Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma.
10 Aug 1975-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 250, Iss: 15, pp 6151-6159
TL;DR: Proteoglycan monomer (D1) and aggregate (A1) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma and contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred.
About: This article is published in Journal of Biological Chemistry.The article was published on 1975-08-10 and is currently open access. It has received 543 citations till now. The article focuses on the topics: Proteoglycan & Benzamidine.
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TL;DR: A murine tumor previously classified as a poorly differentiated chondrosarcoma is studied, and it is shown at the ultrastructural level that the tumor matrix is a homogeneous, nonfibrillar material, resembling basement membrane.
Abstract: We have studied a murine tumor previously classified as a poorly differentiated chondrosarcoma. Although the cells in this tumor are surrounded by large quantities of extracellular matrix material, the matrix fails to react with stains specific for the sulfated glycosaminoglycans present in normal cartilage. Here we show at the ultrastructural level that the tumor matrix is a homogeneous, nonfibrillar material, resembling basement membrane. Neither the proteoglycan matrix granules nor collagen fibrils characteristic of cartilage are present in the tumor matrix. Amino acid analyses of whole tumor tissue, enzyme-solubilized tumor components, and the protein extracted from lathyritic tumors confirmed that the tumor matrix is a basement membrane collagen. The collagenous protein extracted from the tumor by nonenzymatic means contains three unique polypeptides larger than the alpha-chain components of the other types of collagen. These studies indicate that the tumor is not a type of chondrosarcoma but a basement membrane producing tumor.
544 citations
TL;DR: Preliminary studies with collagen and chitosan matrixes encapsulated in "hollow" beads suggest that cell growth and morphology are profoundly influenced by the composition of the cellular environment.
Abstract: Methodology is described for the culture of avian and mammalian chondrocytes in ionotrophically gelled "semi-solid" and "hollow" alginate beads. Chondrocytes grown in "semi-solid" gels exhibited a spherical shape as opposed to a fibroblastic morphology observed in monolayer culture. In the "semi-solid" beads, the cells grew as small clumps and as large aggregates. The aggregates were round or elliptical in appearance and surrounded by a dense Alcian Blue positive halo. Preliminary studies with collagen and chitosan matrixes encapsulated in "hollow" beads suggest that cell growth and morphology are profoundly influenced by the composition of the cellular environment. Chondrocyte structure and function in the "semi-solid" and "hollow" beads were partially characterized by light microscopy, histochemical and biochemical means. The encapsulation methodology is readily applicable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture.
533 citations
TL;DR: Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion.
Abstract: Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.
531 citations
01 Jan 1980
TL;DR: The segregation of the proteoglycans into a separate category is based on a few specific characteristics, including the fact that the d-glucuronic-acid-containing repeating disaccharide of chondroitin, N-acetylchondrosine, has recently been identified as a component of thyroglobulin.
Abstract: With the possible exception of hyaluronic acid, the connective tissue polysaccharides are all synthesized by their parent cells as components of proteoglycans. In these substances, a number of polysaccharide chains are covalently linked to a protein core; e.g., in the proteoglycan of bovine nasal cartilage, which is the prototype of molecules of this kind, close to 100 chondroitin sulfate chains, with a molecular weight of approximately 20,000, and slightly fewer keratan sulfate chains are linked to a core protein (mol. wt. 200,000) which constitutes 7–8% of the entire molecule. In many respects, the proteoglycans are similar to other protein-bound complex carbohydrates, and the conspicuous polysaccharide component per se does not distinguish the proteoglycans from the class of glycoproteins; e.g., there are members of the glycoprotein class, such as the blood group substances, which have a high relative content of carbohydrate consisting of a substantial number of monosaccharide units. Rather, the segregation of the proteoglycans into a separate category is based on a few specific characteristics: (1) each polysaccharide consists of repeating disaccharide units in which a hexosamine, d-glucosamine, or d-galactosamine is always present; (2) all connective tissue polysaccharides except keratan sulfate contain a uronic acid, either d-glucuronic acid or its 5-epimer, l-iduronic acid, or both; (3) ester sulfate groups are present in all members of the group except in hyaluronic acid; in addition, N-sulfate groups are found in heparin and heparan sulfate. Although certain other bipolymers are known to contain ester sulfate, e.g., some epithelial mucins (Horowitz, 1977), these compounds are clearly distinguishable from the connective tissue polysaccharides by the other criteria indicated above. It may also be mentioned that the d-glucuronic-acid-containing repeating disaccharide of chondroitin, N-acetylchondrosine, has recently been identified as a component of thyroglobulin (Spiro, 1977); however, since the disaccharide is present as a single unit, thyroglobulin may not be considered a proteoglycan.
393 citations
TL;DR: The 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties, consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue.
385 citations
References
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Journal Article•
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
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TL;DR: The results show that the polyacrylamide gel electrophoresis method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
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TL;DR: It has been found possible to distinguish betweenHeparin, heparin derivatives, and other polyuronides of connective tissue by comparing the effect of chlorides on the color yield in both procedures by modifying Dische's carbazole reaction for uronic acid in the presence of borate.
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TL;DR: Micromethods have been developed and 35S-labeled chondroitin sulfates A, B, and C in a given mixture have been precisely and rapidly determined by measuring radioactivity alone.
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