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.

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 Characterization of protein–polysaccharides of articular cartilage from mature and immature pigs

1969 ◽  
Vol 114 (4) ◽  
pp. 871-876 ◽  
Author(s):  
Kenneth D. Brandt ◽  
Helen Muir
Keyword(s):  
Articular Cartilage ◽  
Sodium Acetate ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Analytical Data ◽  
Age Groups ◽  
Extraction Procedure ◽  
Gel Filtration ◽  
Calcium Acetate ◽  
Keratan Sulphate

Protein–polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6·8 with iso-osmotic sodium acetate followed by 0·63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein–polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein–polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein–polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein–polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein–polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein–polysaccharides as in the larger.

scholarly journals Evidence for interactions between the 30 kDa N- and 50 kDa C-terminal tryptic fragments of human lactotransferrin

1986 ◽  
Vol 236 (3) ◽  
pp. 839-844 ◽  
Author(s):  
D Legrand ◽  
J Mazurier ◽  
J P Aubert ◽  
M H Loucheux-Lefebvre ◽  
J Montreuil ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Alpha Helix ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Short Peptide ◽  
Tryptic Fragment ◽  
Tryptic Digest ◽  
Helical Content ◽  
Computer Based ◽  
Peptide Segments

Gel filtration of a mild tryptic digest of diferric human lactotransferrin carried out in presence of 10% (v/v) acetic acid led to the isolation of two fragments, an N-terminal tryptic fragment having an Mr of 30,000 and a C-terminal tryptic fragment having an Mr of 50,000 [Legrand, Mazurier, Montreuil & Spik (1984) Biochim. Biophys. Acta 787, 90-96]. Both fragments possess a degree of organization lower than that of the native protein, as shown by the decrease of about 30% of the alpha-helical content observed by c.d. The two fragments are able to re-associate in neutral solutions, as shown by the isolation, by gel chromatography, of a re-associated 80 kDa N,C-tryptic complex having the chromatographic behaviour of the native lactotransferrin. Computer-based comparison of the measured c.d. spectrum of the mixture of N-tryptic and C-tryptic fragments (molar ratio 1:1) with the spectrum calculated by assuming one molecule of each fragment, shows that the alpha-helix content of lactotransferrin is restored. These results strongly suggest the existence of non-covalent and reversible interactions between the two lobes of lactotransferrin. In addition it was demonstrated that short peptide segments (residues 19-24, 45-58 and 264-276) are involved in the secondary-structure modifications referred to above.

scholarly journals Heterogeneity of protein–polysaccharides of porcine articular cartilage. The chondroitin–sulphate proteins associated with collagen

1971 ◽  
Vol 123 (5) ◽  
pp. 747-755 ◽  
Author(s):  
K. D. Brandt ◽  
Helen Muir
Keyword(s):  
Articular Cartilage ◽  
Sodium Acetate ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Alkali Treatment ◽  
Molar Ratio ◽  
Sodium Acetate Solution ◽  
Acetate Solution ◽  
Keratan Sulphate ◽  
Collagenase Digestion

Pig articular cartilage, from which protein–polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris–HCl buffer after bacterial collagenase digestion, followed by the same urea–sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein–polysaccharide preparation obtained previously. The protein–polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein–polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea–sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein–polysaccharides in all three extracts although somewhat shorter than in protein–polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein–polysaccharides in cartilage.

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.

Quantitation of uncombined gonadotropin subunits within and released by the rat anterior pituitary

1988 ◽  
Vol 119 (2) ◽  
pp. 203-212
Author(s):  
H. Edward Grotjan
Keyword(s):  
Anterior Pituitary ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Absolute Amount ◽  
Β Subunit ◽  
Α Subunit ◽  
Molar Ratios ◽  
Tissue Extracts ◽  
Triton X

Abstract. The release of uncombined gonadotropin subunits by rat anterior pituitaries during an in vitro incubation as well as intracellular concentrations were assessed. Uncombined subunits and native gonadotropins were quantitated by radioimmunoassays after samples were subjected to gel filtration on Sephadex G-100 Superfine. Small, but detectable, amounts of uncombined rat LH β subunit were released under basal conditions. GnRH increased the absolute amount of rLHβ released but did not alter the rLHβ/rLH molar ratio (≈ 0.02). Tissue extracts prepared in aqueous buffer (100 000 × g supernatants) and 0.5% Triton X-100 extracts of the 100 000 × g pellets from the initial homogenization ('pellet extracts') contained larger quantities of uncombined rLHβ as well as elevated rLHβ/rLH molar ratios (≈ 0.10 and ≈ 0.20, respectively). Significant amounts of uncombined rLHα were released and were present in both tissue and pellet extracts. However, when FSH β subunit was quantitated in tissue extracts after gel filtration, all of the immunoreactive materials eluted in the position of native rFSH (FSHβ/rFSH molar ratio < 0.0025). Pellet extracts contained significant amounts of rLHβ, rLH and rLHα but lesser amounts of rFSH suggesting that intracellular gonadotropins are not completely extracted when homogenization is performed in aqueous buffers. Thus, rat anterior pituitaries contain and release significant amounts of the uncombined α subunit, relatively small amounts of uncombined rLHβ and extremely small amounts of uncombined FSHβ, if any.

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.

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