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At least 19 recordsLinked to original sources
PubMed1975

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.

Animals↗
PubMed1983

Binding of fibronectin and its proteolytic fragments to glycosaminoglycans. Exposure of cryptic glycosaminoglycan-binding domains upon limited proteolysis.

Binding of intact plasma fibronectin and its proteolytic fragments to glycosaminoglycans immobilized on agarose beads was systematically compared at different ionic strengths. In low ionic strength buffer, intact fibronectin bound to heparin and high sulfated heparan sulfate, but not to low sulfated heparan sulfate, dermatan sulfate, chondroitin sulfates A and C, or hyaluronic acid. Fractionation of the thermolysin digest of fibronectin on the glycosaminoglycan-Sepharoses at low ionic strength revealed that three groups of fragments, i.e. Mr = 150,000-140,000, 24,000, and 16,000 (150K-140K, 24K, and 16K) fragments, were capable of binding to glycosaminoglycans with different specificities and affinities. The 150K-140K fragments exhibited the same specificities as intact fibronectin, binding only to heparin and high sulfated heparan sulfate. However, the 24K fragment bound not only to these two glycosaminoglycans but also to low sulfated heparan sulfate and other glycosaminoglycans as well. The 16K fragments were also capable of binding to most glycosaminoglycans with lower affinity than the 24K fragment. These results suggest that the binding sites in the 24K and 16K fragments are cryptic in the intact protein, but are exposed after limited proteolysis. The binding of fibronectin and its fragments to glycosaminoglycans is dependent on the ionic strength. At physiologic ionic strength, only heparin-Sepharose could bind intact fibronectin. Similarly, only heparin-Sepharose could bind the 150K-140K and 24K fragments, but not the 16K fragments, at the same ionic condition. Other glycosaminoglycan-Sepharoses did not retain significant amounts of any of the fibronectin fragments, suggesting that the affinity of plasma fibronectin and its fragments to heparan sulfate and other glycosaminoglycans, except heparin, is not strong enough to achieve stable mono- (or di-) valent binding under physiologic conditions.

Animals↗
PubMed1988

Extraction and analysis of glycosaminoglycans from intima-media of single rabbit aortae: effect of balloon catheter de-endothelialization on the content and profile of glycosaminoglycans.

A novel procedure is described for extracting, and simultaneously 3H-labelling, glycosaminoglycans from the intima-media of a single rabbit aorta. The procedure was used to compare contents and types of glycosaminoglycans isolated from uninjured (control) aortae, and partially re-endothelialized aortae at 11 weeks after de-endothelialization by a balloon catheter. Briefly, the isolated delipidated tissue was digested in 0.8 M NaOH containing NaB3H4 at room temperature. The neutralized digest was then degraded by a non-specific proteinase during dialysis. The 3H-labelled glycosaminoglycan fraction was recovered after a gel filtration step. The yield (10.5 micrograms of glycosaminoglycan/mg of dry, delipidated tissue) was within the range reported previously for rabbit aorta. Although the de-endothelialized (DEA) and re-endothelialized areas (REA) of all injured aortae contained a significantly thickened intima, the glycosaminoglycan concentration (DEA, 11.2 micrograms/mg; REA, 11.6 micrograms/mg) did not differ significantly from that of control aorta. The profile of [3H]glycosaminoglycan types was determined by the serial use of glycosaminoglycan-selective methods: greater than 86% of 3H was released as small molecular weight products by this analytical scheme. The glycosaminoglycan profile for control tissue compared well with several previous reports. Compared with control aortae, both DEA and REA contained relatively less chondroitin sulphate, whereas DEA contained more hyaluronic acid and REA contained more heparan sulphate. Future use of this procedure will improve measurement of the changes to the extracellular matrix which take place after injury to the vessel wall and which may precede atherogenesis.

Animals↗
PubMed1989

Regulation of serum glycosaminoglycan sulfotransferase activities: inhibition by sulfated glycosaminoglycans and activation by polyamines and basic peptides including a polylysine-containing segment of the c-Ki-ras 2 protein.

The regulatory mechanisms for the glycosaminoglycan sulfotransferases in fetal calf serum were investigated. The enzymes examined were those which transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate to 1) position 6 of the internal N-acetylgalactosamine units of chondroitin, 2) position 6 of galactose units of keratan sulfate, and 3) position 2 (an amino group) of glucosamine units of heparan sulfate. The former two enzymes were activated by spermidine, spermine, protamine, and poly L-lysine. All the enzymes were strongly inhibited by heparin and dextran sulfate, whereas only the chondroitin 6-O-sulfotransferase was inhibited by sulfated galactosaminoglycans. The inhibition of this enzyme by the sulfated glycosaminoglycans was abolished by polylysine, indicating that the activation by polylysine is partly due to the neutralization of endogenous acidic inhibitors, including sulfated glycosaminoglycans. Affinity chromatographic studies demonstrated that heparin specifically binds to the three enzymes, which have anionic isoelectric points, and that chondroitin 6-sulfate, spermine, and polylysine bind to the former two enzymes under physiological conditions. Thus, the activation by spermine and polylysine as well as the inhibition by sulfated glycosaminoglycans also appears to occur through their binding to the enzymes. Studies with synthetic lysine oligomers and an affinity-purified (approximately 700-fold) fraction containing the former two enzymes indicated that the pentamer is the minimum unit required for the activation. A synthetic peptide, containing six consecutive lysines at the carboxy terminus of the human c-Ki-ras 2 protein, also regulated the two enzyme activities at micromolar concentrations. The possible physiological implications of the observed effects of these regulatory substances on the glycosaminoglycan sulfotransferases are discussed in relation to glycosaminoglycan synthesis during the proliferation, differentiation, and transformation of cells. The possibility of sulfated glycosaminoglycans being enzyme regulators is also discussed.

Animals↗
PubMedDate not supplied

Influence of exogenous glycosaminoglycans on growth and glycosaminoglycan synthesis of cultured human diploid fibroblasts (WI-38).

Human diploid fibroblast (WI-38) in monolayer culture were treated with exogenous glycosaminoglycans for short (up to 4 days) or long (several weeks and months) periods, and the effects on growth and glycosaminoglycan synthesis, as measured by the incorporation of 35S-sulfate and 14C-glycosamine into cell-bound and cell-released (medium) glycosaminoglycans, were determined. Short- and long-term exposure to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronic acid at concentrations up to 100 microgram/ml did not affect cell growth, while heparin (between 20 and 100 micrograms/ml), heparan sulfate (above 100 micrograms/ml) or hyaluronic acid (2500 micrograms/ml) exerted significant growth-inhibitory effects. While short-term or long-term influence (each at 100 micrograms/ml) of chondroitin-4-sulfate, chondroitin-6-sulfate and hyaluronic acid resulted in a slight inhibition of incorporation of both radioactive precursors into cell-bound glycosaminoglycans, heparin (between 20 and 500 micrograms/ml) or heparan sulfate (at 100 or 500 micrograms/ml) significantly stimulated 14C-glycosamine incorporation into cell-bound glycosaminoglycans, what appeared to be predominantly into the hyaluronic acid fraction. Following long-term treatment with heparin at 20, 50 or 100 micrograms/ml, incorporation rates of both 14C-glucosamine and 35S-sulfate into both cell-bound and cell-released (medium) glycosaminoglycans were elevated, suggesting a general stimulation of glycosaminoglycan synthesis. Possible mechanisms for the action of these compounds (especially heparin) were discussed, e.g. an interaction with specific cell surface-associated sites.

Cell Division↗
PubMed2002

Capillary electrophoresis for the analysis of glycosaminoglycans and glycosaminoglycan-derived oligosaccharides.

Glycosaminoglycans are a family of polydisperse, highly sulfated complex mixtures of linear polysaccharides that are involved in many life processes. Defining the structure of glycosaminoglycans is an important factor in elucidating their structure-activity relationship. Capillary electrophoresis has emerged as a highly promising technique consuming an extremely small amount of sample and capable of rapid, high-resolution separation, characterization and quantitation of analytes. Numerous capillary electrophoresis methods for analysis of intact glycosaminoglycans and glycosaminoglycan-derived oligosaccharides have been developed. These methods allow for both qualitative and quantitative analysis with a high level of sensitivity. This review is concerned with separation methods of capillary electrophoresis, detection methods and applications to several aspects of research into glycosaminoglycans and glycosaminoglycan-derived oligosaccharides. The importance of capillary electrophoresis in biological and pharmaceutical samples in glycobiology and carbohydrate biochemistry and its possible applications in disease diagnosis and monitoring chemical synthesis are described.

