Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate.

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.     

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.     

The sulphation of chondroitin sulphate in embryonic chicken cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [(35)S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1.5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3'-phosphate 5'-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.     

Studies on protein–polysaccharides from pig laryngeal cartilage. Extraction and purification

1. Protein-polysaccharides of chondroitin sulphate were extracted from fresh laryngeal cartilage at pH6.8 by two procedures. Procedure I consisted of brief low-speed homogenization in 0.15m (iso-osmotic) sodium acetate and procedure II consisted of longer homogenization followed by prolonged extraction in 10% calcium chloride solution. 2. The protein-polysaccharides in both extracts were isolated and purified by precipitation with 9-aminoacridine hydrochloride. They were free from serum proteins, collagen and nucleic acids and also of degradative enzymes. The absence of such enzymes was shown by viscosity measurements on solutions of protein-polysaccharides incubated for up to 24hr. at pH4 and 6.8. 3. Mannose, glucose or fucose were not detected by paper chromatography and only traces of sialic acid were present. 4. The yield with procedure II was twice that with procedure I and the products differed in their protein and glucosamine contents. 5. Hyaluronic acid was unlikely to have been precipitated at an acid pH, so the glucosamine was attributed to keratan sulphate, as serum proteins were absent. There was no free keratan sulphate in the preparation. 6. Both preparations were heterogeneous in the ultracentrifuge, showing at least three components.     

Control of chondroitin sulphate biosynthesis. beta-D-Xylopyranosides as substrates for UDP-galactose: D-xylose transferase from embryonic-chicken cartilage.

Embryonic-chicken epiphyseal cartilage was incubated in vitro with a variety of beta-xylosides and the amount of [3H]acetate incorporation into chondroitin sulphate was determined under conditions when normal protein core production was inhibited by cycloheximide. The ability of the different beta-xylosides to relieve thea cycloheximide-mediated inhibition of chondroitin sulphate synthesis was influenced by the nature of the aglycan group of te xyloside. beta-Xylosides with apolar and uncharged aglycan groups were most effective and produced a severalfold stimulation of chondroitin sulphate biosynthesis. beta-Xylosides with charged aglycan groups were less effective initiators of chondroitin sulphate synthesis. The rate of galactose transfer from UDP-galactose to each of the beta-xylosides, catalysed by a cell-free microsomal preparation from embryonic cartilage, was measured. This study showed that the nature of the aglycan group of the beta-xyloside was a factor determining the capacity of the xyloside to act as an acceptor for galactosyltransferase I, the enzyme that catalyses the first galactose transfer reaction of chondroitin sulphate synthesis. The aglycan group of the xyloside also appeared to influence other steps leading to chondroitin sulphate chain initiation in vitro.     

Incorporation of sulphate by chick-embryo corium. Nature and products of the process in vitro.

1. The incorporation of sulphate into the trichloroacetic acid-precipitable fraction of 9-day chick-embryo corium, incubated in Krebs-Ringer phosphate buffer, pH7, is dependent on the sulphate concentration of the medium. Uptake of sulphate is linear with time for 3.5-4hr. and is maximal at 37.5 degrees in the presence of air or oxygen. d-Glucose stimulates the incorporation of sulphate but l-glutamine has no effect. 2. Incorporation of sulphate by the chick corium is enzymic and apparently involves the synthesis of active sulphate (adenosine 3'-phosphate 5'-sulphatophosphate) and the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to acceptor glycosaminoglycuronoglycan. This proposal on the nature of the process is suggested by the similarity between the energy of activation calculated for sulphate-incorporation in the chick-corium preparation and the energy requirement reported for sulphate-activation with purified yeast enzymes. 3. The 9-day chick-embryo corium is composed principally of fibroblasts; there are no histologically demonstrable mast cells. The young fibroblast is apparently responsible for the incorporation of sulphate into glycosaminoglycuronoglycans tentatively identified as chondroitin sulphate(s), heparan sulphate and heparin-like material.     

