations in culture. The matrix proteins remained anchored to the culture dish and were characterized after removal of cellular material with sodium dodecyl sulfate. Sequential enzyme digestion demonstrated the presence of at least three components, including glycoprotein(s), elastin, and collagen. Prolonged extraction of the matrix with detergent under reducing conditions solubilized a fucosylated glycoprotein having an apparent molecular weight of 250,000 and two other proteins with molecular weights of 72,000 and 45,000, respectively. Sublines derived from discrete colonies of smooth muscle cells synthesized all of the matrix components, and the proportion of collagen secreted by some sublines increased with time in culture. The biosynthesis of a mixed extracellular matrix and the relationships among the component proteins were therefore studied in one system producing milligram quantities of material.The extracellular matrix proteins are of great importance to the functioning of an organism. Present culture models for their biosynthesis are not altogether satisfactory because most cells tend to lose their differentiated properties when they are removed from their normal environment (1)-for example, 3T3 and 3T6 cells, which are often used for studies of collagen biosynthesis, have largely lost their ability to synthesize this protein (2, 3). However, it has recently been shown that primary avian tendon cells synthesize physiological amounts of collagen under the correct culture conditions (1).The current interest in the proteins comprising the extracellular matrix (4-7) involves not only collagen and elastin but also the high molecular weight glycoproteins which probably play a major role in the organization and properties of the matrix. The microfibrillar protein of the elastic fiber has an apparent molecular weight of 270,000 (8) and has been suggested to be involved in the organization of the fiber (7). Fibronectin (LETS protein) has also been shown to be a predominantly matrix protein (9), to bind to collagen (10), and possibly to act as a bridge in the binding of cells to collagen (11). Our understanding of the biosynthesis, processing, and interactions of these proteins has been retarded not only by the lack of culture systems that produce them rapidly in physiological amounts but also by the lack of quantitative data on the matrix components. There is little information in the literature as to the actual quantities of material secreted by cultured cells, and no studies have been reported on the biochemical analysis of a mixed matrix produced in vitro.In this report we describe a culture system in which large amounts of an extracellular matrix are formed in a relatively short time. Smooth muscle cells cultured from rat heart form a multilayered structure containing milligram quantities of connective tissue proteins in a crosslinked, insoluble form within 2 weeks, even after many generations in ...

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Section: Resultsmentioning

confidence: 99%

Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells.

Jones¹,

Scott‐Burden²,

Gevers³

1979

Proc. Natl. Acad. Sci. U.S.A.

150

107

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“…The indirect precipitation method described here can be used to identify soluble elastin in biological systems in which other methods of characterization are difficult or impossible. The use of gel electrophoresis in sodium dodecyl sulphate enables one to estimate the molecular weight of the precipitated proteins, and, if [14C]proline is used as a label, hydroxy[14C]proline analyses can be performed on the labelled proteins in different regions of the gel (Murphy et al, 1972;Rosenbloom & Cywinski, 1976). Sykes & Chidlow (1974) reported that the lower limit of sensitivity of their assay for identifying tropoelastin was in the low microgram range.…”

Section: Resultsmentioning

confidence: 99%

“…Tropoelastin labelled with [3H]valine was prepared by incubating thoracic aortae from 17-day embryonic chicks with IO,uCi of [3H]valine/ml (New England Nuclear Corp., Boston, MA, U.S.A.) for 2h at 37°C (Murphy et al, 1972). The aortae were then homogenized in 5 M-guanidine hydrochloride, and the solubilized material was dialysed against 5.5Murea.…”

Section: Methodsmentioning

confidence: 99%

“…were added to the dissolved precipitates and supernatants, and the samples placed in a boiling-water bath for 2 min. They were then dialysed against 0.1 % sodium dodecyl sulphate/0.01 M-sodium phosphate, pH7.4, and the proteins subjected to polyacrylamidedisc-gel electrophoresis in sodium dodecyl sulphate as previously described (Murphy et al, 1972).…”

Section: Methodsmentioning

confidence: 99%

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Radioimmunological identification of tropoelastin

Christner¹,

Dixon²,

Cywinski³

et al. 1976

Biochemical Journal

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Antiserum was prepared in sheep against insoluble elastin isolated from embryonic-chick aortae. In an indirect immunoprecipitation test, the antiserum reacted quantitatively with small amounts of radioactively labelled purified tropoelastin prepared from embryonic-chick aortae. The antiserum did not cross-react with chick procollagen, and the antiserum uas used to identify radioactively labelled tropoelastin secreted by chick aorta cells in suspension culture.

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“…The major arteries from 17-day embryos were dissected as previously described (Murphy et al, 1972), and freshly isolated cells were prepared from this tissue by a method similar to that described by . In a typical experiment arteries from approx.…”

Section: Isolation Of Matrix-free Cells From Embryonic-chick Arteriesmentioning

confidence: 99%

Synthesis of procollagen by matrix-free cells from embryonic-chick arteries

Schofield

Harwood

Jackson

1977

Biochemical Journal

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Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [(14)C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[(14)C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-alpha-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.

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Identification of a soluble intermediate during synthesis of elastin by embryonic chick aortae

Cited by 50 publications

References 12 publications

Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells.

Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells.

Radioimmunological identification of tropoelastin

Synthesis of procollagen by matrix-free cells from embryonic-chick arteries

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