scholarly journals The distribution of sulphate residues in the chondroitin sulphate chain

1971 ◽  
Vol 125 (3) ◽  
pp. 903-908 ◽  
Author(s):  
Åke Wasteson ◽  
Ulf Lindahl
Keyword(s):  
Chondroitin Sulphate ◽  
Gel Chromatography ◽  
Neutral Sugar ◽  
Charge Heterogeneity ◽  
Linkage Region ◽  
Molar Ratios ◽  
Before And After ◽  
Dowex 1 ◽  
Chondroitin Sulphate Chain ◽  
Testicular Hyaluronidase

1. Electrophoresis of chondroitin sulphate, before and after partial degradation with testicular hyaluronidase, revealed charge heterogeneity of the degraded but not of the intact polymer. 2. Hyaluronidase-treated chondroitin sulphate was fractionated by gel chromatography. Two subfractions which were essentially monodisperse with regard to molecular weight (values of 8600 and 4800, respectively) were separated further by chromatography on Dowex 1. The resulting subfractions differed considerably with respect to their sulphate/disaccharide molar ratios. 3. Amino acid and neutral-sugar analyses of the Dowex 1 subfractions showed that the less sulphated fragments contained the carbohydrate–protein linkage region, whereas the high-sulphated fragments essentially lacked this constituent. It was concluded that chondroitin sulphate contains relatively less sulphate in the vicinity of the carbohydrate–protein linkage region than in the more peripheral portion of the polysaccharide chain.

scholarly journals Proteoglycans of hyaline cartilage: Electron-microscopic studies on isolated molecules

1975 ◽  
Vol 151 (1) ◽  
pp. 157-166 ◽  
Author(s):  
J Thyberg ◽  
S Lohmander ◽  
D Heinegård
Keyword(s):  
Hyaline Cartilage ◽  
Chondroitin Sulphate ◽  
Costal Cartilage ◽  
Side Chain ◽  
Gel Chromatography ◽  
Electron Microscopic ◽  
The Core ◽  
Central Filament ◽  
Microscopic Studies ◽  
Chondroitin Sulphate Chain

Proteoglycan monomers from guinea-pig costal cartilage, bovine nasal and bovine tracheal cartilage were observed in the electron microscope after being spread in a monomolecular layer with cytochrome c. The proteoglycan molecule appeared as an extended central core filament to which side-chain filaments were attached at various intervals. The molecules from the three sources displayed great ultrastructural similarities. On average, the core filament was about 290 nm long, there were about 25 side-chain filaments per core filament, the side-chain filaments were about 45 nm long, and the distance between the attachment points of the side-chain filaments to the core filament was about 11 nm. With regard to the overall size of the molecules, no evidence of distinct subpopulations was obtained. Good correlation was found between ultrastructural data for the proteoglycan molecules and chemical data obtained by enzyme digestions and gel chromatography. Together these data strongly support the interpretation of the electron-microscopic pictures as indicating a central filament corresponding to the protein core and side-chain filaments corresponding to the chondroitin sulphate chain clusters of the proteoglycan monomers.

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 Degradation of heparin in mouse mastocytoma tissue

1971 ◽  
Vol 125 (4) ◽  
pp. 1119-1129 ◽  
Author(s):  
Sören Ögren ◽  
Ulf Lindahl
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Supernatant Fraction ◽  
Gel Chromatography ◽  
Neutral Sugar ◽  
Pulse Labelling

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GEH) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GEH was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights (¯Mw) of approx. 60000–70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate–protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [35S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating 35S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting 14C-labelled chondroitin sulphate to the same procedure.

scholarly journals The characterization of a protein–polysaccharide isolated from Kurloff cells of the guinea pig

1970 ◽  
Vol 118 (5) ◽  
pp. 783-790 ◽  
Author(s):  
M. F. Dean ◽  
Helen Muir
Keyword(s):  
Chondroitin Sulphate ◽  
Molecular Size ◽  
Gel Chromatography ◽  
Oestrogen Treatment ◽  
Molar Ratios ◽  
Low Concentrations ◽  
Cell Induction ◽  
Toxic Constituent

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm−1. Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein–polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline β-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein–polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein–polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein–polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein–polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm−1. Although active incorporation of [35S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.

scholarly journals An unsulphated region of the rat chondrosarcoma chondroitin sulphate chain and its binding to monoclonal antibody 3B3

1991 ◽  
Vol 273 (1) ◽  
pp. 237-239 ◽  
Author(s):  
J R Baker ◽  
J E Christner ◽  
S L Ekborg
Keyword(s):  
Monoclonal Antibody ◽  
Structure Function ◽  
Chondroitin Sulphate ◽  
Significant Proportion ◽  
Physiological Significance ◽  
High Concentration ◽  
Chondroitinase Abc ◽  
Linkage Region ◽  
Unsaturated Disaccharide ◽  
Chondroitin Sulphate Chain