Solution conformation of glycosaminoglycans: assignment of the 300-MHz 1H-magnetic resonance spectra of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate, and investigation of an alkali-induced conformation change.

Complete assignments are given for the 1H nuclear magnetic resonance (NMR) spectra at 300 MHz of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate in deuterium oxide solution, supported by spin decoupling and computer simulation. Coupling constants and chemical shifts are as expected from spectra of the model glycosides, methyl beta-D-glucopyranosiduronate, methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and methyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, when allowance is made for systematic influences on chemical shifts of interglycosidic linkages and sulphate substitution. As reported elsewhere, addition of alkali causes the hyaluronate spectrum to sharpen considerably. This is taken to indicate that segmental motion is enhanced by disruption of some system of inter-residue bonding on ionisation of hydroxy groups. Concomitant changes in chemical shifts are seen mainly for H-2 of the glucuronate residue, and the CH3 and H-2 of the acetamidodeoxyglucose residue. Similar effects are not seen for chondroitin sulphates, either in line widths or chemical shifts. Comparison of the spectra of hyaluronate, chondroitin sulphates, and the model glycosides, indicates that proton chemical shifts are sensitive to the conformation differences between the polysaccharides in alkaline solution, but do not detect the differences in neutral solution that are known from NMR relaxation to be present. The altered configuration and/or substitution pattern of the acetamidodeoxyhexose residue in hyaluronate compared with chondroitin sulphates appears to have a critical influence on overall conformation in both alkaline and neutral solution.     

The presence of a novel extracellular hyaluronidase in squid cranial cartilage

A new type of hyaluronidase was isolated from squid cranial cartilage. The enzyme seems to be localised extracellularly, since it is extracted from the tissue by 0.5 M sodium acetate, pH 7.0, in the presence of proteinase inhibitors. Degradation studies suggest that the enzyme belongs to the family of endoglycosidases generating oligosaccharides of rather large size. The best activity of the enzyme was observed at pH 7.0 and 37 degrees C and the optimum buffer for digestion was 0.15 M Tris acetate. It is inactive in sodium phosphate, morpholine acetate and HEPES buffers. The enzyme degrades aggrecan, hyaluronan, chondroitin sulphate and oversulphated chondroitin sulphate.     

Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S.

A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.     

Effects of sulphate and pH on the plant-availability of phosphate adsorbed on goethite

The adsorption of phosphate on metal (hydr)oxides may be influenced by the pH and by the adsorption of other ions. In this study, the influence of sulphate and pH on phosphate adsorption on goethite and the availability to plants of adsorbed phosphate was examined. Maize plants were grown on suspensions of goethite with adsorbed phosphate, containing the same total amount of phosphate and either 0.11 mM or 2.01 mM sulphate at pH 3.7, 4.6 or 5.5. The uptake of phosphorus by the plants increased with the larger sulphate concentration and decreasing pH. Mean P uptake in the treatment with 2.01 mM sulphate and pH 3.7 was 55 µmol plant^-1, whereas in the treatment with 0.11 mM sulphate and pH 5.5 it was 2 µmol plant^-1. Batch adsorption experiments using³² P and speciation modelling of ion adsorption showed that in the presence of sulphate, the phosphate concentration in solution strongly increased with decreasing pH, due to competitive adsorption between sulphate and phosphate on goethite. Modelled phosphate concentrations in solution in the uptake experiment were all below 0.6 µM and correlated well with the observed P uptake. This correlation indicates that the strong influence of the sulphate concentration and pH on the plant-availability of adsorbed phosphate results from the competition between sulphate and phosphate for adsorption on goethite.     

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties.

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.     

