The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells

Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic "exchange-adsorption" model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val⁶,Ala⁷]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.     

Aggregation of hydroxyapatite crystals

A system to study the aggregation of hydroxyapatite crystals was developed. The effect of several factors (Ca²⁺ × P_i product, Ca²⁺ /P_i ratio, pH, and various substances) were tested. Pb²⁺, Zn²⁺, Mg²⁺ and methyleneblue had only small effects; citrate inhibited aggregation. Pyrophosphate was a strong inhibitor and the diphosphonates disodium ethane-1-hydroxy-1,1-diphosphonate and disodium duchloromethylene diphosphonate were even more potent. The monophosphonate pentanemonophosphonate had no effect. Potent inhibition also occurred with glycosaminoglycans: heparin > hyaluronic acid > dermatan sulfate > chondroitin 4-sulfate > chondroitin 6-sulfate. Urine also showed high inhibitory activity. The inhibition of heparin but not that of hyaluronic acid, PP_i or urine was abolished by egg white lysozyme. The effects described might be relevant in the normal mineralization process as well as in the mechanisms leading to pathological calcification, such as urinary stone formation.     

Binding of heparin fractions and other polysulfated polysaccharides to plasma fibronectin: effects of molecular size and degree of sulfation of polysaccharides

Heparin was divided into four fractions on fibronectin-Sepharose. The higher affinity fraction for fibronectin was larger in molecular size, higher in sulfate content and higher in affinity for anti-thrombin III. Together with these heparin fractions, the following three series of heparin samples were examined to compare the affinity for fibronectin-Sepharose: four fractions separated on Sephadex G-100; five fractions separated on antithrombin III-Sepharose, and six partially and completely N-desulfated heparins. The result showed that the affinity of heparin for fibronectin was dependent exclusively on its molecular size, and that an appropriate level of sulfate content in heparin (1.9-2.4 mol/disaccharide) was essential for the affinity. The sulfated preparations of glycosaminoglycans (heparan sulfate, dermatan sulfate and chondroitin 4-sulfate) and neutral polysaccharides (amylose and dextran) having higher sulfate content than heparin were found to display higher affinity for fibronectin than heparin. This suggested that highly sulfated polysaccharides showed potent affinity irrespective of their polysaccharide structure. The sulfated chondroitin 4-sulfate having a sulfate content and molecular size comparable to those of heparin was inferior to heparin with respect to affinity. A competitive dissociation experiment indicated that heparin and other polysulfated polysaccharides share a common binding site on the fibronectin molecule.     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Local bradykinin formation is controlled by glycosaminoglycans

Bradykinin is a potent inflammatory mediator that induces vasodilation, vascular leakage, and pain sensations. This short-lived peptide hormone is liberated from its large precursor protein high molecular weight kininogen (HK) through the contact system cascade involving coagulation factor XII and plasma kallikrein. Although bradykinin release is well established in vitro, the factors and mechanisms controlling bradykinin generation in vivo are still incompletely understood. In this study we demonstrate that binding of HK to glycosaminoglycans (GAGs) of the heparan and chondroitin sulfate type efficiently interferes with bradykinin release in plasma and on endothelial surfaces. Proteolytic bradykinin production on endothelial cells is restored following degradation of cell surface GAG through heparinase. Alternatively, application of HK fragments D3 or light chain, which compete with uncleaved HK for cell binding, promote kininogen proteolysis and bradykinin release. Intravital microscopy revealed that HK fragments increase bradykinin-mediated mesentery microvascular leakage. Topical application of D3 or light chain enhanced bradykinin generation and edema formation in the mouse skin. Our results demonstrate that bradykinin formation is controlled by HK binding to and detachment from GAGs. Separation of the precursor from cell surfaces is a prerequisite for its efficient proteolytic processing. By this means, fragments arising from HK processing propagate bradykinin generation, revealing a novel regulatory level for the kallikrein-kinin system.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Amyloid-beta interactions with chondroitin sulfate-derived monosaccharides and disaccharides. implications for drug development

In Alzheimer's disease, the major pathological features are diffuse and senile plaques that are primarily composed of the amyloid-beta (A beta) peptide. It has been proposed that proteoglycans and glycosaminoglycans (GAG) facilitate amyloid fibril formation and/or stabilize the plaque aggregates. To develop effective therapeutics based on A beta-GAG interactions, understanding the A beta binding motif on the GAG chain is imperative. Using electron microscopy, fluorescence spectroscopy, and competitive inhibition ELISAs, we have evaluated the ability of chondroitin sulfate-derived monosaccharides and disaccharides to induce the structural changes in A beta that are associated with GAG interactions. Our results demonstrate that the disaccharides GalNAc-4-sulfate(4S), Delta UA-GalNAc-6-sulfate(6S), and Delta UA-GalNAc-4,6-sulfate(4S,6S), the iduronic acid-2-sulfate analogues, and the monosaccharides d-GalNAc-4S, d-GalNAc-6S, and d-GalNAc-4S,6S, but not d-GalNAc, d-GlcNAc, or Delta UA-GalNAc, induce the fibrillar features of A beta-GAG interactions. The binding affinities of all chondroitin sulfate-derived saccharides mimic those of the intact GAG chains. The sulfated monosaccharides and disaccharides compete with the intact chondroitin sulfate and heparin GAGs for A beta binding, as illustrated by competitive inhibition ELISAs. Therefore, the development of therapeutics based on the model of A beta-chondroitin sulfate binding may lead to effective inhibitors of the GAG-induced amyloid formation that is observed in vitro.     

Nuclear magnetic resonance spectra of heparin in admixture with dermatan sulfate and other glycosaminoglycans. 2-D spectra of the chondroitin sulfates

Characteristics of the 1H-n.m.r. spectra of heparin admixed with other glycosaminoglycans are described with respect to the identification of the latter as possible contaminants of pharmaceutical heparins. Chemical shift differences are sufficiently large, particularly with the aid of resolution enhancement, to allow for the detection of dermatan sulfate, chondroitin 4- or 6-sulfate, hyaluronic acid, or heparan sulfate as a minor constituent in the presence of heparin. The acetamidomethyl resonance region (delta 1.95-2.15) is especially useful in this context, both for identification and quantitative estimation. Whereas dermatan sulfate is a common contaminant of pharmaceutical heparin preparations, in some instances comprising 10-15 percent of the polymer mixture, the other glycosaminoglycans, by contrast, were not detected in such preparations. Two-dimensional heterocorrelation and homo-correlation n.m.r. experiments have provided 1H- and 13C-chemical shift data that complete or verify (or both) previous information available for heparin, dermatan sulfate, and chondroitin 4- and 6-sulfates (chondroitins A and C).     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.