X-ray diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate (Short Communication)

Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating β-d-(1→4) and α-d-(1→4).     

Sulphate release from keratan sulphate and heparan sulphate by bovine N-acetylglucosamine-6-sulphate sulphatase

The functions of sulphated monosaccharides within glycosaminoglycans(GAGs) and glycoproteins are being studied intensely, but progressis hindered by an inability to selectively desulphate glycoconjugates.We recently identified an N-acetylglucosamine-6-sulphate sulphatase(NG6SS) from bovine kidney that can remove sulphate from N-acetylglucosamine-6-sulphate(GlcNAc-6-SO₄) within oligosaccharides and glycoproteins. However,the potential ‘endosulphatase’ activity of the NG6SStoward GAGs is not known. To test for this possibility, [³H]glucosamine-,[³H]galactose- and ³⁵SO_4- labelled keratan sulphate (KS) wereseparately prepared by metabolic radiolabelling of bovine cornea.NG6SS quantitatively removed sulphate from KS without releaseof sugar fragments. The enzyme had a K_m of 4.7 mM toward freeGlcNAc-6-SO₄, but its K_m for commercially available bovine cornealKS was found to be 9.1 µM. Analyses of both KS and heparansulphate after treatment with NG6SS demonstrated significantloss of sulphate from GlcNAc-6-SO₄ in both GAGs. These findingsmay be relevant for future studies aimed at defining the function(s)of GlcNAc-6-SO₄ residues in GAGs and understanding the catabolismof GAGs, especially in regard to sulphatidoses, such as SanfilippoD syndrome in humans, which involves a deficiency of NG6SS activity catabolism endosulphatase glycosaminoglycans sulphation     

Quantitative immunohistochemical evidence of a functional gradient of chondroitin 4-sulphate/dermatan sulphate, developmentally regulated in the predentine of rat incisor

A quantitative examination was carried out on the early and mature stages of dentinogenesis in the rat incisor, using a post-embedding immunogold labelling with an anti-chondroitin 4 sulphate/dermatan sulphate antibody (2B6). At a very early stage of predentine formation, before polarizing odontoblasts have established junctional complexes, immunolabelling was weak. In contrast, when polarized odontoblasts established distal junctional complexes, immunolabelling in predentine was uniform and threefold denser than in initial predentine. The same gold particle density was found in the non-mineralized mantle dentine. During circumpulpal dentine formation, a gradient was seen in predentine, a larger number of gold particles being scored in the proximal zone compared with the distal region adjacent to the mineralization front. In circumpulpal dentine, some labelling was found within the lumen of the tubules and in the bordering dentine around the tubules. A few particles were also detected in intertubular matrix after demineralization. Together, these data provide evidence for a developmentally regulated gradient during the transition between mantle and circumpulpal dentine, and also in a more mature part of the tooth, a functional gradient that probably plays a role in the process of mineralization. © 1998 Chapman & Hall     

Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture.

35SO42(-)- and [3H]leucine-labelled proteoglycans were isolated from the medium and cell layer of human skin fibroblast cultures. Measures were taken to avoid proteolytic modifications during isolation by adding guanidinium chloride and proteolysis inhibitors immediately after harvest. The proteoglycans were purified and fractionated by density-gradient centrifugation, followed by gel and ion-exchange chromatography. Our procedure permitted the isolation of two major proteoglycan fractions from the medium, one large, containing glucuronic acid-rich dermatan sulphate chains, and one small, containing iduronic acid-rich ones. The protein core of the latter proteoglycan had an apparent molecular weight of 47000 as determined by polyacrylamide-gel electrophoresis, whereas the protein core of the former was considerably larger. The major dermatan sulphate proteoglycan of the cell layer was similar to the large proteoglycan of the medium. Only small amounts of the iduronic acid-rich dermatan sulphate proteoglycan could be isolated from the cell layer. Instead most of the iduronic acid-rich glycans appeared as free chains. The heparan sulphate proteoglycans found in the cell culture were largely confined to the cell layer. This proteoglycan was of rather low buoyant density and seemed to contain a high proportion of protein. The major part of the heparan sulphate proteoglycan from the medium had a higher buoyant density and contained a smaller amount of protein.     

Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta.

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 X 10(6) (PG-25), 0.30 X 10(6) (PG-35) and 0.88 X 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.     

