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 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 Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule

1975 ◽  
Vol 145 (1) ◽  
pp. 53-62 ◽  
Author(s):  
L Jansson ◽  
S Ogren ◽  
U Lindahl
Keyword(s):  
Uronic Acid ◽  
Group Analysis ◽  
Deae Cellulose ◽  
Bovine Liver ◽  
Sugar Residue ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Liver Capsule ◽  
Polysaccharide Chain ◽  
Equilibrium Ultracentrifugation

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.

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 Isolation of 35S- and 3H-labelled proteoglycans from cultures of human embryonic skin fibroblasts

1979 ◽  
Vol 183 (3) ◽  
pp. 669-681 ◽  
Author(s):  
L Cöster ◽  
I Carlstedt ◽  
A Malmström
Keyword(s):  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Cscl Density Gradient ◽  
Embryonic Skin ◽  
Sepharose 4B ◽  
Phosphate Buffered Saline

35SO42– and [3H]-leucine-labelled proteoglycans were isolated from the medium of a fibroblast culture, from an EDTA extract of the monolayer, and from consecutive dithiothreitol and guanidine hydrochloride extracts of the cells. Proteoglycans of different sizes were isolated from the extracts by gel chromatography on Sepharose 4B. In the medium and the EDTA extract the largest proteoglycans contained only 35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparan [35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparin [35S]sulphate. The galactosaminoglycan-containing proteoglycans of the various extracts were separated into a larger component, containing chondroitin sulphate-like side chains, and a smaller component, containing dermatan sulphate. The larger proteoglycan of the medium showed reversible association-dissociation behaviour when chromatographed on Sepharose CL2B in phosphate-buffered saline and 4M-guanidine hydrochloride respectively. This property remained after removal of extraneous proteins by CsCl-density-gradient centrifugation in guanidine hydrochloride. The association was markedly increased by the addition of high-molecular-weight hyaluronic acid.

scholarly journals Glycosaminoglycans of cat invertebral disc

1971 ◽  
Vol 122 (5) ◽  
pp. 647-652 ◽  
Author(s):  
W. F. Butler ◽  
C. M. Wels
Keyword(s):  
Water Content ◽  
Vertebral Column ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Intervertebral Discs ◽  
Chloride Salt ◽  
Lumbar Region ◽  
Dermatan Sulphate ◽  
Mean Values ◽  
Significant Difference

The glycosaminoglycan contents of samples from cat intervertebral discs were examined by using cetylpyridinium chloride salt elution techniques. The values obtained related to the region of the vertebral column from which they were derived, to the area of the disc, and to water content. In wet tissue there was a significant difference between regions of the vertebral column and between areas of the disc and findings agreed with previous histological reports. The greater part of the glycosaminoglycans present consisted of chondroitin sulphate and dermatan sulphate with smaller amounts of hyaluronic acid; little keratan sulphate was found. The maximum amounts of chondroitin sulphate and dermatan sulphate occurred in the 0.5m-magnesium chloride fractions usually, but moved towards higher molar concentrations in samples derived from some sites, particularly in the lumbar region. Mean values for the water content of the areas of the disc were: nucleus pulposus, 82.4%; inner anulus, 65.6%; outer anulus, 50.5%. The water content was directly related to the amounts of chondroitin sulphate and dermatan sulphate.

scholarly journals Properties of fractionated chondroitin sulphate from ox nasal septa

1971 ◽  
Vol 122 (4) ◽  
pp. 477-485 ◽  
Author(s):  
Åke Wasteson
Keyword(s):  
Molecular Weight ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Equilibrium Studies ◽  
The Individual ◽  
A Charge ◽  
The Relationship

