Oxygen-centred free radicals can efficiently degrade the polypeptide of proteoglycans in whole cartilage

1984 ◽  
Vol 4 (12) ◽  
pp. 1017-1026 ◽  
Author(s):  
Roger T. Dean ◽  
Clive R. Roberts ◽  
Luigi G. Forni
Keyword(s):  
Free Radicals ◽  
Chondroitin Sulphate ◽  
Molecular Size ◽  
Amino Groups ◽  
Nasal Cartilage ◽  
Co60 Source ◽  
Hydroperoxy Radical ◽  
Radical Fragmentation ◽  
Intracellular Proteolysis ◽  
Radical Attack

Bovine nasal cartilage slices, biosynthetically labelled in their proteoglycan with35SO4, were used as substrate for the attack of free radicals generated on exposure to a Co60 source (which allows study of single radical species), and by chemical and enzymatic means. Systems generating hydroxyl (OH•) and superoxide (02•-) radicals degraded the proteoglycan efficiently, while the hydroperoxy radical (HO2•) was less efficient; addition of appropriate radical scavengers inhibited degradation. The radioactive products were heterogeneous in molecular size, but with doses up to 3600 Gy were the same size range as intact chondroitin sulphate. They contained free amino groups, and more were liberated by aminopeptidase M digestion, implying that at least a small peptide was present. Thus a major site of radical attack may be the polypeptide chain. We suggest that free-radical fragmentation of polypeptides may be important both in extracellular catabolism and in intracellular proteolysis.

scholarly journals The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation

1977 ◽  
Vol 167 (3) ◽  
pp. 639-646 ◽  
Author(s):  
P J Roughley
Keyword(s):  
Cathepsin B ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Cathepsin G ◽  
Proteolytic Degradation ◽  
Single Chain ◽  
Structural Variations ◽  
Nasal Cartilage ◽  
Core Proteins ◽  
Average Size

1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.

scholarly journals Immobilisation and Release of Radical Scavengers on Nanoclays for Chemical Reinforcement of Proton Exchange Membranes

Membranes ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 208
Author(s):  
Alia Akrout ◽  
Aude Delrue ◽  
Marta Zatoń ◽  
Fanny Duquet ◽  
Francesco Spanu ◽  
...  
Keyword(s):  
Composite Membranes ◽  
Proton Exchange Membranes ◽  
Ceo2 Nanoparticles ◽  
Proton Exchange ◽  
Radical Scavenger ◽  
Radical Scavengers ◽  
Amino Groups ◽  
High Proton Conductivity ◽  
High Proton ◽  
Radical Attack

Mechanical and chemical stability of proton exchange membranes are crucial requirements for the development of fuel cells for durable energy conversion. To tackle this challenge, bi-functional nanoclays grafted with amino groups and with embedded radical scavengers, that is, CeO2 nanoparticles were incorporated into Aquivion® ionomer. The composite membranes presented high proton conductivity and increased stability to radical attack compared to non-modified Aquivion membranes, demonstrating the effectiveness of the approach based on radical scavenger immobilisation and release from clay nanocontainers.

Observations on the morphology of the proteinpolysaccharide complex of bovine nasal cartilage and its relationship to collagen

1966 ◽  
Vol 165 (1001) ◽  
pp. 440-449 ◽  
Keyword(s):  
Nitric Acid ◽  
Aqueous Extract ◽  
Chondroitin Sulphate ◽  
Carbohydrate Chain ◽  
Average Particle Size ◽  
Heavy Fraction ◽  
Bismuth Nitrate ◽  
Average Particle ◽  
Nasal Cartilage ◽  
Intact Cartilage

The investigation has been carried out by electron microscope examination of intact cartilage and of various fractions of an aqueous extract of that tissue. The materials were stained, either with an 0·5 % solution of bismuth nitrate in 0·1 m nitric acid in water at a pH of 1·2, or with a similar solution of bismuth nitrate in 0·1 m nitric acid in 90 % acetone. It is considered that in both circumstances the bismuth was bound exclusively to the sulphate group of chondroitin sulphate and keratosulphate. The protein and chondroitin sulphate moieties of the proteinpolysaccharide complex were isolated after alkaline treatment. The former contains 15 % keratosulphate and it is only this part which stains. Both the protein and the chondroitin sulphate were visualized as discrete groups of particles in which the average particle diameter was 23 Å. It is suggested that each group represents a network of keratosulphate or chondroitin sulphate A ( CSA ) chains respectively, cross-linked by bismuth ions. And that each individual particle represents a segment of a carbohydrate chain which has assumed a coiled configuration owing to the neutralization of its net charge by bismuth. The light fraction of the aqueous extract of cartilage contains the proteinpolysaccharide complex alone. After precipitation with aqueous bismuth nitrate its appearance was essentially similar to that of its CSA and protein fractions, except that the average particle size was 47 Å. After precipitation by bismuth nitrate in acetone, on the other hand, it was seen as single rows of particles, the length of the rows varying from 1100 Å to 1500 Å, and the particles having an average diameter of 30 Å. Each row is interpreted as a proteinpolysaccharide macro-molecule, and it is considered that each particle represents an individual carbohydrate chain, whereas the intervals between the particles represent the unstained protein moiety. The indi­viduality of the macromolecules in these circumstances is ascribed to the low dielectric constant of acetone. The relationship of the proteinpolysaccharide complex to collagen was studied in the heavy fraction of the aqueous extract of cartilage and in intact cartilage. It was observed that the heavy fraction consists of proteinpolysaccharide and collagen and that a pro­portion of the complex is bound to collagen. In each collagen period, proteinpolysaccharide macromolecules were attached transversely round the circumference of the fibre over the a and b 1 bands. A similar relationship was noted in intact cartilage.