Sensory Nerve Endings Shown by Nitro Blue Tetrazolium in Isolated Muscle Spindles of the Cat

Muscle spindles were isolated from freshly removed cat lumbrical muscles in oxygenated Ringer's solution and placed in a solution containing 10 ml of 0.1% nitro blue tetrazolium and 10 ml of 0.2 M phosphate buffered sodium succinate, pH 7.6. Spindles were incubated in this solution at 37 C for 4-12 hr, returned to Ringer's for 30 min at room temperature, fixed in 10% formal-Ringer's for 30 min, and stored indefinitely in distilled water. With this technique the patterns of sensory innervation can be clearly visualized by the deposition of diformazan. The stained preparations may be mounted in glycerol and teased further for whole mount inspection or they may be embedded in Epon and serially sectioned for more detailed study.     

Effect of neonatal head X-irradiation on growth of costal and tibial cartilage in rats: a histochemical and electron microscopic study

Long-Evans rats were exposed to a single dose of head X-irradiation (600 rads) at 2 days of age. Experimental and sham irradiated rats were sacrificed at 14, 20-21, 23, 41-45, and 70-71 days. Tibial epiphyseal width and the number of cells in the epiphyseal plate were determined. Histochemical and electron microscopic studies were carried out on both costal and epiphyseal cartilage. Histochemical techniques revealed a reduction in chondroitin sulfate at 14 days in both costal and epiphyseal cartilage of X-irradiated rats. Epiphyseal cartilage demonstrated recovery subsequently, and this was followed by a normal decrease of chondroitin sulfate with increasing age, but costal cartilage did not recover. Collagen synthesis was also reduced in both costal and epiphyseal cartilage, but not as dramatically as chondroitin sulfate. Except for some electron dense cells and reduced scalloping of the cell membrane, costal chondrocytes from irradiated rats did not show major ultrastructural alterations. In contrast, epiphyseal chondrocytes demonstrated radiation induced alterations in organelles, in enhanced glycogen deposition, and in retardation of chondrocyte maturation. Extracellularly in both costal and epiphyseal cartilage of irradiated rats, collagen density and matrix granules were reduced, while calcification of the matrix was enhanced. Beyond 45 days, the effects of irradiation were markedly reduced. Comparisons of the histochemical results with metabolic studies carried out previously in cartilage from the same animals indicated a more direct concordance of the histochemical results with the pattern of physical growth and supported the usefulness of morphologic and histochemical techniques in the analysis of the growth disorder in the head-irradiated rat.     

The interaction of collagen and acid mucopolysaccarides. A model for connective tissue

1. The interaction of acid mucopolysaccharides of connective tissue with solubilized collagen of native, or near-native, structure was investigated by free solution electrophoresis at pH7.0. 2. Complex-formation was detected by the appearance of a third peak in the ascending limb only, indicating reversible association. 3. Complex-formation was destroyed by prior heating of solubilized collagen, indicating a probable requirement for high molecular weight or internal structure of the protein. 4. Hyaluronate and chondroitin sulphate of mol.wt. 50000 gave complexes with soluble collagen at I 0.4, whereas heparin and chondroitin sulphate of mol.wt. 15000-18000 did not. All mucopolysaccharides yielded complexes at I 0.1. The stability of the complex appears mainly dependent on electrostatic forces and is increased with increase in chain length of the polysaccharide. 5. Solubilized collagen interacted to yield gels with the ;native' chondroitin sulphate-protein macro-molecule from cartilage. 6. A schematic model for the interaction of collagen and chondroitin sulphate-protein macromolecules shows parallel-ordered interaction of collagen fibrils with chondroitin sulphate side chains of the chondroitin sulphate-protein macromolecule. The biological implications of this model are discussed, particularly in relation to the ordered structures and the ionic-network properties of the intercellular components of connective tissue.     

Lack of chondroitin sulphate epitope in the proliferating zone of the growth plate of chicken tibia

Summary Monoclonal antibodies specific to chondroitin sulphate (CS-56) and keratan sulphate (AH12) were used to localize proteoglycans in the proximal tibial articular cartilage and growth plate of broiler chickens. There was no CS-56 labelling in the proliferative zone of the growth plate. In contrast, intense labelling with this antibody was observed in the transitional and hypertrophic zones of the growth plate and the articular cartilage. This was confirmed by extracting chondroitin sulphate fractions from different zones of the growth plate and articular cartilage, and examining their antigenicities to CS-56 by ELISA inhibition assay. It was suggested that the maturation of chondrocytes in the growth plate is related to the production of chondroitin sulphate with CS-56 epitope, which may be a prerequisite for normal endochondral bone formation in the chicken tibia. The role of chondroitin sulphate recognized by CS-56 in the articular cartilage is unknown.     

