Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

The proteoglycans of the canine intervertebral disc

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.     

Degradation of heparin in mouse mastocytoma tissue

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 GE(H)) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GE(H) was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights ( M(w)) 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 [(35)S]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 (35)S-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 (14)C-labelled chondroitin sulphate to the same procedure.     

Isolation and chemical study of the glycosaminoglycans from squid cornea

1. Oversulphated chondroitin sulphate (ca 93% of tissue glycosaminoglycans) with average molecular weight 72,500, chondroitin sulphate (5%) and small amounts of lowsulphated chondroitin sulphate were isolated from squid cornea. 2. The sulphation pattern of oversulphated chondroitin sulphate was delta di-4S (52%), delta di-diSD (28%), delta di-6S (9%) and delta di-OSCS (11%) and that of chondroitin sulphate 49, 1, 20 and 30% respectively. 3. All glycosaminoglycans contained neutral monosaccharides, glucose being the predominant neutral monosaccharide in oversulphated chondroitin sulphate and chondroitin sulphate and fucose in low-sulphated chondroitin sulphate. 4. Although L-iduronic acid was not detected, the digestion of oversulphated chondroitin sulphate with chondroitinases ABC and AC gave unexpected results.     

Identification of oversulphated galactosaminoglycans in intestinal-mucosal mast cells of rats infected with the nematode worm Nippostrongylus brasiliensis.

The oversulphated galactosaminoglycans synthesized by rat mucosal mast cells were isolated from the small intestine of animals infected with the nematode Nippostrongylus brasiliensis, which causes proliferation of these cells. The 35S-labelled polysaccharides were degraded by digestion with chondroitinase ABC, and the structures of the disaccharide products were determined by cleavage with mercuric acetate followed by electrophoretic characterization of the resultant sulphated monosaccharides. It was concluded that about half of the disulphated disaccharide units in the polysaccharide consisted of chondroitin sulphate E-type structures [GlcA-GalNAc(4,6-di-OSO3)], in which both sulphate groups were located on the N-acetylgalactosamine unit. The remainder consisted of isomeric structures with one sulphate group on the N-acetylgalactosamine residue and one on the hexuronic acid unit and presumably represented the dermatan sulphate-type sequence [IdoA(2-OSO3)-GalNAc(4-OSO3)].     

Proteins and glycosaminoglycans in the intercellular matrix of the human cumulus-oophorus and their effect on conversion of proacrosin to acrosin.

Human cumuli-oophori were cultured in vitro in the presence of radioactive protein and polysaccharide precursors. The time course of the cumulus cell secretion was traced by histoautoradiography. Matrix solubilization, and sodium dodecyl sulphate polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that proteoglycan (Mr greater than 1,700,000) was the main cumulus cell product that was prevailingly deposited in the cumulus intercellular matrix and partly released into the culture medium. It was capable of accelerating the conversion of proacrosin to acrosin and this activity was abolished by enzymatic removal of chondroitin sulphate, the predominant glycosaminoglycan of this proteoglycan fraction. None of the other fractions, including a proteoglycan of Mr 80,000-90,000, containing heparan sulphate, accelerated the conversion of proacrosin to acrosin under the conditions used. The results suggest that chondroitin sulphate is the active component of the high-Mr proacrosin activator of the human cumulus-oophorus.     

Studies on the protein-bound chondroitin sulphate of bovine cortical bone

A fraction containing chondroitin sulphate, isolated from bovine cortical bone under mild conditions, was separated by ion-exchange chromatography into three fractions with apparent homogeneity on electrophoresis and ultracentrifugation. Two of these appeared to consist of chondroitin sulphate bound to a glycoprotein ;core' that had similarities to the bone sialoprotein described previously. The differences in composition of the two fractions were considered to be due to variation in the number or lengths of the polysaccharide chains. The presence of xylose and the alkali-lability of the bond between protein and polysaccharide suggested the presence of a xylosylserine linkage. The third fraction had the properties of a relatively pure chondroitin sulphate which contained a small amount of peptide. These fractions differed considerably from the protein-polysaccharide complexes of epiphysial and other cartilages, and their relevance to the possible role of glycosaminoglycans is discussed.     

Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycansubstrates was examined using viscosimetric measurements andgradient polyacrylamide gel electrophoresis (PAGE). Heparinlyase I (heparinase, EC 4.2.2.7 [EC] ) and heparin lyase II (no ECnumber) both acted on heparin in a random endolytic fashion.Heparin lyase II showed an ideal endolytic action pattern onheparan sulphate, while heparin lyase I decreased the molecularweight of heparan sulphate more slowly. Heparin lyase III (heparitinase,EC 4.2.2.8 [EC] ) acted endolytically only on heparan sulphate anddid not cleave heparin. Chondroitin ABC lyase (chondroitinaseABC, EC 4.2.2.4 [EC] ) from Proteus vulgaris acted endolytically onchondroitin-6-sulphate (chondroitin sulphate C) and dermatansulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate(chondroitin sulphate A) at a reduced rate, decreasing its molecularweight much more slowly. Two chondroitin AC lyases (chondroitinaseAC, both EC 4.2.2.5 [EC] ) were examined towards chondroitin-4- and-6-sulphates. The exolytic action of chondroitin AC lyase Afrom Arthrobacter aurescens on both chondroitin-4- and -6-sulphateswas demonstrated viscosimetrically and confirmed using bothgradient PAGE and gel permeation chromatography. ChondroitinAC lyase F from Flavobacterium heparinum (Cytophagia heparinia)acted endolytically on the same substrates. Chondroitin B lyase(chondroitinase B, no EC number) from F.heparinum acted endolyticallyon dermatan sulphate giving a nearly identical action patternas observed for chondroitin ABC lyase acting on dermatan sulphate. action pattern chondroitin lyase glycosaminoglycan heparin lyase.     

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core.

Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues.     

Alterations in human gingival glycosaminoglycan pattern in inflammation and in phenytoin induced overgrowth

Glycosaminoglycans were extracted from normal, inflamed and phenytoin induced overgrowth of human gingival tissue by proteolysis and alcohol precipitation. Extracts were run in a Dowex-1 column and the fractions were treated with mucopolysaccharidases. Cellulose acetate electrophoresis was carried out with or without enzyme digestion for identification of individual glycosaminoglycans. Glycosaminoglycans were found to be decreased in inflammation but were observed to increase in the overgrowth. Hyaluronic acid was found to be increased in both the pathological conditions. Dermatan sulphate, chondroitin sulphate and heparan sulphate were observed to be decreased in inflammation. In overgrowth, dermatan sulphate and chondroitin sulphate were found to increase while the presence of heparan sulphate was not significant. The changes in the pattern of individual glycosaminoglycan in the two varied conditions are discussed.Abbreviations GAG glycosaminoglycan - MPS mucopolysaccharide - DS dermatan sulphate - HS heparan sulphate - CS chondroitin sulphate - HA hyaluronic acid - KS keratan sulphate     

Properties of fractionated chondroitin sulphate from ox nasal septa

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 [eta]=5.0x10(-6)xM(1.14), 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 (>10(4)) was s(0) (20,w)=2.3x10(-2)xM(0.44).     

Altered proteoglycan synthesis by micromelial limbs induced by 6-aminonicotinamide. Appearance of abnormal forms of cartilage-characteristic proteoglycan (PG-H).

Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.     

Evidence for the existence of a multichain proteoglycan of heparan sulphate

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.     

Fractionation of proteoglycans from bovine corneal stroma.

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.     

Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II.

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.     

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.     

Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].     

The nature of the protein moieties of cartilage proteoglycans of pig and ox.

Proteoglycans extracted with 4M-guanidinium chloride from pig laryngeal cartilage and bovine nasal septum were purified by density-gradient centrifugation in CsCl under 'associative' followed by 'dissociative' conditions [Hascall & Sajdera (1969) J. Biol. Chem. 244, 2384-2396]. Proteoglycans were then digested exhaustively with testicular hyaluronidase, which removed about 80% of the chondroitin sulphate. The hyaluronidase was purified until no proteolytic activity was detectable under the conditions used for digestion. The resulting 'core' proteins of both species were fractionated by a sequence of gel-chromatographic procedures which gave four major fractions of decreasing hydrodynamic size. Those that on electrophoresis penetrated 5.6% (w/v) polyacrylamide gels migrated as discrete bands whose mobility increased with decreasing hydrodynamic size. The unfractionated 'core' proteins had the same N-terminal amino acids as the intact proteoglycan, suggesting that no peptide bonds had been cleaved during hyaluronidase digestion. Alanine predominated as the N-terminal residue in all the fractions of both species. Fractions were analysed for amino acid, amino sugar, uronic acid and neutral sugar compositions. In pig 'core' proteins, the glutamic acid content increased significantly with hydrodynamic size, but in bovine 'core' proteins this trend was less marked. Significant differences in amino acid composition between fractions suggested that in each species there was more than one variety of proteoglycan. The molar proportions of xylose to serine destroyed on alkaline beta-elimination were equivalent in most fractions, indicating that the serine residues destroyed were attached to the terminal xylose of chondroitin sulphate chains. The ratio of serine residues to threonine residues destroyed on beta-elimination, was similar in all fractions of both species. Since the fractions of smallest hydrodynamic size contained less keratan sulphate than those of larger size, it implies that in the former the keratan sulphate chains were shorter than in the latter.     

D(+)catechin enhances heparan sulphate content in rat liver

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.