scholarly journals Properties of heparan sulphate and chondroitin sulphate from young and old human aortae

1969 ◽  
Vol 114 (1) ◽  
pp. 89-96 ◽  
Author(s):  
G. Manley ◽  
R. N. Mullinger ◽  
P. H. Lloyd
Keyword(s):  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Total Amino Acid ◽  
Cross Linking ◽  
Molecular Weights ◽  
Consistent Increase ◽  
Salt Gradient ◽  
Alkali Hydrolysis ◽  
Dowex 1

1. Glycosaminoglycans were liberated from old and young human ascending aortae by digestion with papain. Heparan sulphate and chondroitin sulphate were separated by the different solubilities of their complexes with cetylpyridinium chloride in solutions of sodium chloride. Final fractionation was achieved by salt-gradient column chromatography on Dowex 1 (Cl−form). 2. Heparan sulphate from old aortae showed a slight, but consistent, increase in sulphation compared with heparan sulphate from young aortae. 3. The major amino acids associated with aortic heparan sulphate and chondroitin sulphate were serine, glycine, glutamic acid and aspartic acid. Heparan sulphate and chondroitin sulphate from old aortae contained about twice as much total amino acid as heparan sulphate and chondroitin sulphate from young aortae. Alkali hydrolysis resulted in the destruction of more serine in chondroitin sulphate from old, compared with young, aortae. 4. Molecular weights of glycosaminoglycans from old and young aortae were found to be similar, and in the region of 35000. 5. It is suggested that there is an increased degree of protein–glycosaminoglycan cross-linking in old aortae.

scholarly journals Human Serum and Urine Glycosaminoglycans in Health and in Patients with Chronic Renal Failure

1992 ◽  
Vol 29 (2) ◽  
pp. 190-195 ◽  
Author(s):  
L Bower ◽  
C Warren ◽  
G Manley
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Normal Urine ◽  
Glycosaminoglycan Metabolism ◽  
Normal Controls

Quantitation of uronic acid precipitable by cetylpyridinium chloride (CPC) and electrophoretic separation of glycosaminoglycans were performed on sera from patients with chronic renal failure and compared to normal controls. Serum CPC-precipitable uronic acid (CpUA) levels in patients with renal failure were significantly higher (mean 13·7 mg/L, range 7·1–23·6 mg/L) than normal controls (mean 9·6 mg/L, range 5·1–13·9 mg/L) due to increased concentrations of low sulphated chondroitin sulphate. A positive correlation between serum CpUA and creatinine was found in renal failure patients. Urine CpUA excretion was raised in renal failure patients compared to normal controls with an increased excretion of chondroitin sulphate (Ch-S) of reduced electrophoretic mobility. Heparan sulphate (HS), a major glycosaminoglycan in normal urine, was absent from the urine of these patients. The possible origin of urine glycosaminoglycans and the role of the kidney in glycosaminoglycan metabolism are discussed.

Glycoproteins and glycosaminoglycans of cultured normal human epidermal keratinocytes

1983 ◽  
Vol 61 (1) ◽  
pp. 325-338
Author(s):  
K.W. Brown ◽  
E.K. Parkinson
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Sodium Dodecyl ◽  
Immunological Methods ◽  
Epidermal Keratinocytes ◽  
Immunoperoxidase Staining ◽  
Human Epidermal Keratinocytes ◽  
Molecular Weights ◽  
Keratinocyte Cell ◽  
Normal Human

[3H]glucosamine has been used to label metabolically keratinocyte cell-surface glycoconjugates. The major labelled bands identified on sodium dodecyl sulphate/polyacrylamide gels had apparent molecular weights of greater than 250 000, and 150 000-80 000. Most of these components were trypsin-sensitive, indicating that the label was protein-bound. Some of the labelled components were shown to be proteoglycans and the labelled glycosaminoglycans released from them by trypsin were identified as hyaluronic acid (54%), heparan sulphate (33%) and chondroitin sulphate (13%). Specific immunological methods (immunoperoxidase staining and immunoprecipitation) showed that keratinocytes produced fibronectin. Immunoperoxidase staining showed keratinocytes produce only small ‘stitches’ of fibronectin at cell edges; no large fibrils were seen nor any staining over or between cells.

scholarly journals The glycosaminoglycans of human tracheobronchial cartilage

1970 ◽  
Vol 120 (4) ◽  
pp. 777-785 ◽  
Author(s):  
R. M. Mason ◽  
F. S. Wusteman
Keyword(s):  
Molecular Weight ◽  
Potassium Hydroxide ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Exchange Chromatography ◽  
Chemical Heterogeneity ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Age Dependent ◽  
Dowex 1

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5–1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.

scholarly journals Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils

1991 ◽  
Vol 275 (1) ◽  
pp. 67-73 ◽  
Author(s):  
S R Nelson ◽  
M Lyon ◽  
J T Gallagher ◽  
E A Johnson ◽  
M B Pepys
Keyword(s):  
Light Chain ◽  
Amyloid Fibrils ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Cellulose Acetate Electrophoresis ◽  
Amyloid A ◽  
Isolation And Characterization ◽  
Cscl Density Gradient ◽  
A Minor

Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.