Carbohydrate Conformation↗
PubMed1996

Drug-induced glycosaminoglycan storage: dose-dependent changes in the pattern of accumulated glycosaminoglycans in cultured bovine and human fibroblasts.

The present study determines the amounts and patterns of glycosaminoglycans stored in cultured corneal fibroblasts after treatment with tilorone and three related compounds. The compounds have immunomodulatory properties and have been shown to impair the lysosomal degradation of glycosaminoglycans as a side effect. This side effect has been described as drug-induced mucopolysaccharidosis because the induced lysosomal storage of glycosaminoglycans leads to cellular lesions resembling those in patients with inherited mucopolysaccharidosis. In the present study, the dose-dependency of glycosaminoglycan storage was analyzed after treatment (96 hr) of bovine corneal fibroblasts. The investigated drug concentrations ranged from low concentrations inducing cytological lesions typical of drug-induced mucopolysaccharidosis to high concentrations at the borderline of cytotoxicity. The intracellular amounts of dermatan sulfate, heparan suflate, and chondroitin sulfate were quantified by densitometric scanning of Alcian Blue-stained bands after electrophoresis. All investigated compounds induced a predominant dermatan sulfate storage (3-4-fold accumulation) at low drug concentrations. With rising drug concentrations, a shift of the pattern of stored glycosaminoglycans was observed, characterized by the additional accumulation of heparan sulfate (up to 5-fold of control levels). In cultured human fibroblasts, tilorone also caused a marked dermatan sulfate storage, reaching maximum values at 5 microM and marked heparan sulfate storage at 20 microM. The present data provide evidence: (a) that selective dermatan sulfate accumulation is a characteristic feature of drug-induced glycosaminoglycan storage in cultured bovine and human fibroblasts, if these cells are treated with low concentrations (< or = 5 microM), that are likely to reflect the situation in vivo; and (b) that additional heparan sulfate storage is induced in vitro only by treatment with high concentrations that induce nonspecific cellular lesions.

Acridines↗
PubMed1996

Glycosaminoglycans in nerve injury: 1. Low doses of glycosaminoglycans promote neurite formation.

This study has shown that glycosaminoglycans added to the culture medium may affect neurite formation in SH-SY5Y neuroblastoma cells. The most effective glycosaminoglycans are heparin and COS 8, a preparation with low anticoagulant activity. Promotion of neuritogenesis was remarkable at concentrations as low as 10(-8) and 10(-10). When added at 10(-4) M both agents are inhibitory. Chondroitin-4 sulfate, dermatan sulfate, and heparan sulfate were also effective, the doses required were, however, as high as 10(-4) M for promoting and 10(-4) M for inhibiting neuritogenesis. Thereby low doses of glycosaminoglycans promote, while higher doses inhibit neurite formation. The effects were observed when neuritogenesis was promoted in neuroblastoma cultures either by deprivation of serum or by addition of retinoic acid, in the former case neuritogenesis occurred within 48 hr; in the latter, in 14 days. PC12 pheochromocytoma cells neuritogenesis was triggered by adding NGF to the culture medium. We have also observed that glycosaminoglycan supplementation to the culture medium lowered the quantity of NGF required to form neurites by PC12 cells. Glycosaminoglycans at the dose of 10(-8) M allow the formation of PC12 neurites even in presence of 1 ng/ml NGF, a dose that normally is ineffective.

Animals↗
PubMed2002

Analysis of glycosaminoglycan-derived disaccharides in biologic samples by capillary electrophoresis and protocol for sequencing glycosaminoglycans.

