The lack of effect of glucosamine sulphate on aggrecan mRNA expression and 35S-sulphate incorporation in bovine primary chondrocytes

Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and ³⁵S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 μM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited ³⁵S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or ³⁵S-sulphate incorporation.     

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.     

Gas chromatographic analysis of hexosamines in glycoproteins

A gas chromatographic method for the analysis of hexosamines in glycoproteins was described which uses the alditol acetate derivatives of the sugars. A polyamide (Poly A 103) liquid phase was used which effectively separates glucosamine, galactosamine, and mannosamine from each other. Mannosamine served as internal standard to facilitate accurate quantitation of glucosamine and galactosamine.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Mitogenicity and binding properties of beta-galactoside-binding lectin from chick-embryo kidney.

THe beta-galactoside-binding lectin binds to glucosamine, mannosamine and galactosamine in addition to beta-galactoside, as determined by the inhibition of haemagglutination. Haemagglutination is further extended to examine the interaction of the binding sites for hexosamines and beta-galactosides, indicating that the binding of hexosamine and beta-galactoside is competitive. The lectin also shows strong mitogenic activity toward lymphocytes from mouse lymph node, as determined by the stimulation of thymidine incorporation.     

High-throughput analysis of hexosamine using a colorimetric method

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives—glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride—were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.     

Chondrocyte-mediated catabolism of aggrecan: evidence for a glycosylphosphatidylinositol-linked protein in the aggrecanase response to interleukin-1 or retinoic acid.

The control of chondrocyte-mediated degradation of aggrecan has been studied in rat chondrosarcoma cells and bovine cartilage explants treated with either IL-1 or retinoic acid. The capacity of glucosamine to inhibit the aggrecanase-mediated response (J. D. Sandy, D. Gamett, V. Thompson, and C. Verscharen (1998) Biochem. J. 335, 59-66) has been extended to an investigation of the effect of other hexosamines. Mannosamine inhibits the aggrecanase response to both IL-1 and RA at about one-tenth the concentration of glucosamine in both rat cell and bovine explant systems. This effect of mannosamine appears to be due to its capacity to inhibit the synthesis of glycosylphosphatidylinositol (GPI)-linked proteins by chondrocytes since the GPI synthesis inhibitor 2-deoxyfluoroglucose (2-DFG) also inhibited the aggrecanase response to IL-1b and RA in rat cells. Moreover, phosphatidylinositol-specific phospholipase C (PIPLC) treatment of rat cells markedly inhibited the aggrecanase response to IL-1b and RA. These inhibitory effects of mannosamine, 2-DFG, and PIPLC in rat cells did not appear to be due to an interference with general biosynthetic activity of the cells as measured by [3H]proline incorporation into secreted proteins. We suggest that the aggrecanase response by chondrocytes to IL-1 and RA is dependent on the activity of a GPI-anchored protein on the chondrocyte cell surface.     

Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils.

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48--61% chondroitin 4-sulphate, 20--30% dermatan sulphate, 10--15% heparin and 7--9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells.

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.     

Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage

The enzymatic processes underlying the degradation of aggrecan in cartilage and the corresponding changes in the biomechanical properties of the tissue are an important part of the pathophysiology of osteoarthritis. Recent studies have demonstrated that the hexosamines glucosamine (GlcN) and mannosamine (ManN) can inhibit aggrecanase-mediated cleavage of aggrecan in IL-1-treated cartilage cultures. The term aggrecanase describes two or more members of the ADAMTS family of metalloproteinases whose glutamyl endopeptidase activity is known to be responsible for much of the aggrecan degradation seen in human arthritides. In this study we examined the effect of ManN and GlcN on aggrecanase-mediated degradation of aggrecan induced by IL-1alpha and the corresponding tissue mechanical properties in newborn bovine articular cartilage. After 6 days of culture in 10 ng/ml IL-1 plus ManN, mechanical testing of explants in confined compression demonstrated that ManN inhibited the IL-1alpha-induced degradation in tissue equilibrium modulus, dynamic stiffness, streaming potential, and hydraulic permeability, in a dose-dependent fashion, with peak inhibition ( approximately 75-100% inhibition) reached by a concentration of 1.35 mM. Aggrecan from explants cultured in IL-1 was found by Western analysis to be almost entirely processed down to the G1-NITEGE(373) end product. Addition of ManN or GlcN was found to produce 75-90% inhibition of this cleavage, but the proportion of aggrecan remaining in the tissue which was cleaved at aggrecanase sites in the chondroitin sulfate (CS)-rich region (Glu(1501) and Glu(1687)) was higher than with IL-1 alone. This result suggests that the preservation of mechanical properties by hexosamines in explants is primarily due to inhibition of cleavage at the Glu(373) site in the interglobular domain. While the precise mechanism by which hexosamines function in this system is unclear, the present analysis suggests that the mechanical properties examined may be predominantly a function of electrostatic repulsion due to the charged CS chains in the tightly packed repetitive sequences of the CS-1 region.     

Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [³⁵S]sulphate (and in some cases [6-³H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [³H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.     

The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-(14)C]glucose and [(35)S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride-cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-(14)C]glucose were found to be 0.79+/-0.16 and 3.2+/-0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-(14)C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [(35)S]sulphate incorporation and were found to be in good agreement with those obtained from [U-(14)C]glucose labelling.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

The effect of scurvy on hexosamine-containing substances in healing wounds in guinea pigs

1. Granulation tissue from healing tendonectomy wounds in guinea pigs was analysed and the effects of inanition and ascorbic acid deficiency on this tissue were investigated. 2. Inanition produced no significant effect on either the glucosamine or the galactosamine content of the tissue. Ascorbic acid deficiency decreased the galactosamine content without affecting the glucosamine content. 3. Fractionation of papain-digested granulation tissue gave three major fractions, which behaved respectively as glycopeptide, hyaluronic acid and a sulphated glycosaminoglycan mixture. At least half of the sulphated glycosaminoglycan mixture behaved as dermatan sulphate. 4. Inanition produced no consistent effect on the fractions examined. In ascorbic acid deficiency, a decrease in the sulphated glycosaminoglycan fraction was observed, which accounted for the decreased galactosamine content of the tissue. This was accompanied by a decrease in hyaluronic acid and a slight increase in the glycopeptide fraction.     

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.     

Purification and some properties of arylsulphatases A and B from rabbit kidney cortex.

Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N--acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.     

Types of oligosaccharide sulphation, depending on mucus glycoprotein source, corpus or antral, in rat stomach.

Radiolabelled mucus glycoprotein was obtained from tissue and a culture medium each of the corpus and antrum of rat stomach incubated with [35S]sulphate in vitro. Gel-filtration analysis of oligosaccharides liberated by alkaline-borohydride treatment from glycoproteins indicated that 35S-labelled oligosaccharides from the corpus vary considerably with respect to chain length whereas those from antral mucus glycoprotein are composed of small oligosaccharides. Examination of the reduced radiolabelled products obtained by HNO2 cleavage of the hydrazine-treated oligosaccharides indicated sulphate esters of N-acetylglucosamine to be present at three locations on a carbohydrate unit: [35S]sulphated monosaccharide (2,5-anhydromannitol 6-sulphate), [35S]sulphated disaccharide [galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate] and [35S]sulphated trisaccharide [fucosyl(alpha 1-2)-galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate]. Sulphated disaccharide and trisaccharide, possibly originating from the N-acetyl-lactosamine and fucosyl-N-acetyl-lactosamine sequences respectively, were detected in the corpus, especially as large oligosaccharides, but were present in the antrum in only very small amounts. The sulphated monosaccharide, however, most probably originating from 6-sulphated N-acetylglucosamine residues at non-reducing termini, was present in all oligosaccharide fractions in both the corpus and antrum.     

Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [(35)S]sulphate or [(14)C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [(35)S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [(35)S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60-80% of the [(35)S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (K(av.)) of the proteoglycan monomer on Sepharose CL-2B was 0.28-0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2-D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [(35)S]sulphate and [(14)C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60-70%), with 9-11% keratan sulphate in the monomer proteoglycan; (3) K(av.) values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH(4)-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.