Role of mitochondria in calcification, Mitochondrial activity distribution in the epiphyseal plate and accumulation of calcium and phosphate ions by chondrocyte mitochondria

Histologically homogeneous sections corresponding to resting, columnar and hypertrophic zones of the epiphyseal plate of calf scapula were homogenized and assayed for mitochondrial, lysosomal and others important to calcification activities. These activities were found to be significantly higher in the columnar and hypertrophic zones as compared with those in the resting zone. Mitochondria obtained from the hypertrophic zone of the epiphyseal plate of calf scapula or calf costal chondral junction were resolved into a “light” and a “heavy” population by isopycnic centrifugation presumably due to difference in the content of granule forming calcium phosphate. Finally mitochondria from resting cartilage can give rise only to a “light” mitochondrial population, unless they are allowed to accumulate calcium and phosphate ions from the medium during respiration where a “light” and a “heavy” population results upon centrifugation.     

Anion binding to liver alcohol dehydrogenase

1. Complex formation at the general anion-binding site of the liver alcohol dehydrogenase subunit has been characterized by transient-state kinetic methods, using NADH as a reporter ligand. Equilibrium dissociation constants for anion binding at the site are reported. They conform basically to the lyotropic series of affinity order, with exceptionally tight binding of sulphate. The particular specificity for sulphate might be a general characteristic of anion-binding enzymic arginyl sites. 2. Anionic species of phosphate and pyrophosphate buffer solutions do not interact significantly with the general anion-binding site over the pH range 8-10. At lower pH, phosphate binding becomes significant due to complex formation with the monovalent H2PO4 species. The latter interaction corresponds to a dissociation constant of about 60 mM, indicating that phosphate binding is comparatively weak also at low pH. 3. It is concluded that previously reported pH dependence data for coenzyme binding to liver alcohol dehydrogenase cannot be much affected by coenzyme-competitive effects of buffer anion binding. Kinetic parameter estimates now determined for NADH binding in weakly buffered solutions agree within experimental precision with those obtained previously from measurements made in buffer solutions of 0.1 M ionic strength.     

Glycosaminoglycans in early chick embryo

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.     

Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion,precolumn derivatization,and fluorescence detection

A sensitive and selective HPLC method for the determination of the disaccharides of chondroitin sulfate in horse and dog plasma was validated. Chondroitin sulfate is degraded by chondroitinase ABC to three primary unsaturated disaccharides, (1) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose, (2) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and (3) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose, when treated with chondroitinase. Plasma samples (0.5 ml) were treated with 50 mU of chondroitinase ABC in 50 microl of 1 mM sodium phosphate buffer (pH 7.0) at 37 degrees C for 6 h. The samples were extracted with 25% trifluoroacetic acid in ethanol. The resultant samples were derivatized with 1% dansylhydrazine in ethanol at 40 degrees C for 3 h. The chromatographic conditions consisted of fluorescence detection (excitation at 350 nm and emission at 530 nm), mu-Bondapack NH(2) (300 x 3.9 mm), and mobile phase of acetonitrile:100 mM acetate buffer, pH 5.6 (76:24), pumped at 1.0 ml/min. The standard curves for each chondroitin disaccharide showed linearity over the selected concentration range (r > or = 0.99). The intraday percentage relative standard deviation was < or =9.5% and the interday precision was < or =6.9% or less. The relative intraday and interday error ranged from -7.3 to 6.6% for each chondroitin disaccharide in the plasma. The extraction recovery was found to be in the range of 90-96%. The validated method accurately quantitated the disaccharides of chondroitin sulfate after administration to dogs and horses.     

Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity.

The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo.