Presence Of Chondroitin Sulfate Research Articles

Evaluating specific adhesion of Plasmodiumfalciparum-infected erythrocytes to immobilised hyaluronic acid with comparison to binding of mammalian cells

A feature of infection with Plasmodium falciparum is the ability of parasite-infected erythrocytes to adhere to vascular endothelial cells and accumulate in vital organs, associated with severe clinical disease. Hyaluronic acid was recently identified as a receptor for adhesion and has been implicated in mediating the accumulation of parasites in the placenta. Here, we report in vitro assays to measure specific adhesion of infected erythrocytes to hyaluronic acid that is distinct from binding to chondroitin sulphate A, another glycosaminoglycan implicated as a receptor in placental malaria. In this study, specific adhesion of mature stage infected erythrocytes to hyaluronic acid of high purity immobilised on plastic surfaces was abolished by pre-treating hyaluronic acid with a specific hyaluronate lyase from Streptomyces, whereas the same treatment of chondroitin sulphate A had little effect. Adhesion to hyaluronic acid could not be explained by the presence of chondroitin sulphate A or other glycosaminoglycans in the hyaluronic acid preparations. Chinese hamster ovary cells bound in a similar manner in the assays and confirmed that hyaluronic acid was appropriately immobilised for cell adhesion. In contrast to parasites, these cells did not adhere to chondroitin sulphate A. The adsorption of hyaluronic acid onto plastic surfaces was also confirmed by the use of a specific hyaluronic acid-binding protein. Fixing cells with glutaraldehyde at the completion of adhesion assays reduced the number of parasites remaining adherent to hyaluronic acid, but not to chondroitin sulphate A or CD36. These findings have important implications for understanding and evaluating interactions between P. falciparum and hyaluronic acid that may be involved in disease pathogenesis.

1H and 13C HR-MAS NMR investigations on native and enzymatically digested bovine nasal cartilage.

Rheumatic diseases are accompanied by a progressive destruction of the cartilage layer of the joints. Despite the frequency of the disease, degradation mechanisms are not yet understood and methods for early diagnosis are not available. Although some information on pathogenesis could be obtained from the analysis of degradation products of cartilage supernatants, the most direct information on degradation processes would come from the native cartilage as such. We have used 1H as well as 13C HR-MAS (high resolution magic angle spinning) NMR spectroscopy to obtain suitable line-widths of NMR resonances of native cartilage. 1D and 2D NMR spectra of native cartilage were compared with those of enzymatically-treated (collagenase and papain) samples. In the 1H NMR spectra of native cartilage, resonances of polysaccharides, lipids and a few amino acids of collagen were detectable, whereas the 13C NMR spectra primarily indicated the presence of chondroitin sulfate. Treatment with papain resulted only in small changes in the 1H NMR spectrum, whereas a clear diminution of all resonances was detectable in the 13C NMR spectra. On the other hand, treatment with collagenase caused the formation of peptides with an amino acid composition typical for collagen (glycine, proline, hydroxyproline and lysine). It is concluded that the HR-MAS NMR spectra of cartilage may be of significance for the investigation of cartilage degradation since they allow the fast evaluation of cartilage composition and only very small amounts of sample are required.

Regulation of epiblast cell movements by chondroitin sulfate during gastrulation in the chick

Avian gastrulation is dependent on the ingression of outer layer cells into the interior of the embryo by means of a transient structure referred to as the primitive streak. As the growing streak progresses through the central area pellucida of the blastoderm, selective de-epithelialization of epiblast cells results in the initial migratory cells of the primitive mesoderm and endoderm. Here, we have examined the possibility that extracellular matrix molecules of the epiblast basal lamina influence the selection of streak-specific epiblast cells. By using whole embryo culture, we have found that removal of chondroitin sulfate glycosaminoglycans at gastrulation stages leads to defective streak formation. In situ hybridization with streak-specific markers in these embryos reveals ectopic patterns of gene expression, suggesting that differentiation of primitive streak precursors in the pregastrula epiblast is independent of normal streak morphogenesis. In addition, in vitro assays with chondroitin sulfate containing matrices suggest that specific cells of the epiblast are inhibited from joining the streak during gastrulation. Taken together, these results indicate that the presence of chondroitin sulfate in the epiblast basal lamina facilitates the allocation of cells to the primary germ layers by preventing ectopic axis formation. © 2000 Wiley-Liss, Inc.

