Extraction and purification of proteoglycans from mature bovine bone

Proteoglycans were extracted in good yields from the mineralized matrix of ground bovine bone, by using a two-step extraction procedure. Proteoglycans (8% of total), not associated with the bone mineral, were extracted at − 20 degrees C with 4M-guanidinium chloride containing proteinase inhibitors. Proteoglycans associated with the mineral, which accounted for 60% of the total, were then solubilized when EDTA was added to the extraction solvent. They were fractionated and purified in the presence of 4M-guanidinium chloride by CsCl-density-gradient centrifugations followed by chromatography on Sepharose CL-4B. Further purification was obtained by chromatography on DEAE-cellulose and hydroxyapatite in the presence of 7 M-urea. Three populations of proteoglycans and additional glycosaminoglycan peptides were obtained. The molecular dimensions of both intact molecules and of their side chains as well as their amino acid composition were different, indicating that they represent separate molecular entities. The main proteoglycan self-aggregated in the absence of 4M-guanidinium chloride or 7 M-urea, a property that was abolished when the proteoglycan core protein was fragmented.

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Isolation and characterization of two sialoproteins present only in bone calcified matrix

Biochemical Journal ◽

10.1042/bj2320715 ◽

1985 ◽

Vol 232 (3) ◽

pp. 715-724 ◽

Cited By ~ 364

Author(s):

A Franzén ◽

D Heinegård

Keyword(s):

Sialic Acid ◽

Core Protein ◽

Deae Cellulose ◽

Sedimentation Coefficient ◽

Dry Weight ◽

Enzyme Linked Immunosorbent Assay ◽

Bovine Bone ◽

Guanidinium Chloride ◽

The Core ◽

Isolation And Characterization

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

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Characterization and N-terminal sequence of human platelet proteoglycan

Biochemical Journal ◽

10.1042/bj2551007 ◽

1988 ◽

Vol 255 (3) ◽

pp. 1007-1013 ◽

Cited By ~ 30

Author(s):

J P Périn ◽

F Bonnet ◽

P Maillet ◽

P Jollès

Keyword(s):

Core Protein ◽

Proteinase Inhibitors ◽

Human Platelet ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Exchange Chromatography ◽

Terminal Sequence ◽

Guanidinium Chloride ◽

Cscl Density Gradient ◽

Yolk Sac Tumour

Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.

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The chemical constitution of the proteoglycan of human intervertebral disc

Biochemical Journal ◽

10.1042/bj1570753 ◽

1976 ◽

Vol 157 (3) ◽

pp. 753-763 ◽

Cited By ~ 30

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.

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The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis

Biochemical Journal ◽

10.1042/bj1670629 ◽

1977 ◽

Vol 167 (3) ◽

pp. 629-637 ◽

Cited By ~ 111

Author(s):

P J Roughley ◽

A J Barrett

Keyword(s):

Ionic Strength ◽

Cathepsin D ◽

Core Protein ◽

Degradation Products ◽

Gradient Centrifugation ◽

Limited Proteolysis ◽

Extraction Procedure ◽

Cathepsin G ◽

Nasal Cartilage ◽

Cscl Density Gradient

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.

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Purification and partial characterization of a tumour-metastasis-associated high-Mr glycoprotein from rat 13762NF mammary adenocarcinoma cells

Biochemical Journal ◽

10.1042/bj2420779 ◽

1987 ◽

Vol 242 (3) ◽

pp. 779-787 ◽

Cited By ~ 11

Author(s):

P A Steck ◽

S M North ◽

G L Nicolson

Keyword(s):

Cellular Localization ◽

Proteinase Inhibitors ◽

Cell Clone ◽

Gradient Centrifugation ◽

Mammary Adenocarcinoma ◽

Metastatic Tumour ◽

Guanidinium Chloride ◽

Tumour Metastasis ◽

Adenocarcinoma Cells ◽

Cscl Density Gradient

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.

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Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

Biochemical Journal ◽

10.1042/bj2090497 ◽

1983 ◽

Vol 209 (2) ◽

pp. 497-503 ◽

Cited By ~ 63

Author(s):

N Uldbjerg ◽

A Malmström ◽

G Ekman ◽

J Sheehan ◽

U Ulmsten ◽

...

Keyword(s):

Molecular Weight ◽

Uterine Cervix ◽

Proteinase Inhibitors ◽

Gradient Centrifugation ◽

Average Molecular Weight ◽

Deae Cellulose ◽

Sedimentation Equilibrium ◽

Dermatan Sulphate ◽

Guanidinium Chloride ◽

Isolation And Characterization

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

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Absence of covalently linked core protein from newly synthesized hyaluronate

Biochemical Journal ◽

10.1042/bj2070445 ◽

1982 ◽

Vol 207 (3) ◽

pp. 445-457 ◽

Cited By ~ 25

Author(s):

R M Mason ◽

C d'Arville ◽

J H Kimura ◽

V C Hascall

Keyword(s):

Core Protein ◽

Cell Layer ◽

Gradient Centrifugation ◽

Primary Cultures ◽

Guanidinium Chloride ◽

Protein Pool ◽

Cscl Density Gradient ◽

Chain Synthesis ◽

Covalently Linked ◽

Zwitterionic Detergent

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.

