Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage

Abstract

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 X 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 X 10(-8) cm2 X S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 X 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.

Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions

“…High-buoyant-density proteoglycans (Al Dl, p > 1.58 g/ml) from bovine femoral-head cartilage (15-18-month-old animals), obtained fresh from the abattoir, were prepared essentially as described by Lyon et al (1983), but the proteinase-inhibitor mixture also contained 0.01 M-N-ethylmaleimide.…”

“…Proteoglycan aggregates (Al fraction) from bovine femoral-head cartilage (15-18-month-old animals), were prepared essentially as described by Lyon et al (1983), but the proteinase-inhibitor mixture also contained 0.01 M-N-ethylmaleimide. The aggregates were extensively dialysed against 5 mM-sodium phosphate, pH 7.4, containing 0.01 M-EDTA at 6 'C.…”

“…These proteoglycans have a range of Mr values from 106 to 3 x 106 and an M. of 2.3 x 106. Studies on articular-cartilage high-buoyantdensity proteoglycans from bovine femoral-head (Lyon et al, 1983), bovine condylar (Swann et al, 1979) and chick-limb-bud cartilage (Shogren et al, 1982) indicate that these proteoglycans are smaller [Mw values 1.16 x 106, 1.3 x 106 and (1.05-1.96) x 106 respectively].…”

Aggrecan, the Primary Weight-Bearing Cartilage Proteoglycan, Has Context-Dependent, Cell-Directive Properties in Embryonic Development and Neurogenesis: Aggrecan Glycan Side Chain Modifications Convey Interactive Biodiversity

“…Homogenisation procedures, which are commonly used in the extraction of proteins from other soft connective tissues, cannot be used for the isolation of aggrecan in an intact form since their high shear forces fragment the aggrecan. The cartilage extract can then be subjected to anion exchange chromatography on support matrices derivatised with anionic ligands such as diethylaminoethyl (DEAE) or sulphopropyl [ 229 ], dissociative size exclusion chromatography in 4-M GuHCl containing buffers using open pore gel chromatographic media such as Sephacryl or Sepharose CL2B [ 230 ], or density gradient equilibrium isopycnic ultracentrifugation in high concentrations of CsCl [ 231 ]. This latter procedure relies on aggrecan’s high buoyant density in CsCl gradients of ≥1.55 g/mL to isolate aggrecan; while extracted proteins typically have buoyant densities of 1.3–1.35 g/mL, HA has a buoyant density of 1.4–1.45 g/mL.…”

“…This latter procedure relies on aggrecan’s high buoyant density in CsCl gradients of ≥1.55 g/mL to isolate aggrecan; while extracted proteins typically have buoyant densities of 1.3–1.35 g/mL, HA has a buoyant density of 1.4–1.45 g/mL. Density gradient ultracentrifugation can be conducted in the presence of 4M GuHCl to ensure aggrecan is isolated free of other interactive components also present in the cartilage extract [ 231 , 232 ].…”