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

1987 ◽  
Vol 242 (3) ◽  
pp. 779-787 ◽  
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.

scholarly journals Characterization and N-terminal sequence of human platelet proteoglycan

1988 ◽  
Vol 255 (3) ◽  
pp. 1007-1013 ◽  
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.

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

1985 ◽  
Vol 232 (1) ◽  
pp. 111-117 ◽  
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.

scholarly journals Interactions between different corneal proteoglycans

1978 ◽  
Vol 173 (3) ◽  
pp. 935-939 ◽  
Author(s):  
P Speziale ◽  
M S Speziale ◽  
L Galligani ◽  
C Balduini
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Original Sample ◽  
Chemical Analyses ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Cscl Density Gradient ◽  
Two Peaks ◽  
Fraction Density

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.

scholarly journals Extraction and purification of proteoglycans from mature bovine bone

1984 ◽  
Vol 224 (1) ◽  
pp. 47-58 ◽  
Author(s):  
A Franzén ◽  
D Heinegård
Keyword(s):  
Core Protein ◽  
Proteinase Inhibitors ◽  
Extraction Procedure ◽  
Deae Cellulose ◽  
Bovine Bone ◽  
Guanidinium Chloride ◽  
Extraction Solvent ◽  
Extraction And Purification ◽  
Cscl Density Gradient ◽  
Mineralized Matrix

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.

scholarly journals The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion

1983 ◽  
Vol 213 (2) ◽  
pp. 427-435 ◽  
Author(s):  
I Carlstedt ◽  
H Lindgren ◽  
J K Sheehan
Keyword(s):  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Cervical Mucus ◽  
Trypsin Digestion ◽  
Radius Of Gyration ◽  
Guanidinium Chloride ◽  
Disulphide Bonds ◽  
Tentative Model ◽  
The Relationship ◽  
Mucus Glycoproteins

Human cervical-mucus glycoproteins (mucins) were extracted with 6 M-guanidinium chloride in the presence of proteinase inhibitors and purified by isopycnic density-gradient centrifugation. The whole mucins (Mr approx. 10 × 10(6] were degraded into ‘subunits’ (Mr approx. 2 × 10(6] by reduction of disulphide bonds. Trypsin digestion of the ‘subunits’ produced glycopeptides with Mr approx. 380000, which appear to be rod-like with a length of approx. 105 nm. The relationship between the radius of gyration and the Mr value obtained by light-scattering for whole mucins, ‘subunits’ and ‘domains’ suggest that cervical-mucus glycoproteins are linear flexible macromolecules composed of, on the average, four or five ‘domains’/subunit and four subunits/whole mucin macromolecule. The shape-dependent particle scattering function for the whole mucins and the ‘subunits’ are in accordance with that of a linear flexible chain. No evidence for a branched or a star-like structure was found. A tentative model for cervical mucins is proposed.

scholarly journals Structural differences between heparan sulphates of proteoglycan involved in the formation of basement membranes in vivo by Lewis-lung-carcinoma-derived cloned cells with different metastatic potentials

1992 ◽  
Vol 288 (1) ◽  
pp. 215-224 ◽  
Author(s):  
H Nakanishi ◽  
K Oguri ◽  
K Yoshida ◽  
N Itano ◽  
K Takenaga ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Lung Carcinoma ◽  
Lewis Lung Carcinoma ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Dry Weight ◽  
Basement Membranes ◽  
Metastatic Tumour ◽  
Lewis Lung ◽  
Cscl Density Gradient

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased content of the three repeating disaccharides having 6-O-sulphated glucosamine residues.

scholarly journals Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver

1991 ◽  
Vol 273 (2) ◽  
pp. 415-422 ◽  
Author(s):  
M Lyon ◽  
J T Gallagher
Keyword(s):  
Molecular Mass ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Exchange Chromatography ◽  
Guanidinium Chloride ◽  
Apparent Homogeneity ◽  
Core Proteins ◽  
Sds Page ◽  
Triton X

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

scholarly journals Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural ‘architecture’

1991 ◽  
Vol 276 (3) ◽  
pp. 667-675 ◽  
Author(s):  
D J Thornton ◽  
J K Sheehan ◽  
H Lindgren ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Gel Phase ◽  
Mucus Glycoproteins

Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as ‘heavy’ and ‘light’ fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the ‘light’ fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].

scholarly journals Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

1983 ◽  
Vol 209 (2) ◽  
pp. 497-503 ◽  
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.

scholarly journals Isolation and characterization of human cervical-mucus glycoproteins

1983 ◽  
Vol 211 (1) ◽  
pp. 13-22 ◽  
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%).

Sign in / Sign up

Export Citation Format

Share Document