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Rooster comb hyaluronate—protein, a non-covalently linked complex

Biochemical Journal ◽

10.1042/bj2350117 ◽

1986 ◽

Vol 235 (1) ◽

pp. 117-123 ◽

Cited By ~ 13

Author(s):

C P Tsiganos ◽

D H Vynios ◽

D L Kalpaxis

Keyword(s):

Protein Fraction ◽

Core Protein ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Deae Cellulose ◽

Solvent Mixture ◽

Exchange Chromatography ◽

Cscl Density Gradient ◽

Covalently Linked

Hyaluronate from rooster comb was isolated by ion-exchange chromatography on DEAE-cellulose from tissue extracts and papain digests. The preparations were labelled with [14C]acetic anhydride and subjected to CsCl-density-gradient centrifugation in 4 M-guanidinium chloride in the presence and absence of 4% ZwittergentTM 3-12. A radioactive protein fraction was separated from the hyaluronate when the zwitterionic detergent was also present. The protein could also be separated from the glycosaminoglycan by chromatography on Sepharose CL-6B eluted with the same solvent mixture. The protein fraction contained three protein bands of Mr 15,000-17,000 as assessed by polyacrylamide-gel electrophoresis in 0.1% SDS, and seemed to lack lysozyme activity. No evidence of other protein or amino acid(s) covalently linked with the hyaluronate was obtained. The hyaluronate-protein complex may be re-formed upon mixing the components, the extent of its formation depending on the conditions used. The results show that, as in chondrosarcoma [Mason, d'Arville, Kimura & Hascall (1982) Biochem. J. 207, 445-457] and teratocarcinoma cells [Prehm (1983) Biochem. J. 211, 191-198] the rooster comb hyaluronate also is not linked covalently to a core protein.

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Metabolism of rat bone proteoglycans in vivo

Biochemical Journal ◽

10.1042/bj2160589 ◽

1983 ◽

Vol 216 (3) ◽

pp. 589-596 ◽

Cited By ~ 53

Author(s):

C W Prince ◽

F Rahemtulla ◽

W T Butler

Keyword(s):

Extraction Procedure ◽

Total Radioactivity ◽

Mineralized Tissue ◽

Guanidinium Chloride ◽

Disaccharide Composition ◽

A Minor ◽

Papain Digestion ◽

Rat Bone

Former evaluations of the role of proteoglycans in mineralization have neglected to address the possibility that the metabolism of proteoglycans may be of significance in this regard. This problem was studied by using radiolabeling in vivo of rat calvaria with [35Sulphate for 2-72 h and a sequential extraction procedure to yield two pools of newly synthesized proteoglycans: one obtained from non-mineralized tissue by extraction with guanidinium chloride (GdmCl) and another obtained only after demineralization with EDTA. Total radioactivity in calvaria was maximal after 12 h of incorporation, but by 36 h had declined to a level that was about 55-65% of maximum. Radioactivity in the GdmCl extract declined steadily after 12 h, whereas that in the EDTA extract remained constant until 36 h, when it began to increase. Each extract contained a minor proteoglycan that eluted at the void volume (Vo) of a Sepharose CL-6B column. Unlike in the EDTA extract, this proteoglycan gradually disappeared from the GdmCl extract. Each extract also contained a major, smaller proteoglycan, with a Kav. of 0.24 and 0.36 in the GdmCl and EDTA extracts respectively. Papain digestion of each extract yielded glycosaminoglycan chains with Kav. values of 0.32 and 0.50 on CL-6B in the GdmCl and EDTA extracts respectively. Digestion of each extract with chondroitinase ABC and chondroitinase AC showed that the glycosaminoglycans were of similar disaccharide composition, with about 85% being 4-sulphated and the remainder 6-sulphated and/or iduronic acid-containing. These data suggest that about 45% of the newly synthesized proteoglycans are removed from the tissue during the course of mineralization.

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The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities

Biochemical Journal ◽

10.1042/bj2320111 ◽

1985 ◽

Vol 232 (1) ◽

pp. 111-117 ◽

Cited By ~ 25

Author(s):

M T Bayliss ◽

P J Roughley

Keyword(s):

Hyaluronic Acid ◽

Articular Cartilage ◽

Proteinase Inhibitors ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Human Articular Cartilage ◽

Adult Human ◽

Cscl Density Gradient ◽

Proteoglycan Aggregates ◽

Hyaluronic Acid Binding

Proteoglycan was extracted from adult human articular cartilage from both the knee and the hip, and A1 preparations were prepared by CsCl-density-gradient centrifugation at starting densities of 1.69 and 1.5 g/ml. Irrespective of whether the cartilage was diced to 1 mm cubes or sectioned to 20 micron slices there was always a lower proportion of both protein and proteoglycan aggregate in the A1 preparation prepared at 1.69 g/ml. Furthermore, the addition of exogenous hyaluronic acid to the extracts before centrifugation did not improve the yield of aggregate at 1.69 g/ml. These results were not affected by the presence of proteinase inhibitors in the extraction medium. It appears that adult human articular cartilage contains a high proportion of low-density proteoglycan subunits and hyaluronic acid-binding proteins that make most of the re-formed proteoglycan aggregates of a lower density than is usually encountered with younger human and mammalian hyaline cartilages.

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