scholarly journals The spin trapping of pyrimidine nucleotide free radicals in a Fenton system

1989 ◽  
Vol 261 (3) ◽  
pp. 831-839 ◽  
Author(s):  
W D Flitter ◽  
R P Mason
Keyword(s):  
Free Radicals ◽  
Hydroxyl Radical ◽  
Spin Trap ◽  
Gamma Ray ◽  
Thiobarbituric Acid ◽  
Spin Trapping ◽  
Pyrimidine Nucleotide ◽  
Gamma Ray Irradiation ◽  
Radical Attack ◽  
Fenton System

The reaction of the hydroxyl radical, generated by a Fenton system, with pyrimidine deoxyribonucleotides was investigated by using the e.s.r. technique of spin trapping. The spin trap t-nitrosobutane was employed to trap secondary radicals formed by the reaction of the hydroxyl radical with these nucleotides. The results presented here show that hydroxyl-radical attack on thymidine, 2-deoxycytidine 5-monophosphate and 2-deoxyuridine 5-monophosphate produced nucleotide-derived free radicals. The results indicate that .OH radical attack occurs predominantly at the carbon-carbon double bond of the pyrimidine base. The e.s.r. studies showed a good correlation with previous results obtained by authors who used x- or gamma-ray irradiation to generate the hydroxyl radical. A thiobarbituric acid assay was also used to monitor the damage produced to the nucleotides by the Fenton system. These results showed qualitative agreement with the spin-trapping studies.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species

1981 ◽  
Vol 199 (1) ◽  
pp. 81-87 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Limb Bud ◽  
Binding Region ◽  
Human Cartilage ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycans ◽  
Rabbit Articular Cartilage ◽  
Hyaluronic Acid Binding

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.

scholarly journals Fractionation and characterization of proteoglycans isolated from chondrocyte cell cultures

1981 ◽  
Vol 197 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Sven Björnsson ◽  
Dick Heinegȧrd
Keyword(s):  
Cell Culture ◽  
Chondroitin Sulphate ◽  
Calf Serum ◽  
Void Volume ◽  
Cartilage Tissue ◽  
Foetal Calf Serum ◽  
Nasal Cartilage ◽  
Core Proteins ◽  
Minor Portion ◽  
A Minor

Chondrocyte cultures were established from foetal bovine tracheal cartilage and maintained in Ham's F12 medium with or without 10% (v/v) foetal calf serum. The proteoglycans were isolated and characterized. (1) The proteoglycans from cultures both with and without serum distributed in associative or dissociative CsCl gradients like proteoglycans from cartilage tissue. (2) The amino acid composition, protein contents and glucosamine/galactosamine ratios were grossly identical with those of the tissue derived proteoglycans. (3) Sedimentation coefficients (s0) for the monomers were 21.0S and 22.7S from cultures without and with serum respectively. The s0 values obtained for aggregates were 72.3S and 93.2S respectively. The limiting viscosity numbers [η] were 248ml/g and 298ml/g respectively. These data corresponded well to those obtained for the tissue-derived proteoglycans. (4) The sizes of the core proteins and chondroitin sulphate chains respectively were the same for both types of cell-culture proteoglycans and similar to those of the tissue proteoglycans. Both the keratan sulphate-rich region and the hyaluronic acid-binding region were identified. The latter, however, was not resistant to limit digestion with trypsin, in contrast with the fragment derived from the bovine nasal cartilage. (5) About 70% of the cell-culture proteoglycans chromatographed in the void volume on a Sepharose 2B column, whereas reduced and alkylated samples (monomers) chromatographed completely included in the column. The two link proteins present in A1 preparations of cartilage proteoglycans were also present in A1 preparations of cell-culture proteoglycans. (6) A minor portion (10%) of the 35S-labelled proteoglycans in the cultures was associated with the cells. Reduced and alkylated samples were larger compared with the monomers in the medium, and chromatographed partly (25%) excluded on the Sepharose 2B column. A larger proportion (50%) of the non-reduced samples chromatographed in the void volume of the column.

scholarly journals Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain

1987 ◽  
Vol 245 (1) ◽  
pp. 229-234 ◽  
Author(s):  
T Krusius ◽  
V N Reinhold ◽  
R K Margolis ◽  
R U Margolis
Keyword(s):  
Ion Exchange ◽  
Chondroitin Sulphate ◽  
Gel Filtration ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Size Fractions ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.

scholarly journals Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

1982 ◽  
Vol 203 (3) ◽  
pp. 691-698 ◽  
Author(s):  
Harold D. Keiser
Keyword(s):  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Antigenic Determinants ◽  
Density Gradient Ultracentrifugation ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Immunoprecipitation Reaction ◽  
Hyaluronic Acid Binding

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.

scholarly journals Extended and globular protein domains in cartilage proteoglycans

1987 ◽  
Vol 245 (3) ◽  
pp. 763-772 ◽  
Author(s):  
M Paulsson ◽  
M Mörgelin ◽  
H Wiedemann ◽  
M Beardmore-Gray ◽  
D Dunham ◽  
...  
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Globular Domain ◽  
Protein Core ◽  
Nasal Cartilage ◽  
Cdna Sequences ◽  
Multidomain Structure ◽  
Variable Extent ◽  
Cartilage Proteoglycans

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3′-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].

scholarly journals Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1978 ◽  
Vol 175 (3) ◽  
pp. 913-919 ◽  
Author(s):  
Dick Heinegård ◽  
Stefan Lohmander ◽  
Johan Thyberg
Keyword(s):  
Electron Microscopy ◽  
Hyaluronic Acid ◽  
Electron Micrographs ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Side Chain ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Central Filament ◽  
Proteoglycan Aggregates

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.

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