Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta

Summary The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate.Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possibly also present at a high concentration in the endothelium. Staining of sections after treatment with 4m guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.     

The proteoglycan skeleton of the Kurloff body as evidenced by Cuprolinic Blue staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosome organelle, metachromatic towards Toluidine Blue, of a blood cell unique to the guinea pig and called the Kurloff cell. Splenic Kurloff cell from oestrogen-treated guinea pig cells were examined after staining with Cuprolinic Blue, a cationic phthalocyanine-like dye, in the presence of MgCl₂ in a critical electrolyte concentration method. Better results were obtained when the fixation-staining by the glutaraldehyde Cuprinolinic Blue MgCl₂ mixture was preceded by a glutaraldehyde pre-fixation. On light microscopy, Kurloff bodies generally exhibited an overall pink and glassy metachromasia, sometimes with additional darker metachromatic small dots at their peripheries. At the ultrastructural level, the metachromatic central matrix of the Kurloff body usually exhibited, as a major feature, a typical network pattern of ribbon-like or stellate electron-dense precipitates suggesting the presence of a skeleton of Cuprolinic Blue-reactive filamentous structures. Taking into account their high anionicity (as shown by the stability of the dye binding in the presence of 0.3 m MgCl₂) and their susceptibility to chondroitinase ABC, these anionic structures were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell.     

The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity.

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Further characterization of a large proteoglycan in human lung alveoli

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, heavily staining, generally large, filaments have been demonstrated in human lung alveoli. In some lung specimens they are abundant, while in others they are very scanty. The filaments are seen: around bundles of collagen fibrils, at places which seem electron microscopically almost empty, associated with basement membranes around elastin, and sometimes associated with individual collagen fibrils. After poststaining tiny threads--connecting the filaments--could sometimes be observed. The filaments are resistant to treatment with nitrous acid, heparitinase or pronase after prefixation. After digestion with chondroitinase ABC, chondroitinase AC or pronase without prefixation, the filaments are no longer detectable. The tiny threads are chondroitinase ABC resistant. It is concluded that the Cuprolinic Blue-positive filaments represent proteoglycans which contain chondroitin sulfate and/or glucuronic acid-rich dermatan sulfate. The possible role of these proteoglycans in tissue repair is discussed.     

Uneven Distribution and Size of Rabbit Lens Capsule Proteoglycans

To document the ultrastructural distribution of lens capsule proteoglycans, rabbit lens capsules were fixed and stained overnight in 50mM sodium acetate, pH 5.6, containing 2.5% glutaraldehyde, 0.2% Cuprolinic Blue and 0.2M MgCl2. They were rinsed, stained with 1% aqueous sodium tungstate, embedded in Epon, sectioned (60nm), and examined with an electron microscope at 60kV.Proteoglycan–Cuprolinic Blue complexes mainly appeared as networks of small electron-dense filaments throughout the posterior and anterior capsules. The posterior capsule was a single layer with a network of small proteoglycan filaments gradually decreasing in size from the humoral side (90×10nm) to the lenticular side (30×8nm). The humoral side of the anterior capsule had a thin lamina (400nm) containing large (180×40nm), very electron-dense proteoglycan–Cuprolinic Blue complexes plus small proteoglycans. Below this lamina, the complexes were only seen as filaments slightly smaller than those in the corresponding area of the posterior capsule.Cuprolinic Blue binding of the anterior and posterior lens capsules revealed differences in the size and distribution of their sulphated proteoglycans which do not correspond to the patterns of their immunoreactivity with anti-heparan sulphate proteoglycan. The humoral lamina in the anterior capsules, with large proteoglycan structures, might be a distinct structural and functional compartment.     

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.     