Spontaneous precipitation of calcium carbonate in the presence of chondroitin sulfate

The kinetics of spontaneous precipitation of vaterite (CaCO 3) from an aqueous solution in the presence of chondroitin sulfates (CSA, CSB, CSC) was investigated by the constant composition method. The presence of chondroitin sulfate in the supersaturated solution resulted in a reduction of the crystal growth rate by 23–65%. Induction times preceding vaterite precipitation were inversely proportional to the solution's supersaturation and a surface energy of 52 mJ m −2 was calculated according to the classical nucleation theory. Chondroitin sulfate influences the particle size distribution of the vaterite crystals formed and stabilizing this mineral phase, preventing the transformation to calcite. The apparent order found from kinetics data was n>2, thus suggesting a surface nucleation mechanism.

Regulation of epiblast cell movements by chondroitin sulfate during gastrulation in the chick

Avian gastrulation is dependent on the ingression of outer layer cells into the interior of the embryo by means of a transient structure referred to as the primitive streak. As the growing streak progresses through the central area pellucida of the blastoderm, selective de-epithelialization of epiblast cells results in the initial migratory cells of the primitive mesoderm and endoderm. Here, we have examined the possibility that extracellular matrix molecules of the epiblast basal lamina influence the selection of streak-specific epiblast cells. By using whole embryo culture, we have found that removal of chondroitin sulfate glycosaminoglycans at gastrulation stages leads to defective streak formation. In situ hybridization with streak-specific markers in these embryos reveals ectopic patterns of gene expression, suggesting that differentiation of primitive streak precursors in the pregastrula epiblast is independent of normal streak morphogenesis. In addition, in vitro assays with chondroitin sulfate containing matrices suggest that specific cells of the epiblast are inhibited from joining the streak during gastrulation. Taken together, these results indicate that the presence of chondroitin sulfate in the epiblast basal lamina facilitates the allocation of cells to the primary germ layers by preventing ectopic axis formation.

Study of inhibition mechanisms of glycosaminoglycans on calcium oxalate monohydrate crystals by atomic force microscopy.

Atomic force microscopy (AFM) was applied to the (-101) faces of calcium oxalate monohydrate (COM) crystals grown from calcium oxalate (CaOx) solutions. Microstructures of many spiral hillocks with step height of 1 nm were observed on the faces. Then using AFM in situ, we analysed the re-growth process of the spiral steps on the face of COM seed-crystals in CaOx growth solutions that contained growth inhibitors of glycosaminoglycans and studied their inhibition mechanisms on COM crystals. The total morphology of the faces of COM seed crystals re-grown in the CaOx growth solutions was assessed by scanning electron microscopy (SEM). In the growth solution without glycosaminoglycans (control experiment) or with chondroitin sulphate (ChS), AFM images and SEM micrographs of the faces of the re-grown seed crystals showed two-dimensional (2D) nucleation although 2D nucleation was delayed in the presence of ChS. However, the addition of dermatan sulphate (DS) to the growth solution resulted in isotropic growth by a step flow mode and spiral mechanism. With regard to the main inhibition mechanisms of two glycosaminoglycans (ChS and DS) on COM crystals, it can be concluded from these results that ChS delays 2D nucleation and DS inhibits 2D nucleation.

Amyloid Precursor-like Protein 2 Promotes Cell Migration toward Fibronectin and Collagen IV

Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.

Growth factors increase pericellular proteoglycans independently of their mitogenic effects on A10 rat vascular smooth muscle cells

Proliferation of vascular smooth muscle cells with the accumulation of proteoglycans in the extracellular matrix is one of the significant changes found in atherosclerotic lesions. In order to clarify the relationship between pericellular proteoglycan and cell growth, we established a simple method for quantitatively estimating the amount of pericellular proteoglycans and investigated the effects of various growth factors on the synthesis of pericellular proteoglycans by cultured A10 rat smooth muscle cells. Analysis of trypsin accessible [ 35SO 4]-labeled material in the pericellular area of the A10 cell culture by Q-sepharose anion-exchange chromatography showed two peaks. One peak, eluted at 0.55 M NaCl, disappeared after treatment with 2 mU/ml of heparitinase, indicating that heparan sulfates (HS) were present. The other peak, which eluted at 0.65 M NaCl, disappeared with 20 mU/ml of chondroitinase ABC, indicating the presence of chondroitin sulfates and dermatan sulfates (CS/DS). We estimated the effects of several growth factors on the synthesis of the pericellular proteoglycans by measuring heparitinase- and chondroitinase-ABC-sensitive radioactivities. Although PDGF-AB significantly stimulated cell proliferation and the synthesis of pericellular CS/DS, its dose-dependent effect on the cell growth did not coincide with that on the proteoglycan synthesis. IGF-I (1 nM) increased pericellular CS/DS but not the cell number, while basic FGF (1 nM) and EGF (1 nM) increased the cell number but not pericellular CS/DS. All the growth factors we examined had no effect on the synthesis of pericellular HS. These results indicate that growth factors increase pericellular proteoglycans independently of their mitogenic effects.