The chondroitin sulphate chains of proteoglycans are not uniformly sulphated. Commonly, regions of under- and over-sulphation are found. It is probable that variability in chondroitin sulphation has physiological significance, although such structure-function relationships largely remain unexplored. Chondroitin sulphate from rat chondrosarcoma proteoglycan has been found to possess no oversulphated residues. It is primarily chondroitin 4-sulphate, although a significant proportion of unsulphated disaccharides (14%) are also present. It appears that some unsulphated disaccharides are concentrated only at the point of attachment to the linkage region (i.e. it is the major unsaturated disaccharide remaining attached to chondrosarcoma proteoglycan core produced by chondroitinase ABC digestion). This proteoglycan core binds monoclonal antibody (MAb) 3B3. Although 3B3 principally binds to 6-sulphated ‘stubs’ of proteoglycan cores [Couchman, Caterson, Christner & Baker (1984) Nature (London) 307, 650-652], given a high concentration of unsulphated ‘stubs’, it can alternatively bind to these residues. It is also evident that caution must be exercised in using MAb 3B3 to identify chondroitin 6-sulphated proteoglycans.

scholarly journals Heterogeneity of protein–polysaccharides of porcine articular cartilage. The sequential extraction of chondroitin sulphate–proteins with iso-osmotic neutral sodium acetate

1971 ◽  
Vol 121 (2) ◽  
pp. 261-270 ◽  
Author(s):  
Kenneth D. Brandt ◽  
Helen Muir
Keyword(s):  
Sodium Acetate ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Gel Filtration ◽  
Progressive Increase ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Large Molecules ◽  
Molar Ratios ◽  
Neutral Sodium

Protein–polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein–polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein–polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein–polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein–polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein–polysaccharides of earlier extracts even though these contained some large molecules.

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.

Localisation and characterization of acid mucopolysaccharides in the early chick blastoderm

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 169-178
Author(s):  
Ch. Vanroelen ◽  
L. Vakaet ◽  
L. Andries
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Middle Layer ◽  
Ventral Side ◽  
Chick Blastoderm ◽  
Cellulose Acetate Electrophoresis ◽  
Acid Mucopolysaccharides ◽  
Extracellular Compartment ◽  
Early Specialization ◽  
Testicular Hyaluronidase

Acid mucopolysaccharides in the extracellular compartment of early chick blastoderms (16 h of incubation) were labelled with tritiated glucosamine and/or ]35S]sulphate. The incorporation pattern was studied autoradiographically. Treatment with testicular hyaluronidase revealed a testicular hyaluronidase-sensitive fraction, mainly at the periphery of Middle Layer and Deep Layer cells, and a testicular hyaluronidase-resistant fraction, mainly at the ventral side of the Upper Layer. A biochemical analysis, utilizing chondroitinase ABC and nitrous acid, followed by cellulose acetate electrophoresis, demonstrated the synthesis of a non-sulphated fraction, i.e. hyaluronic acid and/or chondroitin, and a sulphated fraction, comprising two undersulphated components, i.e. chondroitin sulphate, and heparan sulphate or heparin. The appearance of different AMPS in specific areas of the early chick blastoderm is regarded as an early specialization of the extracellular compartment.

High-yield purification of the complex-forming glycoprotein in urine from normal and abnormal subjects.

1989 ◽  
Vol 35 (4) ◽  
pp. 577-581 ◽  
Author(s):  
H Wakui ◽  
R Kobayashi ◽  
H Itoh ◽  
H Imai ◽  
Y Nakamoto ◽  
...  
Keyword(s):  
Biochemical Properties ◽  
High Yield ◽  
Gel Chromatography ◽  
Tubular Proteinuria ◽  
Charge Heterogeneity ◽  
Purification Method ◽  
Efficient Procedure ◽  
Renal Disorders ◽  
Special Collection ◽  
Urine Specimens

Abstract Ordinary methods for purification of complex-forming glycoprotein (protein HC) or alpha 1-microglobulin, a protein closely related to protein HC, require either large volumes of urine or special collection of urine specimens from patients with tubular proteinuria. Here we describe a fast, efficient procedure for isolating protein HC in high yield from urines from healthy and diseased subjects, with use of Cibacron blue, hydroxylapatite, and gel chromatography. Using this procedure, we also obtained a considerable amount of polymeric protein HC from the urine of a patient with chronic renal failure. The pattern of charge heterogeneity and immunoreactivity with anti-protein HC differed between the polymeric and monomeric forms of protein HC. We also observed a variability in charge heterogeneity of protein HC among patients with renal disorders. These results demonstrate that this purification method is useful for further studies to elucidate the biochemical properties of protein HC and its clinical significance in renal disorders.

scholarly journals Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate

1995 ◽  
Vol 311 (1) ◽  
pp. 233-238 ◽  
Author(s):  
K Lidholt ◽  
I Eriksson ◽  
L Kjellén
Keyword(s):  
Chain Length ◽  
Chondroitin Sulphate ◽  
Major Portion ◽  
Gel Chromatography ◽  
High Affinity ◽  
Polysaccharide Chain ◽  
Polysaccharide Composition ◽  
Length Determination

Proteoglycans (PGs), biosynthetically labelled with [35S]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the 35S-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [35S]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these 35S-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [35S]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) approximately 30,000) than the CS chains in the NA-PGs (M(r) approximately 25,000). CS chains in the hybrid PGs were slightly smaller (M(r) approximately 20,000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20%) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [35S]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.

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