Age-related changes in the synthesis of matrix macromolecules by bovine articular cartilage.
Calf and mature cow articular cartilage was labeled in vitro with [35S]SO4 and [3H]glycine and kinetics of incorporation of both isotopes by cartilage fragments was determined by scintillation spectroscopy. The cartilage fragments were then extracted in sequence with 4M GuHCl (Guanidium chloride) and pepsin. The pepsin digest was adjusted to 1.3 M NaCl and pepsin-solubilized collagen salted out. The 4M GuHCl extract, collagen and pepsin-resistent residue were then freeze-dried. The 4M GuHCl extract was further fractionated by DEAE (Diethylaminoethyl) 52 ion exchange chromatography to obtain protein and PG (Proteoglycan) fractions. The protein fraction was also characterised by SDS-PAGE and PG fraction by Sepharose C1-2B chromatography under associative conditions in the presence and absence of an exogenous HA (Hyaluronic acid). The GAG (Glycosaminoglycan) side chains of the PG samples were analysed by Sephadex G-200 column chromatography and their composition determined by paper chromatography after chondroitinase ABC digestion. Linear incorporation of both isotopes was observed from 1 to 18 hours of incubation and roughly equal amounts of [35S]SO4 counts were found on per cell bases in both cartilages although less [3H]glycine was incorporated by cow chondrocytes. It was also found that calf chondrocytes synthesize much greater proportion of the collagen whereas the cow cells synthesize PGs of smaller hydrodynamic sizes, bearing shorter GAG side chains that are enriched in KS (Keratan sulfate) and Ch-6S (Chondroitin-6 sulfate isomer). A failure of cow 35S-PGs monomers to interact with an exogenous HA in the presence of other extracted components was also demonstrated. The relevance of these findings for the mechanism of cartilage damage in aging and osteoarthritis is discussed.
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Cartilage Research Laboratory U-18 of INSERM, Paris, France.
Aprile
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Mitrovic
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Swann
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DA
D000375:Aging / Q000502:physiology*; D000818:Animals; D002358:Cartilage, Articular / Q000502:physiology*; D002417:Cattle; D003094:Collagen / Q000096:biosynthesis; D005109:Extracellular Matrix / Q000502:physiology*; D006025:Glycosaminoglycans / Q000096:biosynthesis; D046911:Macromolecular Substances; D009924:Organ Culture Techniques; D011509:Proteoglycans / Q000096:biosynthesis
D006025:Glycosaminoglycans; D046911:Macromolecular Substances; D011509:Proteoglycans; D003094:Collagen