scholarly journals Isolation of proteoglycans from human articular cartilage

1978 ◽  
Vol 169 (1) ◽  
pp. 123-132 ◽  
Author(s):  
M T Bayliss ◽  
S Y Ali
Keyword(s):  
Articular Cartilage ◽  
Degradation Products ◽  
Weight Ratio ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Normal Cartilage ◽  
Density Fractions ◽  
Low Protein ◽  
Normal Human

Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.

scholarly journals Age-related changes in the composition and structure of human articular-cartilage proteoglycans

1978 ◽  
Vol 176 (3) ◽  
pp. 683-693 ◽  
Author(s):  
M T Bayliss ◽  
S Y Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Age Groups ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Age Related ◽  
Composition And Structure ◽  
Normal Human ◽  
Cartilage Proteoglycans

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.

scholarly journals Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

1982 ◽  
Vol 206 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Charles J. Malemud ◽  
Victor M. Goldberg ◽  
Roland W. Moskowitz ◽  
Lee L. Getzy ◽  
Robert S. Papay ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Deae Cellulose ◽  
Culture Conditions ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Tissue Slices ◽  
Keratan Sulphate ◽  
Defined Culture

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [35S]sulphate or [14C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [35S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [35S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60–80% of the [35S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (Kav.) of the proteoglycan monomer on Sepharose CL-2B was 0.28–0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2–D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [35S]sulphate and [14C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60–70%), with 9–11% keratan sulphate in the monomer proteoglycan; (3) Kav. values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH4-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.

scholarly journals Structure of proteoglycans from different layers of human articular cartilage

1983 ◽  
Vol 209 (2) ◽  
pp. 387-400 ◽  
Author(s):  
M T Bayliss ◽  
M Venn ◽  
A Maroudas ◽  
S Y Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Articular Surface ◽  
Current Model ◽  
Gel Chromatography ◽  
Slice Thickness ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Keratan Sulphate

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.

scholarly journals The chemical constitution of the proteoglycan of human intervertebral disc

1976 ◽  
Vol 157 (3) ◽  
pp. 753-763 ◽  
Author(s):  
R H Pearce ◽  
B J Grimmer
Keyword(s):  
Chondroitin Sulphate ◽  
Deae Cellulose ◽  
Intervertebral Discs ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Qualitative And Quantitative ◽  
Cscl Density Gradient ◽  
Chemical Constitution ◽  
Normal Human ◽  
Quantitative Analyses

Proteoglycan was prepared from three pools of normal human intervertebral discs by extraction with buffered 4M-guanidinium chloride followed by CsCl-density-gradient ultracentrifugation. Chromatography on agarose (Bio-Gel A-150m) and on DEAE-cellulose suggested a single polydisperse proteoglycan species. The intrinsic viscosities of three preparations were 166, 122 and 168 ml/g. After degradation with 0.5M-KOH containing 0.02M-NaBH4, the glycosaminoglycans were recovered quantitatively and their Ca2+ salts separated into a hexuronate-rich fraction (fraction 1), which was precipitated in 0-45% (v/v) ethanol, and a hexose-rich fraction (fraction2), which was precipitated in 45-70% (v/v) ethanol. Qualitative and quantitative analyses of the glycosaminoglycans revealed fraction 1 to be chondroitin sulphate, and fraction 2 to be keratan sulphate; the latter was contaminated with protein and possibly a small amount of another glycosaminoglycan. For both glycosaminoglycans, plots of log(mol.wt.) against weight fell close to a normal distribution. The mode for chondroitin sulphate was close to 20000; that for keratan sulphate, 10000. A threefold range of molecular weight included the central 16-84% [+/- 1 S.D. of log(mol.wt.)] of the weight of both fractions.

scholarly journals Studies on cathepsin B in human articular cartilage

1978 ◽  
Vol 171 (1) ◽  
pp. 149-154 ◽  
Author(s):  
M T Bayliss ◽  
S Y Ali
Keyword(s):  
Articular Cartilage ◽  
Cathepsin B ◽  
Cathepsin D ◽  
Human Articular Cartilage ◽  
Normal Cartilage ◽  
Elderly Individuals ◽  
Age Related ◽  
Hydrolysis Of ◽  
Increased Activity ◽  
Thiol Proteinase

The thiol proteinase cathepsin B (EC 3.4.22.1), previously called cathepsin B1, was assayed in human articular cartilage by its hydrolysis of the synthetic substrate alpha-N-benzoyl-DL-arginine 2-naphthylamide. The enzyme was activated by cysteine and EDTA and completely inhibited by iodoacetamide and HgCl2. It was also partially inhibited by whole human serum. Human osteoarthrotic cartilage had increased activity when compared with normal cartilage. Cathepsin B activity of normal cartilage was age-related, being high in juveniles and declining to low values in adult and elderly individuals. Cathepsin D and cathepsin B both exhibited a zonal variation through the cartilage depth; the surface cells appeared to contain more activity than those close to the subchondral bone.

scholarly journals Characterization of proteoglycans isolated from associative extracts of human articular cartilage

1993 ◽  
Vol 293 (1) ◽  
pp. 165-172 ◽  
Author(s):  
V Vilím ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Molecular Mass ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Sds Page

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

scholarly journals The synthesis of dermatan sulphate proteoglycans by fetal and adult human articular cartilage

1989 ◽  
Vol 261 (2) ◽  
pp. 501-508 ◽  
Author(s):  
L I Melching ◽  
P J Roughley
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Uronic Acid ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Human Articular Cartilage ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Adult Human

Non-aggregating dermatan sulphate proteoglycans can be extracted from both fetal and adult human articular cartilage. The dermatan sulphate proteoglycans appear to be smaller in the adult, this presumably being due to shorter glycosaminoglycan chains, and these chains contain a greater proportion of their uronic acid residues as iduronate. Both the adult and fetal dermatan sulphate proteoglycans contain a greater amount of 4-sulphation than 6-sulphation of the N-acetylgalactosamine residues, in contrast with the aggregating proteoglycans, which always show more 6-sulphation on their chondroitin sulphate chains. In the fetus the major dermatan sulphate proteoglycan to be synthesized is DS-PGI, though DS-PGII is synthesized in reasonable amounts. In the adult, however, DS-PGI synthesis is barely detectable relative to DS-PGII, which is still synthesized in substantial amounts. Purification of the dermatan sulphate proteoglycans from adult cartilage is hampered by the presence of degradation products derived from the large aggregating proteoglycans, which possess similar charge, size and density properties, but which can be distinguished by their ability to interact with hyaluronic acid.

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