scholarly journals The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage

1984 ◽  
Vol 224 (3) ◽  
pp. 977-988 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Bovine Articular Cartilage ◽  
The Core ◽  
Elevated Rate ◽  
Chain Synthesis ◽  
Chondroitin Sulphate Chain ◽  
Bovine Cartilage

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.

scholarly journals The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans

1982 ◽  
Vol 202 (2) ◽  
pp. 387-395 ◽  
Author(s):  
D Mitchell ◽  
T Hardingham
Keyword(s):  
Actinomycin D ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Chain Elongation ◽  
Chondrosarcoma Cell ◽  
Acceptor Concentration ◽  
The Core ◽  
Chain Synthesis ◽  
Cartilage Proteoglycans ◽  
Elongation Activity

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.

scholarly journals Involvement of the core protein in the first β-N-acetylgalactosamine transfer to the glycosaminoglycan–protein linkage-region tetrasaccharide and in the subsequent polymerization: the critical determining step for chondroitin sulphate biosynthesis

1999 ◽  
Vol 340 (2) ◽  
pp. 353-357 ◽  
Author(s):  
Satomi NADANAKA ◽  
Hiroshi KITAGAWA ◽  
Fumitaka GOTO ◽  
Jun-ichi TAMURA ◽  
Klaus W. NEUMANN ◽  
...  
Keyword(s):  
Culture Medium ◽  
Glucuronic Acid ◽  
Core Protein ◽  
Enzyme System ◽  
Chondroitin Sulphate ◽  
Human Melanoma ◽  
The Core ◽  
A Cell ◽  
Chain Polymerization

α-Thrombomodulin (α-TM) with a truncated glycosaminoglycan-protein linkage tetrasaccharide, GlcAβ1-3Galβ1-3Galβ1-4Xyl, was tested as an acceptor together with a sugar donor, UDP-N-[3H]acetylgalactosamine, using a cell-free enzyme system prepared from the serum-free culture medium of a human melanoma cell line. The truncated tetrasaccharide on α-TM served as an acceptor, whereas the linkage tetrasaccharide-serine did not. Our characterization of the radioactively labelled product by enzymic digestion revealed that the N-[3H]acetylgalactosamine residue was transferred to α-TM through a β1,4-linkage. The substrate competition experiments with the chondro-hexasaccharide and α-TM reinforced our speculation that a common N-acetylgalactosaminyltransferase catalysed the transfer of N-acetylgalactosamine to both the linkage tetrasaccharide and the longer chondroitin oligosaccharides. Moreover, chondroitin polymerization was demonstrated on the tetrasaccharide of α-TM using both UDP-glucuronic acid and UDP-N-acetylgalactosamine as sugar donors. Much longer chains were synthesized on α-TM than on the linkage penta- and hexa-saccharide-serines. Together, these results indicated that the core protein is required for the transfer of the first N-acetylgalactosamine residue through a β1,4-linkage and also for subsequent efficient chain polymerization reactions, and that the critical determining step for chondroitin sulphate biosynthesis is the transfer of the first N-acetylgalactosamine residue.

scholarly journals The relation of RNA synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage

1986 ◽  
Vol 235 (2) ◽  
pp. 499-505 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos
Keyword(s):  
Half Life ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Glycosaminoglycan Synthesis ◽  
First Order ◽  
The Core ◽  
First Order Kinetics ◽  
Bovine Cartilage

Addition of actinomycin D (or cordycepin, an alternative inhibitor of RNA synthesis) to cartilage cultures resulted in a first-order decrease in the rate of incorporation of [35S]sulphate into proteoglycan (half-life = 7.5 +/- 1.1 h). Addition of 1.0 mM-benzyl beta-D-xyloside relieved the initial inhibition of glycosaminoglycan synthesis induced by actinomycin D; however, after a lag of about 10 h the rate of xyloside-initiated glycosaminoglycan synthesis also decreased with apparent first-order kinetics (half-life = 7.1 +/- 1.8 h), which paralleled the decrease in the rate of core-protein-initiated glycosaminoglycan synthesis. The hydrodynamic size of the proteoglycans formed in the presence of actinomycin D remained essentially constant (Kav. 0.21-0.23), whereas the constituent glycosaminoglycan chains were larger than those formed by control cultures, which suggested that the core protein was substituted with fewer but larger glycosaminoglycan chains. Proteoglycans formed in the presence of beta-D-xyloside were significantly smaller (Kav. approximately 0.33) than those synthesized by control cultures, and were further diminished in size after exposure of cultures to actinomycin D. Glycosaminoglycan chains synthesized by these same cultures on to both core-protein and xyloside acceptors were also smaller than those of control cultures. The decrease in synthesis observed after exposure to actinomycin D was not reflected by any significant decrease in the activities of several glycosyltransferases involved in chondroitin sulphate synthesis (galactosyltransferase-I, galactosyltransferase-II, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II).

scholarly journals Proteoglycans of hyaline cartilage: Electron-microscopic studies on isolated molecules

1975 ◽  
Vol 151 (1) ◽  
pp. 157-166 ◽  
Author(s):  
J Thyberg ◽  
S Lohmander ◽  
D Heinegård
Keyword(s):  
Hyaline Cartilage ◽  
Chondroitin Sulphate ◽  
Costal Cartilage ◽  
Side Chain ◽  
Gel Chromatography ◽  
Electron Microscopic ◽  
The Core ◽  
Central Filament ◽  
Microscopic Studies ◽  
Chondroitin Sulphate Chain

Proteoglycan monomers from guinea-pig costal cartilage, bovine nasal and bovine tracheal cartilage were observed in the electron microscope after being spread in a monomolecular layer with cytochrome c. The proteoglycan molecule appeared as an extended central core filament to which side-chain filaments were attached at various intervals. The molecules from the three sources displayed great ultrastructural similarities. On average, the core filament was about 290 nm long, there were about 25 side-chain filaments per core filament, the side-chain filaments were about 45 nm long, and the distance between the attachment points of the side-chain filaments to the core filament was about 11 nm. With regard to the overall size of the molecules, no evidence of distinct subpopulations was obtained. Good correlation was found between ultrastructural data for the proteoglycan molecules and chemical data obtained by enzyme digestions and gel chromatography. Together these data strongly support the interpretation of the electron-microscopic pictures as indicating a central filament corresponding to the protein core and side-chain filaments corresponding to the chondroitin sulphate chain clusters of the proteoglycan monomers.

scholarly journals Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta

1986 ◽  
Vol 240 (2) ◽  
pp. 575-583 ◽  
Author(s):  
R Kapoor ◽  
C F Phelps ◽  
T N Wight
Keyword(s):  
Uronic Acid ◽  
Glucuronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Polymer Concentration ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Structural Units ◽  
The Core ◽  
Covalently Linked

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 × 10(6) (PG-25), 0.30 × 10(6) (PG-35) and 0.88 × 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.

scholarly journals Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen

1984 ◽  
Vol 223 (2) ◽  
pp. 401-412 ◽  
Author(s):  
R M Mason ◽  
J D Lineham ◽  
M A Phillipson ◽  
C M Black
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Primary Cultures ◽  
Normal Subjects ◽  
Dermal Fibroblasts ◽  
Proteoglycan Synthesis ◽  
Dependent Manner ◽  
Glycoprotein Synthesis ◽  
Chondrocyte Cultures

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

scholarly journals Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage

1984 ◽  
Vol 217 (1) ◽  
pp. 259-263 ◽  
Author(s):  
B M Vertel ◽  
W B Upholt ◽  
A Dorfman
Keyword(s):  
Messenger Rna ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Protein Product ◽  
Neonatal Rat ◽  
The Core ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Specific Antisera ◽  
Free Translation

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.

Investigation into the effect of proteoglycan molecules on the tribological properties of cartilage joint tissues

1998 ◽  
Vol 212 (3) ◽  
pp. 177-182 ◽  
Author(s):  
J Pickard ◽  
E Ingham ◽  
J Egan ◽  
J Fisher
Keyword(s):  
Tribological Properties ◽  
Sliding Friction ◽  
Chondroitin Sulphate ◽  
Constant Load ◽  
Compression Tests ◽  
Bovine Articular Cartilage ◽  
Frictional Properties ◽  
Proteoglycan Content ◽  
Bovine Cartilage

The aim of this research was to investigate the role of the hydrophilic properties of the proteoglycan molecules within the cartilage matrix, on the tribological properties of joint tissues in the mixed and boundary lubricating regime. Bovine articular cartilage, bovine meniscus and bovine cartilage that had been degraded to remove the chondroitin sulphate from its proteoglycans were studied in order to investigate differences in their friction and compression responses. The tissues were tested on a sliding friction rig under nominal contact stresses of 0.5 and 4 MPa. The compression tests were carried out under a 0.8 MPa contact stress. The compression tests showed the cartilage and meniscus deforming at the same rates, but the degraded cartilage deformed more quickly to reach its equilibrium position in a shorter period of time. The friction tests carried out at a constant load revealed the friction of the meniscus rising more rapidly with loading time than the cartilage. The degraded cartilage followed an almost identical curve as the untreated cartilage. Although the reduced proteoglycan content of the degraded cartilage substantially altered the biphasic compression response, it did not have an effect on the frictional properties of the tissue.

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