A nitrous acid procedure as a selective histochemical means of eliminating the N-sulphates of glycoconjugates

Summary A nitrous acid procedure has been shown to lead to the elimination of N-sulphates in sections of a series of tissues containing sulphated glycoconjugates. Two groups of sulphated glycoconjugate-containing tissues were used; one contained N-sulphates and other was devoid of such groupings. In the first group of tissues, mast cells of different origins and renal glomeruli in the rat were employed. Xiphoid and tracheal cartilage matrix, submandibular and sublingual gland acini and gastric, duodenal and colonic mucosae were used in the second group. Sections were treated with nitrous acid and then stained with Alcian Blue pH 1.0, high iron diamine or Aldehyde Fuchsin for sulphated glycoconjugates. Such treatment was found to diminish the staining intensities exclusively in N-sulphated glycoconjugate-containing structures such as mast cell granules and renal glomerular basement membrane, providing a means of chemically eliminating N-sulphates of glycoconjugates in tissues.     

Ultrastructural localization of a chondroitinase-sensitive, cuprolinic blue-positive filament in developing mouse lung

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, the lungs of mice ranging from the late fetal stage (17 days of gestation) to the puberal stage (27 days) were surveyed for their proteoglycans. A large Cuprolinic Blue-positive filament is present within the connective tissue of lungs of late fetal and young postnatal mice. It is mostly located at the boundary between large extracellular matrix structures and electron microscopically empty areas, but sometimes also at the surface of fibroblast-like cells. The stainability of the filament disappears after treatment with chondroitinase ABC or chondroitinase AC, but not after treatment with nitrous acid. The Cuprolinic Blue-positive structure appears to be most abundant around 2 days postnatally. From day 10 on, its number decreases dramatically, and it can be no longer observed in the lungs of 27-day-old mice.     

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicer's high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3–5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following, observation of sections treated by a chloroform-methanol mixture.     

Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.     

Proteoglycan and collagen morphology in superficially scarred rabbit cornea

Summary We have examined the changes in collagen and proteoglycan morphology in superficial lamellar keratectomy wounds produced in rabbit corneas. The ultrastructural location within the tisse of keratan sulphate and chondroitin sulphate proteoglycans was demonstrated using the cationic dye Cuprolinic Blue under critical electrolyte conditions. Large proteoglycan filaments (up to 500 nm long) appeared in the early stages of wound healing; these were most common after two weeks' wound healing, after which they decreased both in number and size. At these early stages of scar formation, spaces containing proteoglycans were present amongst bundles of collagen fibrils. As proteoglycans play an important role in controlling corneal hydration, the presence of the large proteoglycan-filled spaces would result in an abnormally high water content which is found in early early scar tissue.     

Changing profiles of proteoglycans in the transition of predentine to dentine.

Proteoglycans and their constituent glycosaminoglycans have been proposed to play important roles in matrix mediated formation of mineralised tissues, such as dentine. This study has examined the changing profile of proteoglycan species during the transition of unmineralised predentine to mineralised dentine. Three-week-old calves teeth were collected and proteoglycans purified from the predentine, the predentine/dentine interface and dentine. Decorin and biglycan, together with related degradation products, were identified in the predentine fraction, alongside degradation products of versican, indicating metabolism of the proteoglycan components within this tissue. Decorin and biglycan were also identified as major proteoglycan species within extracts from the predentine/dentine interface and dentine. Analysis of the glycosaminoglycan constituents within each fraction demonstrated significant changes in their composition. Predentine contained a high proportion of dermatan sulfate (DS) (51.5%), with chondroitin sulfate (CS) (17.8%) and hyaluronan (HA) (30.7%) additionally identified. Within the predentine/dentine interface the proportion of CS increased greatly (62.5%), with corresponding decrease in the proportion of DS (21.4%) and HA (16.1%) also evident. CS only was identifiable within the dentine matrix. A four-fold increase in the level of sulfation was identified for glycosaminoglycans extracted from the predentine/dentine interface compared with the predentine and dentine fraction. The ratio of DeltaDi4S:DeltaDi6S was higher for glycosaminoglycans isolated from the predentine fraction. Glycosaminoglycans extracted from the dentine fraction possessed longer chain lengths than those present in the predentine and predentine/dentine fractions. The results indicate that the proteoglycans within each fraction undergo subtle structural modification, particularly at the onset of mineralisation, indicating an active involvement of these macromolecules in the overall mineralisation process.     

Biosynthesis of sulphated macromolecules by rabbit lens epithelium. I. Identification of the major macromolecules synthesized by lens epithelial cells in vitro

Rabbit lens epithelial cells synthesize and secrete a variety of [35S]sulphate-labeled glycoconjugates in vitro. Associated with the cell layer, and with the medium, was a high molecular weight glycoconjugate(s) that contained heparan sulphate which was apparently covalently linked to sulphated glycoprotein. This component(s) was eluted in the void volume of a Sepharose CL-2B column and could not be fractionated by detergent treatment or extraction with lipid solvents. The cell layer also contained glycosaminoglycans (72% heparan sulphate, 28% chondroitin sulphate), as well as a small proportion of a low molecular weight sulphated glycoprotein. The major 35S-labeled species secreted into the medium were sulphated glycoproteins with approximate molecular weights of 120,000 and 35,000 together with a heparan sulphate proteoglycan. This proteoglycan could be precipitated from the culture medium with 30% saturated (NH4)2SO4 and eluted from Sepharose CL-4B columns at approximately the same position (Kav = 0.15) as heparan sulphate proteoglycans described in the basement membrane of the EHS "sarcoma" (Hassell, J. R., P. G. Robey, H. J. Barrach, J. Wilczek, S. I. Rennard, and G. R. Martin, 1980, Proc. Natl. Acad. Sci. USA, 77:4494-4498) and of the mouse mammary epithelium (David, G., and M. Bernfield, 1981, J. Cell Biol., 91:281-286). Its presence in the culture medium was unanticipated but may be explained by the inability of these cultures to deposit a basement membrane when grown on a plastic surface. The relationship of this heparan sulphate proteoglycan to the lens epithelial basement membrane is the subject of the following paper.     

A histochemical study of anionic sites in the intermediate layer of rat femoral cartilage using polyethyleneimine at different pH levels

Anionic sites in the intermediate layer of young rat hyaline cartilages were examined using a cationic dye, polyethyleneimine (PEI), at different pH levels. Femoral heads were resected and fixed in 2.5% glutaraldehyde and treated with 0.5% PEI at pH 7.4, pH 2.5 or pH 1.0. Some cartilage samples were first digested with chondroitinase ABC or hyaluronidase. The PEI deposits at pH 7.4 appeared to be irregular shapes. Their sizes seemed to be larger than those at pH 2.5 or pH 1.0. The PEI deposits were also found on the surface of collagen fibrils at both pH 7.4 and pH 2.5 even after the chondroitinase ABC digestion, but were not found at pH 1.0. Moreover, they disappeared after hyaluronidase digestion. Accordingly, it is suggested that PEI-positive structures varied depending on pH levels. In addition, hyaluronan may be localized near collagen fibrils, but most sulphated proteoglycans may not. This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Isolation of heparan sulfate proteoglycan from beneath the monolayers of rat hepatocytes and its binding to type IV collagen

Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on beta-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1 x 2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.     

Ultrastructural histochemistry of the surface lamina of normal articular cartilage

Summary Two collagen-poor, ultramicroscopic layers are described at the surface of canine articular cartilage. They are distinguished by staining with an electron-dense cationic dye, Cupromeronic Blue, in a critical electrolyte concentration technique and by digestion with testicular hyaluronidase. The superficial layer, approximately 50 nm thick, stained at low electrolyte concentrations but failed to stain in conditions specific for sulphated glycosaminoglycans. It was hyaluronidase-resistant and may be either glycoprotein or protein in nature. The deeper layer, 100–400 nm thick, stained positively at electrolyte concentrations specific for sulphated glycosaminoglycans but not in conditions specific for keratan sulphate. It was removed by hyaluronidase digestion. This layer probably represents a chondroitin sulphate-rich proteoglycan.These surface layers may be important in the lubrication of the articular surface and in the permeability and compression resistance of the superficial cartilage zone.     

The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC.

Synopsis Treatment of tissue sections with enzymes which degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. BothStreptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of bothStreptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, andStreptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.     

Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Summary Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens containd round granules ( 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl₂ indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC, Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the ³⁵SO₄ labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/ dermatan sulphates comprised about one fifth of the total activity incorporated. The distribution of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.