Isolation and characterization of chondroitin 6-sulfate proteoglycans present in the extracellular matrix of rabbit bone marrow

Abstract The authors' previous studies showed that the glycosaminoglycans present in rabbit bone marrow were composed of chondroitin 6-sulfate (79%) and hyaluronic acid (16%). Immunohistochemically the chondroitin 6-sulfate was demonstrated to be in bone marrow matrix constructing hematopoietic microenvironment. In this study the authors isolated and characterized these glycosaminoglycans in their macromolecular form (i.e., proteoglycans). Bone marrow of 3-month-old rabbits was defatted with organic solvents containing proteinase inhibitors at -20 degrees C, and proteoglycans were extracted from the defatted tissue with 4 mol/L guanidine HCl containing the proteinase inhibitors. After extensive dialysis of the extract against 7 mol/L urea, more than 90% of hexuronate-containing materials was recovered in the urea-soluble fraction. The proteoglycans were purified from the urea-soluble fraction by diethyl aminoethyl (DEAE)-Sephacel chromatography, CsCl density-gradient centrifugation, and Bio-Gel A-5m gel filtration, then were rechromatographed on DEAE-Sephacel and on Bio-Gel A-5m. The proteoglycans were separated into three molecular species with different mol wts that were assessed to be 46,000, 16,000, and 8,300 by sedimentation-equilibrium centrifugation. Amino acid analyses of these proteoglycans revealed that serine and glycine accounted for approximately 60% of the total amino acids common to the three proteoglycans. The glycosaminoglycan side chains of these proteoglycans were converted stoichiometrically into unsaturated 6-sulfated disaccharide by digestion with chondroitinase AC-II, indicating that they were fully sulfated chondroitin 6-sulfate. Their apparent mol wts were estimated by gel filtration on Bio-Gel A-0.5m to be 10,900, 14,400, and 7,700. Computation of these results, taken together with their biochemical composition, revealed that the largest proteoglycan, PG-II, consisted of four chains of the chondroitin 6-sulfate and the core peptide with mol wt of approximately 4,000. The smaller two proteoglycan subpopulations, PG-I and PG-III, consisted of a single glycosaminoglycan chain linked to a small peptide with mol wts of approximately 500 and 1,000, respectively.

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Isolation and characterization of chondroitin 6-sulfate proteoglycans present in the extracellular matrix of rabbit bone marrow

Blood ◽

10.1182/blood.v72.2.745.bloodjournal722745 ◽

1988 ◽

Vol 72 (2) ◽

pp. 745-755

Author(s):

E Okayama ◽

K Oguri ◽

T Kondo ◽

M Okayama

Keyword(s):

Bone Marrow ◽

Soluble Fraction ◽

Proteinase Inhibitors ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sedimentation Equilibrium ◽

Isolation And Characterization ◽

Cscl Density Gradient ◽

Core Peptide ◽

Rabbit Bone

The authors' previous studies showed that the glycosaminoglycans present in rabbit bone marrow were composed of chondroitin 6-sulfate (79%) and hyaluronic acid (16%). Immunohistochemically the chondroitin 6-sulfate was demonstrated to be in bone marrow matrix constructing hematopoietic microenvironment. In this study the authors isolated and characterized these glycosaminoglycans in their macromolecular form (i.e., proteoglycans). Bone marrow of 3-month-old rabbits was defatted with organic solvents containing proteinase inhibitors at -20 degrees C, and proteoglycans were extracted from the defatted tissue with 4 mol/L guanidine HCl containing the proteinase inhibitors. After extensive dialysis of the extract against 7 mol/L urea, more than 90% of hexuronate-containing materials was recovered in the urea-soluble fraction. The proteoglycans were purified from the urea-soluble fraction by diethyl aminoethyl (DEAE)-Sephacel chromatography, CsCl density-gradient centrifugation, and Bio-Gel A-5m gel filtration, then were rechromatographed on DEAE-Sephacel and on Bio-Gel A-5m. The proteoglycans were separated into three molecular species with different mol wts that were assessed to be 46,000, 16,000, and 8,300 by sedimentation-equilibrium centrifugation. Amino acid analyses of these proteoglycans revealed that serine and glycine accounted for approximately 60% of the total amino acids common to the three proteoglycans. The glycosaminoglycan side chains of these proteoglycans were converted stoichiometrically into unsaturated 6-sulfated disaccharide by digestion with chondroitinase AC-II, indicating that they were fully sulfated chondroitin 6-sulfate. Their apparent mol wts were estimated by gel filtration on Bio-Gel A-0.5m to be 10,900, 14,400, and 7,700. Computation of these results, taken together with their biochemical composition, revealed that the largest proteoglycan, PG-II, consisted of four chains of the chondroitin 6-sulfate and the core peptide with mol wt of approximately 4,000. The smaller two proteoglycan subpopulations, PG-I and PG-III, consisted of a single glycosaminoglycan chain linked to a small peptide with mol wts of approximately 500 and 1,000, respectively.

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Purification and characterization of human platelet proteoglycan

Biochemical Journal ◽

10.1042/bj2330073 ◽

1986 ◽

Vol 233 (1) ◽

pp. 73-81 ◽

Cited By ~ 35

Author(s):

M Okayama ◽

K Oguri ◽

Y Fujiwara ◽

H Nakanishi ◽

H Yonekura ◽

...