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

1. gamma-Hexachlorocyclohexane, gamma-pentachlorocyclohexene and delta-pentachlorocyclohexene were converted by houseflies and grass grubs into metabolites that had chromatographic properties identical with those of S-2,4-dichlorophenylglutathione. 2. The metabolism of gamma-hexachlorocyclohexane and the pentachlorocyclohexene isomers was negligible in newly emerged blowflies, but increased over the next 10 days. 3. The metabolism of both gamma-hexachlorocyclohexane and the pentachlorocyclohexene isomers was inhibited by simultaneous dosage with tetrabromophenolphthalein ethyl ester or Bromophenol Blue in both grass grubs and flies, but only the metabolism of pentachlorocyclohexenes in blowflies was stopped by simultaneous dosage with bis-(N-dimethylaminophenyl)methane. NN-Di-n-butyl-p-chlorobenzenesulphonamide had no effect on the metabolism of pentachlorocyclohexenes by blowflies. 4. The use of these inhibitors and colorimetric assays leads to the conclusion that a pentachlorocyclohexene is not a major intermediary metabolite of gamma-hexachlorocyclohexane in these insects.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ''matrix'', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).     

Synthesis of glycosaminoglycans by human embryonic lung fibroblasts. Different distribution of heparan sulphate, chondroitin sulphate and dermatan sulphate in various fractions of the cell culture

Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate ³⁵SO₄²⁻ for various periods of time. ³⁵S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO₂ and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various ³⁵S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The ³⁵S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the ³⁵S-labelled galactosaminoglycans were extensively degraded by periodate oxidation–alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Åberg & Fransson (1975) Biochem. J. 151, 477–489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected.     

The excretion and degradation of chondroitin 4-sulphate administered to guinea pigs as free chondroitin sulphate and as proteoglycan

The excretion and degradation was studied of (35)S-labelled 4-chondroitin sulphate injected into guinea pigs in the form of proteoglycan isolated from cartilage and in the form of free chondroitin 4-sulphate prepared from the same proteoglycan by proteolysis. When the proteoglycan was injected there was a delay of about 15-20min before significant amounts or radioactivity were excreted, whereas after injection of chondroitin 4-sulphate a considerable amount of radioactivity was excreted within 10min and a much higher proportion of the radioactive dose was excreted in 1h or 24h compared with the proteoglycan. In both cases, however, a major part of the radioactivity was not excreted even in 24h. Sterile conditions were used to collect the radioactive material directly from the bladder. When chondroitin 4-sulphate was injected, the molecular sizes of injected and excreted materials were similar, as assessed by gel chromatography on Sephadex G-200, whereas when proteoglycan was injected the molecular size of the excreted labelled material was similar to that of the chondroitin 4-sulphate chains in the original proteoglycan. In neither case did the size of the excreted labelled material change with time over 1h, and low-molecular-weight labelled material was virtually absent. In contrast, when urine was collected for 24h without preservative the labelled material in it was extensively degraded after either the proteoglycan or chondroitin 4-sulphate had been given. Chondroitin 4-sulphate became similarly degraded when incubated with non-sterile urine, but not when the urine was passed through a bacterial filter, suggesting that degradation was caused by contaminating micro-organisms in the experiments in which urine was collected for 24 h. It is concluded that chondroitin 4-sulphate chains of about 18000 molecular weight can be excreted readily as such, whereas intact proteoglycans must be degraded to free glycosaminoglycans first, although both are taken up by the tissues more rapidly than they are excreted.     

Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.     

High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography

Recent literature indicates that specific glycosaminoglycanstructures are involved in various biological processes, suchas anticoagulation, growth factor activation and viral infection.The initial step in the structural analysis of glycosaminoglycansis a definitive compositional analysis of its characteristicdisaccharide repeat structures. Current chromatographic or electrophoreticprocedures may have limitations in analysing glycosaminoglycansamples that are in low abundance, contain novel structuresthat need to be further characterized, or are metabolicallylabelled from radioactive precursors as a result of biosyntheticexperiments. This study presents a new methodology for analysingdisaccharides and oligosaccharides derived from chondroitinsulphate, dermatan sulphate and hyaluronan that fulfils theabove criteria. The procedure involves the separation of reducedforms of these glycoconjugates on a CarboPac PA1 column usingalkaline eluants. This study adopted a strategy which uses specificenzymes to release these disaccharides from their glycosaminoglycanforms. A borohydride reduction reaction was modified to be compatiblewith the buffer conditions commonly used with these enzymesin order to quantitatively reduce the disaccharides to theiralditol forms (thereby stabilizing them to alkaline pH). Chromatographyconditions were established which separated all known disaccharidealditol structures from chondroitin sulphate, dermatan sulphateand hyaluronan with extremely high resolution in a single run.Integrated pulsed amperometry was compared to UV absorbancemeasurement at 232 nm as two sensitive methods for detectingthese reduced disaccharides; most of them could be routinelydetected in the range of 50–500 ng. Data are presentedapplying this method to quantify hyaluronan in a biologicalsample which contains {small tilde}5000 cells and only {smalltilde}10 ng of hyaluronan. Additional data are presented todemonstrate that this procedure will also separate oligosaccharidealditols derived from hyaluronan. borohydride reduction glycosaminoglycans integrated pulsed amperometry     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Hepatocyte growth factor/scatter factor and its interaction with heparan sulphate and dermatan sulphate

