scholarly journals Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus

1988 ◽  
Vol 251 (2) ◽  
pp. 347-356 ◽  
Author(s):  
M R Jahnke ◽  
C A McDevitt
Keyword(s):  
Density Gradient ◽  
Nucleus Pulposus ◽  
Gel Electrophoresis ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Buoyant Density ◽  
Hydrodynamic Size ◽  
Keratan Sulphate

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.

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 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 Evidence that platelet density depends on the alpha-granule content in platelets

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 482-485 ◽  
Author(s):  
BA van Oost ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Density Distribution ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Platelet Rich Plasma ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Adenosine Diphosphate ◽  
Density Gradients

Abstract The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.

scholarly journals Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc

1979 ◽  
Vol 179 (3) ◽  
pp. 573-578 ◽  
Author(s):  
R L Stevens ◽  
P G Dondi ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Core Protein ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Hydrodynamic Size ◽  
Laryngeal Cartilage ◽  
Cartilage Proteoglycans

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.

scholarly journals Mucins secreted by a transformed cell line derived from human tracheal gland cells1

1997 ◽  
Vol 326 (2) ◽  
pp. 431-437 ◽  
Author(s):  
Jean-Marc LO-GUIDICE ◽  
Marc D. MERTEN ◽  
Geneviève LAMBLIN ◽  
Nicole PORCHET ◽  
Marie-Christine HOUVENAGHEL ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Cell Line ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
High Molecular Mass ◽  
Cscl Density Gradient

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose® CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to β-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc α2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520–528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Transgressive, reiterative selection by continuous buoyant density gradient centrifugation of Dunaliella salina results in enhanced lipid and starch content

2015 ◽  
Vol 9 ◽  
pp. 194-203 ◽  
Author(s):  
Leyla T. Hathwaik ◽  
Doug Redelman ◽  
Vera Samburova ◽  
Barbara Zielinska ◽  
David K. Shintani ◽  
...  
Keyword(s):  
Density Gradient ◽  
Dunaliella Salina ◽  
Density Gradient Centrifugation ◽  
Starch Content ◽  
Gradient Centrifugation ◽  
Buoyant Density

scholarly journals Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation

1972 ◽  
Vol 126 (4) ◽  
pp. 791-803 ◽  
Author(s):  
T. E. Hardingham ◽  
Helen Muir
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Specific Radioactivity ◽  
Equilibrium Density ◽  
Gel Chromatography ◽  
Sulphate Content

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

scholarly journals Survival of density subpopulations of rabbit platelets: use of 51Cr-or 111In-labeled platelets to measure survival of least dense and most dense platelets concurrently

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 362-367 ◽  
Author(s):  
ML Rand ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Density Gradient ◽  
Total Population ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Specific Radioactivity ◽  
Rabbit Platelets

Abstract The origin of the density heterogeneity of platelets was studied by measuring the survival of density subpopulations of rabbit platelets separated by discontinuous Stractan density gradient centrifugation. When a total population of 51Cr-labeled platelets was injected into recipient rabbits, the relative specific radioactivity of the most dense platelets decreased rapidly. In contrast, that of the least dense platelets had not changed 24 hr after injection, and then decreased slowly. To distinguish between the possibilities that most dense platelets are cleared from the circulation more quickly than least dense platelets or that platelets decrease in density as they age in the circulation, the concurrent survival of least dense and most dense platelets, labeled with either 51Cr or 111In-labeled total platelet populations, determined concurrently in the same rabbits, were identical, calculated from 1 hr values as 100%. However, the 1-hr recovery of 111In-labeled platelets was slightly but significantly less than that of 51Cr-labeled platelets. Therefore, we studied the survival of 51Cr-labeled least dense and 111In-labeled most dense platelets as well as that of 111In-labeled least dense and 51Cr-labeled most dense platelets. Mean 1-hr recovery of least dense platelets, labeled with either isotope (78% +/- 7%, SD) was similar to that of most dense platelets, labeled with either isotope (77% +/- 8%; SD). Mean survival of least dense platelets was 47.3 +/- 18.7 hr (SD), which was significantly less than that of most dense platelets (76.1 +/- 21.6 hr; SD) (p less than 0.0025). These results indicate that platelets decrease in buoyant density as they age in the circulation and that most dense platelets are enriched in young platelets, and least dense in old. Thus, the events that affect platelets as they age in the circulation contribute to platelet density heterogeneity, although they may not be the sole cause of it.

scholarly journals An interaction between lysozyme and mucus glycoproteins. Implications for density-gradient separations

1979 ◽  
Vol 181 (3) ◽  
pp. 717-724 ◽  
Author(s):  
J M Creeth ◽  
J L Bridge ◽  
J R Horton
Keyword(s):  
Sialic Acid ◽  
Ionic Strength ◽  
Density Gradient ◽  
Blood Group ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Ph Values ◽  
The Common ◽  
Mucus Glycoproteins

1. Some mucus glycoproteins form soluble complexes with lysozyme at neutral pH values. 2. The extent of complex-formation was determined, by an ultracentrifugal difference method, for a range of glycoproteins covering the common blood-group specificities. 3. Interaction was strongest with those glycoproteins of blood-group Lea specificity; these were also richest in sialic acid. 4. Interaction diminished with increase of ionic strength, and was not detectable at I 0.50; however, an asialoglycoprotein was found to retain some activity. The interaction is accordingly primarily, but probably not exclusively, coulombic in origin. 5. The buoyant density of lysozyme in CsCl, CsBr, CsI and Cs2SO4 was determined; the values in the last three salts are anomalously high. This finding accounts for the previously noted difficulty of separating free protein from glycoproteins by single-stage centrifugation in CsBr. 6. Conditions for effective separation of glycoproteins from secretions containing lysozyme by density-gradient centrifugation are reported.

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