Keyword(s):

Soluble Fraction ◽

Proteinase Inhibitors ◽

Chondroitin Sulphate ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Human Platelets ◽

Sedimentation Equilibrium ◽

Urea Solution ◽

Cellulose Acetate Membrane ◽

Cscl Density Gradient

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.

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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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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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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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Purification and characterization of a tetrameric α-macroglobulin proteinase inhibitor from the gastropod mollusc Biomphalaria glabrata

Biochemical Journal ◽

10.1042/bj3160893 ◽

1996 ◽

Vol 316 (3) ◽

pp. 893-900 ◽

Cited By ~ 31

Author(s):

Randall C. BENDER ◽

Christopher J. BAYNE

Keyword(s):

Quaternary Structure ◽

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Serine Proteinase ◽

Biomphalaria Glabrata ◽

Sedimentation Equilibrium ◽

Flight Mass Spectrometry ◽

Isolation And Characterization ◽

Α2 Macroglobulin

The α-macroglobulin proteinase inhibitors (αMs) are a family of proteins with the unique ability to inhibit a broad spectrum of proteinases. Whereas monomeric, dimeric and tetrameric αMs have been identified in vertebrates, all invertebrate αMs characterized so far have been dimeric. This paper reports the isolation and characterization of a tetrameric αM from the tropical planorbid snail Biomphalaria glabrata. The sequence of 18 amino acids at the N-terminus indicates homology with other αMs. The subunit mass of approx. 200 kDa was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and SDS/PAGE. The quaternary structure was determined by sedimentation equilibrium centrifugation and native pore-limit electrophoresis. Evidence for a thioester is provided by the fact that methylamine treatment prevents the autolytic cleavage of the snail αM subunit and results in the release of 4 mol of thiols per mol of snail αM. The snail αM inhibited the serine proteinase trypsin, the cysteine proteinase bromelain and the metalloproteinase thermolysin. The spectrum of proteinases inhibited, together with the demonstration of steric protection of the proteinase active site and a ‘slow to fast’ conformational change after reacting with trypsin, all suggest that the inhibitory mechanism of the snail αM is similar to the ‘trap mechanism’ of human α2-macroglobulin.

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Isolation and characterization of lung connective-tissue glycoproteins

Biochemical Journal ◽

10.1042/bj2030593 ◽

1982 ◽

Vol 203 (3) ◽

pp. 593-601 ◽

Cited By ~ 9

Author(s):

C Lafuma ◽

M Moczar ◽

L Robert

Keyword(s):

Proteinase Inhibitors ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Lung Parenchyma ◽

Isolation And Characterization ◽

Isoelectric Points ◽

And Function

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.

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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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Isolation and characterization of human cervical-mucus glycoproteins

Biochemical Journal ◽

10.1042/bj2110013 ◽

1983 ◽

Vol 211 (1) ◽

pp. 13-22 ◽

Cited By ~ 129

Author(s):

I Carlstedt ◽

H Lindgren ◽

J K Sheehan ◽

U Ulmsten ◽

L Wingerup

Keyword(s):

Density Gradient ◽

Proteinase Inhibitors ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Disc Electrophoresis ◽

Guanidinium Chloride ◽

Molecular Weights ◽

Isolation And Characterization ◽

Mucus Glycoproteins

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

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Comparative study of technique to obtain stem cells from bone marrow collection between the iliac crest and the femoral epiphysis in rabbits

Acta Cirurgica Brasileira ◽

10.1590/s0102-86502009000500011 ◽

2009 ◽

Vol 24 (5) ◽

pp. 400-404 ◽

Cited By ~ 11

Author(s):

Lilian Piñero Eça ◽

Renata Belmonte Ramalho ◽

Isis Sousa Oliveira ◽

Paulo Oliveira Gomes ◽

Paulo Pontes ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Iliac Crest ◽

Mononuclear Cells ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Group A ◽

Rabbit Bone ◽

Group B ◽

Bone Marrow Blood

PURPOSE: To assess the technique for the collection of rabbit bone marrow stem cells from different regions to be used as an experimental model in regenerative medicine. METHODS: Thirty rabbits were allocated into 2 groups: GROUP A, n=8, animals that underwent bone marrow blood (BMB) harvesting from the iliac crest; and GROUP B: including 22 rabbits that underwent BMB harvesting from the femur epiphysis. After harvesting, mononuclear cells were isolated by density gradient centrifugation (Ficoll - Histopaque). The number of mononuclear cells per ml was counted in a Neubauer chamber and cell viability was checked through Tripan Blue method. RESULTS: Harvesting from the iliac crest yielded an average of 1 ml of BMB and 3,6.10(6) cells/ml over 1 hour of surgery, whereas an average of 3ml of BMB and 11,79.10(6) cells./ml were obtained in 30 min from the femur epiphysis with a reduced animal death rate. CONCLUSION: The analysis for the obtention of a larger number of mononuclear cells/ml from rabbit bone marrow blood was more satisfactory in the femur epiphysis than in the iliac crest.

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