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle.

Expression of chondroitin sulfate and keratan sulfate proteoglycans in the path of growing retinal axons in the developing chick.

Previous investigations have identified proteoglycans in the central nervous system during development and have implicated some proteoglycans as axon guidance molecules that act by inhibiting axon extension. The present study investigated the pattern of immunoreactivity for several glycosaminoglycans common to certain proteoglycans relative to growing retinal axons in the developing chick visual system and in retinal explant cultures. Immunostaining for chondroitin-6-sulfate, chondroitin-4-sulfate, and keratan sulfate was observed to colocalize with retinal axons throughout the retinofugal pathway during the entire period of retinal axon growth. The proteoglycan form of collagen IX, however, was only observed in the retina, primarily peripheral to the areas with actively growing axons. The pattern of immunostaining for chondroitin sulfate in tissue sections suggested that the retinal axons might be a source for some of the chondroitin sulfate immunostaining in the developing visual pathway. This was confirmed in that chondroitin sulfate immunostaining was also observed on neurites emanating from cultured retinal explants. These findings indicate that retinal axons grow in the presence of chondroitin sulfate and keratan sulfate proteoglycans and that these proteoglycans in the developing chick visual pathway have functions other than to inhibit axon growth.

Histochemical characterization of the interphotoreceptor matrix in the retina of Octopus bimaculoides

Proteoglycans, located in the interphotoreceptor matrix (IPM) of vertebrate retinas, mediate interactions between the photoreceptors and retinal pigment epithelium. Molluscan retinas also have an IPM located between apposing rhabdomeres. Like the cone matrix sheath of the vertebrate IPM, the octopus IPM is labeled by peanut agglutinin (PNA) and contains retinoid-binding-like proteins. In this study we demonstrate further similarities of the vertebrate/invertebrate IPM and identify specific molecular components in this extracellular compartment of the octopus retina. For light microscopy, paraffin-embedded sections of octopus retinas were stained with dyes specific for acid mucopolysacharides including Alcian blue and colloidal iron. In addition, sections were digested with enzymes specific for hyaluronan, chondroitin sulfate, and sialoglycoconjugates. Digestion of sections with these enzymes and subsequent staining with Alcian blue or colloidal iron demonstrated the presence of chondroitin sulfate and sialoglycoconjugates in the octopus IPM as well as other retinal layers and cells. At the electron-microscope level we treated retinal tissue with Cuprolinic Blue and observed the distribution of sulfated glycosaminoglycans along the rhabdomere edges facing the IPM and in a more central area of the IPM where microvillous processes of supportive cells are located. The octopus IPM may have importance in retinal structure and may be a scaffolding on which molecular components of the IPM are located.

Effects of heparan sulfate analogue or other sulfated polysaccharides on the activation of plasminogen by t-PA or u-PA

Effects of heparan sulfate analogue (HHS-5) and other sulfated polysaccharides on the fibrinolytic system were investigated. Activation rate of native plasminogen (Glu-plg) by tissue plasminogen activator (t-PA), urinary plasminogen activator (u-PA) and single-chain u-PA (scu-PA) was enhanced in the presence of HHS-5 dose dependently up to 10 μg/ml. Activation rate of Glu-plg by sct-PA was more enhanced than that by tct-PA in its presence. HHS-5 enhanced the activation of Glu-plg by t-PA or u-PA compared to the activation of Lys-plg. SDS-PAGE confirmed that the enhancement of the hydrolysis of S-2251 by the mixture of Glu-plg, sct-PA and HHS-5 was due to the facilitation of the conversion of Glu-plg to plasmin. HHS-5 only slightly enhanced the amidolytic activity of sct-PA or tct-PA. The enhancement of the activation of plasminogen by t-PA or u-PA was more significant in the presence of HHS-5 than in the presence of chondroitin sulfate C, dextran sulfate or heparin. It is possible that the enhancement of the activation of Glu-plg by activators in the presence of HHS-5 was due to the conformational change of Glu-plg upon interaction with HHS-5.