scholarly journals Studies on the accessibility of deoxyribonucleic acid in deoxyribonucleoprotein to cationic molecules

1971 ◽  
Vol 122 (4) ◽  
pp. 583-592 ◽  
Author(s):  
Ruth F. Itzhaki
Keyword(s):  
Molecular Weight ◽  
Toluidine Blue ◽  
Deoxyribonucleic Acid ◽  
Cetylpyridinium Chloride ◽  
Gene Repression ◽  
Cross Linking ◽  
Large Molecules ◽  
Molecular Weights ◽  
Complete Precipitation ◽  
Phosphate Groups

The binding of deoxyribonucleoprotein to Toluidine Blue, to cetylpyridinium chloride and to polylysine of various molecular weights was studied to determine the percentage of free DNA phosphate groups in deoxyribonucleoprotein. Binding was measured by addition of these reagents to deoxyribonucleoprotein at a range of concentrations such that complete precipitation of the deoxyribonucleoprotein occurred. With Toluidine Blue the binding corresponded to about 48% of the DNA phosphates in deoxyribonucleoprotein. The dye did not cause appreciable displacement of protein from the DNA. With cetylpyridinium chloride the binding corresponded to about 41% of the DNA phosphates. With polylysine preparations of molecular weight 1250 and 7790 the binding values for deoxyribonucleoprotein were 46 and 38% respectively. The results suggest that the free phosphates lie in stretches sufficiently long to accommodate most of each polylysine molecule. With polylysine of molecular weight 62000 cross-linking of free stretches of DNA on different deoxyribonucleoprotein molecules probably occurs. It is concluded that although most of the free phosphates are probably ‘hidden’ beneath covering histone, corresponding perhaps to runs of non-basic residues in the latter, they are surprisingly accessible to very large molecules. The relevance of this finding to the problem of gene repression is discussed.

Separate Effects of Urinary Chondroitin Sulphate and Heparan Sulphate on the Crystallization of Urinary Calcium Oxalate: Differences between Stone Formers and Normal Control Subjects

1993 ◽  
Vol 85 (1) ◽  
pp. 33-39 ◽  
Author(s):  
D. K. Y. Shum ◽  
M. D. I. Gohel
Keyword(s):  
Calcium Oxalate ◽  
Normal Control ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Urinary Calcium ◽  
Crystal Nucleation ◽  
Control Subjects ◽  
Stone Formers

1. Urinary glycosaminoglycans were recovered from the papain digest of polyanions precipitated sequentially by cetylpyridinium chloride and sodium acetate-saturated ethanol. Those from the early morning urine of 48 stone formers and 43 normal control subjects measured 11 and 16 μg of uronic acid/ml of urine, respectively. 2. Preparative agarose gel electrophoresis of the recovered glycosaminoglycans in barium acetate buffer (pH 5.8) yielded fractions containing purely chondroitin sulphate, co-polymeric chondroitin/dermatan sulphates and heparan sulphate. Identification was based on the susceptibility of the fractions to chondroitinase or nitrous acid treatment. Similar compositions of glycosaminoglycan classes were observed in samples from stone formers and normal control subjects. 3. The fractionated glycosaminoglycans were dissolved in urine ultrafiltrate to assay for nucleation-promoting and growth-inhibiting activities towards crystallization of urinary calcium oxalate. When compared at the same uronic acid concentration, both the urinary chondroitin sulphate isomers and heparan sulphates of stone formers demonstrated the capacity to enhance crystal nucleation from calcium oxalate endogenous in urine ultrafiltrates, whereas only urinary heparan sulphates of normal control subjects demonstrated this capacity. 4. Tissue-derived reference chondroitin sulphate, dermatan sulphate and heparin, when similarly tested, showed negligible crystal nucleation-promoting activity. The tissue-derived heparan sulphate was similar to the urinary heparan sulphates in showing marked crystal nucleation-promoting activity. 5. Crystal-growth inhibitory activity was evident in all urinary glycosaminoglycan fractions studied. In particular, urinary heparan sulphate of normal control subjects showed higher activity than that of stone formers or the chondroitin sulphate isomers of both stone formers and normal control subjects (P <0.005).

scholarly journals Evidence for the existence of a multichain proteoglycan of heparan sulphate

1970 ◽  
Vol 117 (4) ◽  
pp. 699-702 ◽  
Author(s):  
L. Jansson ◽  
U. Lindahl
Keyword(s):  
Potassium Chloride ◽  
Aortic Wall ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Fraction I ◽  
Two Peaks ◽  
Testicular Hyaluronidase

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.

scholarly journals Attempted isolation of a heparin proteoglycan from bovine liver capsule

1970 ◽  
Vol 116 (1) ◽  
pp. 27-34 ◽  
Author(s):  
U. Lindahl
Keyword(s):  
Potassium Chloride ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Bovine Liver ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Liver Capsule ◽  
Neutral Sugars ◽  
Degraded Material ◽  
Heparin Preparation

(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.

scholarly journals Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1

1987 ◽  
Vol 245 (2) ◽  
pp. 543-550 ◽  
Author(s):  
D H Farrell ◽  
D D Cunningham
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Order Rate Constant ◽  
Subsequent Treatment ◽  
Protease Nexin ◽  
Inhibition Of Thrombin

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

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