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P Pritzl

Publications and source records attributed to P Pritzl.

31 records · Page 2Linked to original sources
PubMed1982

Biosynthesis of type V procollagen by A204, a human rhabdomyosarcoma cell line.

The A204 cell line, derived from a human rhabdomyosarcoma, was studied in culture for its capacity to synthesize collagen types and other extracellular matrix proteins. The cells synthesized and secreted into the culture medium collagenous proteins with apparent molecular weights of 220,000 and 150,000. These were identified as the pro alpha 1 and pro alpha 2 chains of type V collagen by immunoprecipitation and by peptide mapping. The pro alpha 1(V) chain was made in excess of a 2:1 ratio for pro alpha 1(V) to pro alpha 2(V), and a fraction of the pro alpha 1(V) chains together with all of the pro alpha 2(V) chains participated in intermolecular disulfide bonding. The chains were extensively glycosylated at hydroxylysyl residues in ascorbate-supplemented cultures. A fraction of the secreted pro alpha 1(V) chains was processed to the p alpha 1(V) form, but further processing in the culture medium was very slow and the type V collagen molecules deposits in the extracellular matrix apparently retained large non-triple helical domains. Since the A204 cell line does not produce other collagen types, it may prove useful in further studies of the biosynthesis of type V procollagen.

Cell Line↗
PubMed1982

Endothelial cells secrete a novel collagen type in vitro independently of prolyl hydroxylation.

Endothelial cells from bovine aorta, vena cava, and cornea secrete a novel collagen in vitro (Sage et al., 1980). Endothelial collagen (EC), which is sensitive to pepsin and to several neutral proteases, exhibited an additional unusual property in its mode of secretion. In the absence of added sodium ascorbate, EC was secreted by both aortic and corneal endothelial cells at levels which were very similar to those observed in cultures supplemented with this vitamin. In contrast, the secretion of type III procollagen, which normally constitutes 75-80% of collagenous protein in the culture medium, was significantly decreased in ascorbate-deficient cultures. Incubation of aortic endothelial cells with alpha, alpha'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylases, reduced the extent of prolyl hydroxylation in total culture medium protein by 98% but also did not affect the secretion of EC. The secretion of EC by endothelial cells appears to be independent of a requirement for prolyl hydroxylation. This property differs markedly from the secretory characteristics of the interstitial procollagens and more closely resembles that described for type IV (basement membrane) procollagen.

2,2'-Dipyridyl↗
PubMed1981

Susceptibility of type V collagen to neutral proteases: evidence that the major molecular species is a thrombin-sensitive heteropolymer, [alpha 1(V)]2 alpha 2(V).

The susceptibility of human type V collagen to several neutral proteases was examined. Thrombin cleaved both the alpha 1(V) and alpha 2(V) chains of this protein at 34 degrees C, producing two pairs of fragments with apparent molecular weights of 95000 and 10000 on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Two-dimensional 125I-labeled peptide mapping of the larger fragments demonstrated that the upper band [which comigrated with alpha 1(I)] was derived from both the alpha 1(V) and alpha 2(V) chains, while the other component [which comigrated with alpha 2(I) was a product of alpha 1(V) alone. Cleavage of type V collagen, containing alpha 3(V) chains, with thrombin produced an analogous pattern with three high molecular weight bands. Chymotrypsin and trypsin cleaved type V collagen at 37 degrees C but not at lower temperatures. Digestion of type V collagen with elastase at 37 degrees C resulted in selective proteolysis of alpha 2(V), leaving alpha 1(V) essentially intact. Pepsin treatment of type V collagen from which alpha 2(V) had been removed by elastase treatment resulted in nearly complete degradation of alpha 1(V). These data support the hypothesis that a major fraction of native type V collagen is a heteropolymer with the chain composition [alpha 1(V)]2 alpha 2(V). Cleavage of type V collagen by thrombin may have physiologic significance in that breakdown of pericellular matrix may be an important step in the response of a tissue to injury.

Chymotrypsin↗
PubMed1981

Characterization of cell matrix associated collagens synthesized by aortic endothelial cells in culture.

Several collagen types have been isolated and characterized from bovine aortic endothelial cells and their associated extracellular matrix. Two collagens, which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha 1(III), alpha 1(V), and alpha 2(V) collagen chains, were isolated by salt precipitation from pepsin digests of cell layer proteins. Two of these chains were further purified by molecular-sieve and ion-exchange chromatography and were identified as alpha 1(III) and alpha 1(V) by one-dimensional peptide maps generated with mast cell protease and cyanogen bromide. In contrast to type III collagen, which was found in both the culture medium and cell layer, type V collagen appeared to be restricted to the cell layer. In addition to their occurrence as cell layer constituents, both types III and V collagens were localized to an extracellular matrix after the cells had been removed from the culture dishes by detergent. Preliminary studies based on peptide maps comparing type III collagen from the cell layer and culture medium provide evidence for structural heterogeneity within this collagen type.

Animals↗
PubMed1981

A new mapping technique for collagen chains.

A new, highly sensitive method for peptide mapping of collagen chains has been developed which utilizes a modification of the radioiodination technique in polyacrylamide gels described by Elder et al. (1977b). Optimal conditions include the use of the Bolton-Hunter reagent to produce 125I-labeled collagen with the enzyme proteinase K, prior to resolution of the cleavage products by two-dimensional electrophoresis and chromatography. Unambiguous results were obtained by restricting comparison among collagens to a given set which was radioiodinated using the same procedure, i.e., in solution, or in a dried or hydrated gel. Iodination of collagens in solution, followed by proteinase K digestion, resulted in highly reproducible maps which were free of background contamination and which permitted characterization of chains with a defined mobility of SDS-PAGE after the iodination procedure. This technique has provided additional evidence that the alpha 1, alpha 2, and alpha 3 chains of type V collagen are structurally unique. In addition, relationships among several fragments from pepsin-treated type IV collagen, which consists of two distinct chains, could be further elucidated.

Collagen↗
PubMed1980

A unique, pepsin-sensitive collagen synthesized by aortic endothelial cells in culture.

A unique collagen, designated EC, has been isolated from the culture medium of adult bovine aortic endothelial cells. After diethylaminoethylcellulose chromatography of [3H]proline-labeled culture medium, three non-disulfide-bonded bacterial collagenase-sensitive components with apparent Mr of 177000 (EC 1), 125000 (EC 2), and 100000 (EC 3) were demonstrated. Molecular sieve chromatography, cyanogen bromide cleavage, and two-dimensional peptide mapping of radioiodinated EC fragments produced by protease digestion suggest that the lower molecular weight components originate from EC 1. Both EC 1 and EC 2 were digested by pepsin within 10 min to products of less than 60000 molecular weight, under conditions which supported only limited proteolysis of other native collagens. A pepsin-resistant fragment of Mr 50000, derived from a digest of EC 2, contained equal amounts of hydroxyproline and proline, suggesting that at least a portion of the endothelial collagen contains a stable, collagen-like triple helix. Comparative mapping using mast cell protease and cyanogen bromide cleavage, followed by polyacrylamide gel electrophoresis, indicates that the primary structure of this collagen differs from that of other known collagen types.

Animals↗
PubMedDate not supplied

Secretory phenotypes of endothelial cells in culture: comparison of aortic, venous, capillary, and corneal endothelium.

Endothelial cells from different tissues display variations in morphology, intercellular junctions, cell surface and growth properties, and in production of basal lamina components, both in vivo and in vitro. We have investigated the spectra of extracellular proteins secreted by bovine endothelial cells cultured from large vessels, cornea, and capillaries. Aortic, venous, and corneal endothelial cells displayed highly similar patterns of protein synthesis as judged by analysis of the culture medium; the major products were fibronectin, a glycoprotein similar or identical to platelet thrombospondin, and Type III procollagen. Ion-exchange chromatography, followed by peptide mapping, confirmed the presence of EC, a novel endothelial collagen previously described in bovine aortic endothelial cell cultures. Minor variations were found in the collagens of the cell layers: Type III, the predominant interstitial collagen, was associated with the basement membrane Types IV and V and, in the case of corneal endothelium, with Type I. In contrast, capillary endothelial cells secreted significantly more collagen than did the aortic, venous, and corneal cells. Approximately 50% of the protein in the culture medium was collagenous and consisted of Types I and III collagen in a ratio of 2:3. These interstitial collagens were the only types detected in capillary cell layers as well. The pattern and overall rate of collagen synthesis by capillary endothelial cells in vitro contrasted significantly with that of the other endothelial cell types and closely resembled that described for cultures of sprouting endothelium. These alterations in secretory phenotype may reflect: 1) a true difference in cell type between capillary and other types of endothelium, 2) differences resulting from cell isolation and initial culture conditions, or 3) a correlation between growth regulation and protein synthesis.

Animals↗