Possible role for chondroitin sulfate in urolithiasis: In vivo studies in an experimental model

The effect of chondroitin sulfate upon the growth of calcium oxalate crystals was measured in vivo by using an experimental model in rats. Adult male Wistar rats were treated by cronical i.p. injections of chondroitin sulfate solutions (1, 5 or 10 mg in 0.3 ml of saline, every 2 days). This treatment led to a dose-dependent increase in the urinary chondroitin sulfate concentration. Urolithiasis was induced by the introduction of a calcium oxalate seed into the bladder of the animals. Urine samples were collected and the calculi formed were removed after 42 days. The chondroitin sulfate concentration have decreased in the lithiasic urines, as compared to controls and higher chondroitin sulfate doses correlated with larger calculi. The presence of chondroitin sulfate in the matrices of stones obtained from chondroitin sulfatetreated animals suggested that there was some adsorption of chondroitin sulfate on to the growth sites of the calcium oxalate crystals. In contrast to the chondroitin sulfate effect observed in vitro, which inhibits the growth of calcium oxalate crystals, our results suggest that in vivo chondroitin sulfate promotes the growth of stones in the urinary tract.

Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain.

Two glycoforms of a secretable human thrombomodulin mutant [TMD1-105 and TMD1-75; Parkinson, Grinnell, Moore, Hoskins, Vlahos & Bang (1990) J. Biol. Chem. 265, 12602-12610] were expressed in human 293 cells and used to study the role of glycosylation in the functions of this endothelial-cell thrombin receptor. Carbohydrate content analysis and intrinsic labelling with [3H]glucosamine and [35S]sulphate showed that TMD1-105 contained a chondroitin sulphate whereas TMD1-75 did not. Other than chondroitin sulphate, the carbohydrate contents of the two glycoforms were identical, indicating similar glycosylation patterns at other O-linked and N-linked sites in the two glycoforms. The properties of TMD1-105 were converted into those of TMD1-75 by chondroitin ABC lyase digestion. Trypsin digestion of labelled TMD1-105 permitted isolation of two overlapping peptides that contained chondroitin sulphate, spanned the entire O-glycosylation domain and had O-glycosylation sites at Ser-492, Ser-498, Thr-500, Thr-504 and Thr-506. The chondroitin sulphate-attachment site was assigned to Ser-492 as this residue is conserved in mouse and bovine thrombomodulin and lies within a sequence Ser-Gly-Ser-492-Gly-Glu-Pro, which has strong similarity to chondroitin sulphate attachment sites in other proteoglycans. Five peptides with N-linked carbohydrate were also isolated and contained glycosylation sites in the lectin-like domain (Asn-47, Asn-115, Asn-116) and in the fourth (Asn-382) and fifth (Asn-409) epidermal growth factor domains. The role of N-linked and simple O-linked carbohydrates in the functions of human thrombomodulin remain unclear. The present studies demonstrate, however, that the presence of chondroitin sulphate in human thrombomodulin has profound effects on all of the anticoagulant properties of this important anticoagulant thrombin receptor.

Glycosaminoglycans, uric acid and calcium oxalate urolithiasis

The interaction between calcium and glycosaminoglycans (GAGs) was studied using a calcium ion-selective electrode. The Ca-binding capacity of GAGs involved 16% of total calcium in the presence of chondroitin sulphate and 28% in the presence of pentosan polysulphate. The action of GAGs on the nucleation of uric acid and sodium urate was examined and inhibitory effects were observed. The action of uric acid as a heterogeneous nucleant of calcium oxalate was studied, and considerable promotion of the heterogeneous nucleation of calcium oxalate by uric acid was found, which could be inhibited by the action of GAGs. From these summarised in vitro results, we conclude that uric can constitute an important risk factor for calcium oxalate urolithiasis through heterogeneous nucleation and the GAGs can play an important role as preventive agents.

Classical and Chondroid Chordoma: A Light-Microscopic, Histochemical, Ultrastructural and Immunohistochemical Analysis of the Various Cell Types

The aim of the present investigation was to characterize the various cell types of classical and chondroid chordomas. Eight cases of classical chordoma, 1 case of sacrococcygeal chordoma with chondroid areas and 2 cases of spheno-occipital chondroid chordoma were studied. Ultrastructurally and immunohistochemically (immunoreactivity for cytokeratins, epithelial membrane antigen [EMA], tissue polypeptide antigen [TPA] and human milk fat globule protein [HMFG]) the 3 cell types (physaliferous, epithelial-like, and spindle-shaped) recognized light-microscopically presented features of epithelial differentiation and rather formed a continuous spectrum than being distinct cell types. The chondroid areas of the chondroid chordomas had similar ultrastructural and immunohistochemical properties except for the lack of immunoreactivity for EMA and HMFG. The results of the critical electrolyte concentration technique according to Scott and Dorling indicated that there was no difference in the sulfated glycosaminoglycan content between classical and chondroid chordomas: all the tumors contained chondroitin sulfate. The presence of chondroitin sulfate, immunoreactivity for vimentin and S-100 protein and areas of cartilaginous differentiation in three cases indicate a relationship both to chondromatous tumors and to normal notochord, from which chordoma is believed to originate.