1. Chondroitin sulphate was isolated from bovine nasal septa by precipitation with cetylpyridinium chloride after digestion of the tissue with papain. 2. The material was divided into two portions, one of which was partially degraded with testicular hyaluronidase. 3. Untreated and hyaluronidase-digested material were fractionated into a total of eleven subfractions by gel chromatography on Sephadex G-200 and Sephadex G-100 respectively. 4. Chemical analyses indicated that the composition of all the fractions was similar to that of chondroitin sulphate. However, electrophoresis revealed a charge-inhomogeneity in the low-molecular-weight fractions obtained after hyaluronidase digestion. 5. The physicochemical properties of the subfractions were investigated by sedimentation-velocity, diffusion and sedimentation-equilibrium studies, osmometry, viscometry and gel chromatography. The individual fractions were essentially monodisperse and showed molecular weights ranging from 2400 to 36000. 6. The relationship between the intrinsic viscosity and the molecular weight was [η]=5.0×10−6×M1.14, indicating that the chondroitin sulphate molecules assume a shape intermediate between that of a random coil and a stiff rod. 7. The relationship between the sedimentation constant and the molecular weight (>104) was s020,w=2.3×10−2×M0.44.

scholarly journals Isolation and some structural analyses of a proteodermatan sulphate from calf skin

1983 ◽  
Vol 213 (2) ◽  
pp. 289-296 ◽  
Author(s):  
T Nakamura ◽  
E Matsunaga ◽  
H Shinkai
Keyword(s):  
Core Protein ◽  
Gradient Centrifugation ◽  
Cetylpyridinium Chloride ◽  
Periodic Acid ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Periodic Acid Schiff ◽  
Cscl Density Gradient ◽  
Calf Skin

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).

scholarly journals Proteoglycan metabolism in the connective tissue of pregnant and non-pregnant human cervix. An in vitro study

1991 ◽  
Vol 275 (2) ◽  
pp. 515-520 ◽  
Author(s):  
M Norman ◽  
G Ekman ◽  
U Ulmsten ◽  
K Barchan ◽  
A Malmström
Keyword(s):  
Connective Tissue ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
In Vitro Study ◽  
Gel Chromatography ◽  
Cervical Tissue ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans

Profound changes occur in the cervix during pregnancy. In particular, the connective tissue is remodelled. To elucidate the mechanisms behind this process, the metabolism of cervical connective tissue was studied using tissue cultures. Cervical biopsies from non-pregnant and pregnant women were incubated with [35S]sulphate. The proteoglycans of the tissue specimens were purified by ion-exchange and gel chromatography and characterized by SDS/PAGE and by enzymic degradation. In the non-pregnant cervix, the incorporation of [35S]sulphate into the proteoglycans was linear for 48 h. During the first 6 h of incubation the accumulation of chiefly one small labelled proteoglycan (apparent Mr 110,000) substituted with dermatan sulphate was recorded. This is in accordance with the known proteoglycan composition of non-pregnant cervical tissue. In addition, small amounts of two larger radioactive dermatan/chondroitin sulphate proteoglycans (apparent Mr values 220,000 and greater than 500,000) were recorded. After longer periods of incubation the proportion of heparan sulphate proteoglycans increased considerably. The pregnant tissue showed a clearly different composition of labelled proteoglycans. An increased accumulation of the two larger dermatan/chondroitin sulphate proteoglycans was seen in addition to the dominant small dermatan sulphate proteoglycan of the non-pregnant cervix. The rate of accumulation of these two proteoglycans was about 3 times higher in the pregnant tissue, whereas that of the small dermatan sulphate proteoglycan was only increased 2-fold. The fact that the concentration of proteoglycans in the pregnant cervix is approximately one-half of that in the non-pregnant cervix indicates that the turnover of proteoglycans in pregnant cervical tissue is significantly increased. The major effect of this profound change of metabolism was a 50% decrease in proteoglycan content and a 2-fold increased proportion of a dermatan sulphate proteoglycan with an apparent Mr of 220,000.

scholarly journals Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans Isolation, fractionation and chemical characterization

1981 ◽  
Vol 197 (2) ◽  
pp. 259-268 ◽  
Author(s):  
R Kapoor ◽  
C F Phelps ◽  
L Cöster ◽  
L A Fransson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Proteinase Inhibitors ◽  
Chondroitin Sulphate ◽  
Periodate Oxidation ◽  
Chemical Characterization ◽  
Gel Chromatography ◽  
Carbohydrate Composition ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.

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.

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