Hepatocyte growth factor (HGF)/scatter factor (SF) is a unique growth factor, in that it binds both heparan sulphate (HS) and dermatan sulphate (DS). The sequences in HS and DS that specifically interact with and modulate HGF/SF activity have not yet been fully identified. Ascidian DS, which uniquely possesses O-sulphation at C-6 (and not C-4) of its N -acetylgalactosamine unit, was analysed for HGF/SF-binding activity in the biosensor. The kinetic analysis revealed a strong, biologically relevant interaction with an equilibrium dissociation constant ( K (d)) of approx. 1 nM. An Erk activation assay also demonstrated stimulation of the MAP kinase pathway downstream of the Met receptor following addition of both HGF/SF and ascidian DS to the glycosaminoglycan-deficient CHO-745 mutant cell line. Furthermore, the activation of Met and the MAP kinase pathway by HGF/SF and ascidian DS leads to a cellular response in the form of migration.     

Isolation of the major chondroitin sulfate/dermatan sulfate and heparan sulfate proteoglycans from embryonic chicken retina

A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.     

Erythrocytes express chondroitin sulphate/dermatan sulphate, which undergoes quantitative changes during diabetes and mediate erythrocyte adhesion to extracellular matrix components

Glycosaminoglycans (GAGs) such as chondroitin sulphate/dermatan sulphate (CS/DS) are complex molecules that are widely expressed on the cell membrane and extracellular matrix (ECM). They play an important role in wide range of biological activities especially during pathological conditions. Diabetes, a metabolic disorder characterized by sustained hyperglycemia, is known to affect GAGs in different tissues and affect erythrocyte adhesion. The present investigation was aimed at exploring the nature of GAGs present in erythrocytes and its role on adhesion of erythrocytes from control and diabetic rats to major extracellular matrix components. GAGs isolated from erythrocytes were demonstrated to be CS/DS and a 2-fold increase was observed in erythrocytes from diabetic rats. Disaccharide composition analysis by HPLC after depolymerization by the enzyme, chondroitinase ABC showed the presence of 4-O sulphated disaccharide units with small amounts of non-sulphated disaccharides, in both control and diabetic erythrocytes. Erythrocytes from diabetic rats, however, showed significantly increased binding to poly-l-ornithine (P-orn), type IV collagen, laminin and fibronectin, which was abrogated on treatment with chondroitinase ABC to various degrees. This study sheds new light on CS/DS in erythrocytes and its likely biological implications in vivo.     

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.     

The degradation of intravenously injected chondroitin 4-sulphate in the rat

The degradation of chondroitin 4-[(35)S]sulphate isolated from chick-embryo cartilage was studied in the rat by experiments on free-range animals, on wholly anaesthetized animals with ureter cannulae, by perfusion of isolated liver, by whole-body radioautography and by isolation of liver lysosomes. After injection into rats 68% of the radioactivity was recovered in the urine after 24h, approximately one-half of this being in the form of low-molecular-weight material, chiefly inorganic sulphate. Cannulation experiments demonstrated that the proportion of low-molecular-weight components excreted in the urine increased with time until, after 12h, virtually all was inorganic sulphate. Whole-body radioautography identified the liver as the major site of radioisotope accumulation after injection of labelled polysaccharide. Perfusion through isolated liver indicated that this organ has the ability to metabolize the polymer with the release of low-molecular-weight products, principally inorganic sulphate. Incubation of a lysosomal fraction prepared from rat liver after injection of chondroitin 4-[(35)S]sulphate gave rise to degradation products of low molecular weight, and experiments in vitro with rat liver lysosomes confirmed that these organelles are capable of the entire degradative process from chondroitin sulphate to free inorganic sulphate.