In vitro mineralization of a three-dimensional collagen matrix by human dental pulp cells in the presence of chondroitin sulphate

These matrices were used as cell culture substrates to investigate the influence of extracellular molecules on mineralization. Pulp cells seeded in type I collagen or type I collagen-chondroitin-4-sulphate sponges were able to grow and were morphologically similar to cells responsible for reparative dentine formation in vivo. In sponges consisting of collagen only, the cells elaborated an abundant new matrix which became organized with time and consisted of collagen fibres surrounded by fibrillar material, but no mineralization was observed. In collagen-chondroitin sulphate sponges, cells deposited less and poorly organized matrix; in these, calcification occurred, increasing with time, and at the ultrastructural level, small needle-like crystals containing calcium and phosphorus were scattered throughout the sponge fibres. These observations suggest that chondroitin sulphate might influence in vitro calcification induced by pulp cells.

Hypothermic preservation of corneas in a hyperkalaemic solution (CPTES): II. Extended storage in the presence of chondroitin sulphate.

Periods of preservation for donor corneas, even for short times, are necessary to facilitate optimum conditions in penetrating keratoplasty. However, current techniques for corneal storage at low temperatures may not provide optimal conditions for maintaining tissue integrity. In particular, the ionic composition of the storage medium has received little attention since it has been assumed throughout that the normal complement of ions in tissue culture media will also be suitable for preservation at reduced temperatures. This study extends our previous investigations on the merits of using CPTES (corneal-potassium-TES), a potassium-rich balanced salt solution containing an impermeant anionic pH buffer (TES), as a storage solution specifically designed to prevent the loss of intracellular potassium and minimise endothelial cell swelling during the time that the normal regulatory processes are switched off. The effect of adding the natural polymer chondroitin sulphate (CS) as a colloid osmotic agent to the hyperkalaemic storage medium is now examined. Corneas stored in CPTES containing 2.5% chondroitin sulphate retained a very high level of structural and functional integrity after three, five, and seven days storage at 0 degrees C; furthermore, stromal swelling was restricted to only 21%. All corneas stored in CPTES + 2.5% CS showed active endothelial function by thinning efficiently at rates that were greater than those previously reported for rabbit corneas stored for similar lengths of time in either M-K medium or K-sol. The zwitterionic buffers TES and HEPES were interchangeable in the hyperkalaemic solution and were non-toxic to corneal endothelium at a concentration of 100 mM. These compounds offer excellent pH buffering in bicarbonate-free medium.

The Effect of Glycosaminoglycans on the Crystallisation of Calcium Oxalate

The effect of glycosaminoglycans on urinary stone formation was evaluated using a mixed suspension, mixed product removal (MSMPR) crystallisation system together with scanning electron microscopy (SEM) to examine the resulting crystals. Chondroitin sulphate was found to decrease the nucleation rate and to promote both the growth rate and suspension density. Results obtained with hyaluronic acid, although inconclusive, are similar to those given by chondroitin sulphate. Heparin sodium salt had a powerful inhibitory effect on both the nucleation rate and the suspension density, the effect increasing in proportion to the heparin concentration. SEM examination showed that the octahedral habit of calcium oxalate dihydrate was modified by the addition of heparin sodium salt and confirmed that the average crystal size in the presence of chondroitin sulphate and hyaluronic acid was significantly greater than the control or that found in the presence of heparin sodium salt.

The presence of chondroitin sulfate in parotid secretory granules and saliva of the rat

The presence of chondroitin sulfate in secretory granules of the rat parotid gland and its saliva was revealed by radioactive sulfate incorporation, followed by isolation and partial characterization of the sulfated species contained within the granules and in the parotid saliva. 35SO4 was incorporated into chromatographically identical macromolecular material both in vitro, in a gland-slice system followed by isolation of granule contents, and in vivo as measured in the pure parotid secretion following intravenous administration of 35SO4=. The majority of the 35SO4= label appeared in a peak in the region where the family of acidic proline-rich proteins elute from a DEAE-Sephadex A-50 column. Papain digestion freed the sulfated material from the bulk of protein present in this peak, leaving sulfate-labelled material that chromatographed on Sepharose CL6B as a single peak corresponding to a molecular weight of 13,000 daltons. The ratio of uronic acid to amino sugar in this sulfated peak was 0.56. The sulfated material was susceptible to degradation by chondroitinase AC. The presence of this chondroitin sulfate in secretory granules and saliva is consistent with previous suggestions that sulfated polyanions may play a role in formation and maturation of secretory granules.