Glycosaminoglycans are biologically significant carbohydrates which either as free chains (hyaluronan) or constituents of proteoglycans (chondroitin/dermatan sulfates, heparin, heparan sulfate and keratan sulfate) participate and regulate several cellular events and (patho)physiological processes. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of glycosaminoglycans. Determination of compositional characteristics, such as disaccharide sulfation pattern, is a useful prerequisite for elucidating the interactions of glycosaminoglycans with matrix effective molecules and, therefore, essential in understanding the biological functions of proteoglycans. The interest in the field of characterization of such biologically important carbohydrates is soaring and advances in this field will signal a new revolution in the area of glycomics equivalent to that of genomics and proteomics. This review focuses on the capillary electrophoresis methods used to determine the disaccharide pattern of glycosaminoglycans in various biologic samples as well as advances in the sequence analysis of glycosaminoglycans using both chromatographic and electrophoretic techniques.

Carbohydrate Sequence↗
PubMed1993

Spectrophotometric method for the assay of glycosaminoglycans and glycosaminoglycan-depolymerizing enzymes.

Undegraded glycosaminoglycans form a complex with 1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphtho-[1,2-d]thiazolium bromide (Stains-all) in solution resulting in a characteristic shift in spectrum. Hyaluronic acid (a nonsulfated glycosaminoglycan) reacts with the dye to form a complex with an absorbance maximum at 650 nm, while sulfated glycosaminoglycans (chondroitin sulfates A and C, dermatan sulfate, keratan sulfate, and heparan sulfate) give rise to an increase in absorbance at 480 nm. Increases in absorbance at the appropriate wavelength are directly proportional to the concentration of glycosaminoglycan interacting with the dye. This phenomenon provided the basis for a sensitive spectrophotometric assay for the quantitative measurement of bacterial and mammalian glycosaminoglycan-depolymerizing enzymes. The basic assay method was adapted for use in 96-well microtiter trays, thus enabling large numbers of assays to be carried out simultaneously.

Biopolymers↗
PubMed1978

An improved determination of total glycosaminoglycans in body fluids by formation of complexes with quinacrine: changes in amniotic fluid total glycosaminoglycans during normal pregnancies and in pregnancies at risk for mucopolysaccharidoses.

Acidic glycosaminoglycans form insoluble complexes with quinacrine and this has been exploited for their analysis in blood, urine and amniotic fluid. The method is specific for glycosaminoglycans including keratan sulphate and the samples do not have to be deproteinized. Values for normal urine, serum and amniotic fluid are presented. Urinary total glycosaminoglycans excreted by patients with mucopolysaccharidoses were also determined. The normal changes in amniotic fluid total glycosaminoglycans have been measured between 14 weeks' gestation and term, and values are given for amniotic fluid total glycosaminoglycans in several pregnancies at risk for mucopolysaccharidoses. It is suggested that this method is a potentially valuable analytical tool in the pre-natal diagnosis of mucopolysaccharidoses.

Amniotic Fluid↗
PubMed2001

Release of glycosaminoglycans in physiological saline and water by wet-spun chitin--acid glycosaminoglycan fibers.

The present study sought to prepare novel chitin- acid glycosaminoglycan fibers that released a portion of the glycosaminoglycans in animal body fluids for use as novel biocompatible dressing materials (artificial skin) in the veterinary and clinical fields. A clear solution of sodium N-acetylchitosan salt (alkaline chitin) mixed with sodium hyaluronate, sodium heparin, sodium chondroitin 4-sulfate, sodium chondroitin 6-sulfate, or sodium dermatan sulfate in 14% aqueous NaOH was spun through a viscose-type spinneret into a 10% aqueous H(2)SO(4) solution saturated with (NH(4))(2)SO(4) at room temperature. The result was chitin fibers containing 5--33% glycosaminoglycans. In a dry state these fibers were white and had a soft feel but they were mechanically weak (0.31--0.69 g/denier tenacity and 3.1--10.6% elongation). Portions (85--97%) of the glycosaminoglycans were released from the fibers by soaking them in physiological saline or distilled water. Scanning electron microscopy analyses revealed a smooth striped surface on the original filaments, and a scaly surface appeared on the chitin filament after soaking.

Animals↗
PubMed1982

Analysis of glycosaminoglycans of flow sorted cells: incorporation of [35S]sulfate and [3H]glucosamine into glycosaminoglycans of B16-F10 cells during the cell cycle.

The incorporation of [35S]sulfate and [3H] glucosamine into cetyl pyridinium chloride (CPC) precipitable glycosaminoglycans was determined in B16-F10 cultured cells sorted with respect to DNA content. Incorporation into surface material was measured indirectly as the difference between the radioactivity of control and trypsin treated cells. Approximately 80% of the total cellular [35S] sulfate labeled CPC precipitable material, but only 5% of that labeled by [3H]glucosamine, was removed by this mild trypsin treatment. Incorporation of [35S]sulfate into the trypsin removable surface material increased progressively from G1 to S to F2 + M in both long-term (48 hours) and short-term (1 hour) labeled cells, while the ratio of surface to total incorporated [35S]sulfate remained relatively constant. Incorporation of [35S]sulfate into total cellular glycosaminoglycans in long- and short-term labeled cells increased as cells progressed from G1 to S to G2 + M; the incorporation of [3H]glucosamine into CPC precipitable material also increased progressively from G1 to S to G2 + M in long-term labeled cells but was greater during S phase relative to G1 or G2 + M in short-term labeled cells. The degree of sulfation of glycosaminoglycans as represented by the ratio of [35S]sulfate to [3H]glucosamine of double labeled cells was relatively constant in long-term labeled cells but was increased during the G1 and G2 + M phases of short-term labeled cells. Comparison of the degree of sulfation of short-term with long-term labeled cells suggests that highly sulfated glycosaminoglycans may be turned over more rapidly during G1 and G2 + M phases of the cell cycle.

Animals↗
PubMedDate not supplied

Effect of short- or long-term treatment with exogenous glycosaminoglycans on growth and glycosaminoglycan synthesis of human fibroblasts (WI-38) in culture.

Short-term (several days) or long-term (several weeks and months) treatment of cultured human diploid fibroblasts (WI-38; phase II) with heparin at 20--500 micrograms/ml inhibited cell proliferation and stimulated glycosaminoglycan synthesis (as measured by the incorporation rates of [35S] sulfate and [14C] glucosamine into cellular and medium glycosaminoglycans). Characterization of the individual glycosaminoglycan types revealed an increased portion of incorporated radioactivity in the heparan sulfate and hyaluronic acid fractions of heparin-treated cells. Treatment with chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate of hyaluronic acid at concentrations up to 500 micrograms/ml exhibited no or slightly inhibitory (especially in the case of hyaluronic acid) effects on growth and glycosaminoglycan synthesis. The average cellular protein and RNA content of short- or long-term heparin (100 micrograms/ml)-treated cells was elevated by about 70--80%. "Senescent" (phase III) WI-38 cells exhibited a relative increase of [35S] sulfate and [14C] glucosamine incorporation into cell-bound and medium heparan sulfate. Possible mechanisms for the action of heparin (for example, interaction with specific cell-surface sites) and a potential role of heparan sulfate in the regulation of cell growth are discussed.

Cell Division↗
PubMed2000

Structural analysis of glycosaminoglycans in animals bearing mutations in sugarless, sulfateless, and tout-velu. Drosophila homologues of vertebrate genes encoding glycosaminoglycan biosynthetic enzymes.

Mutations that disrupt developmental patterning in Drosophila have provided considerable information about growth factor signaling mechanisms. Three genes recently demonstrated to affect signaling by members of the Wnt, transforming growth factor-beta, Hedgehog, and fibroblast growth factor families in Drosophila encode proteins with homology to vertebrate enzymes involved in glycosaminoglycan synthesis. We report here the biochemical characterization of glycosaminoglycans in Drosophila bearing mutations in sugarless, sulfateless, and tout-velu. We find that mutations in sugarless, which encodes a protein with homology to UDP-glucose dehydrogenase, compromise the synthesis of both chondroitin and heparan sulfate, as would be predicted from a defect in UDP-glucuronate production. Defects in sulfateless, a gene encoding a protein with similarity to vertebrate N-deacetylase/N-sulfotransferases, do not affect chondroitin sulfate levels or composition but dramatically alter the composition of heparin lyase-released disaccharides. N-, 6-O-, and 2-O-sulfated disaccharides are absent and replaced entirely with an unsulfated disaccharide. A mutation in tout-velu, a gene related to the vertebrate Exostoses 1 heparan sulfate co-polymerase, likewise does not affect chondroitin sulfate synthesis but reduces all forms of heparan sulfate to below the limit of detection. These findings show that sugarless, sulfateless, and tout-velu affect glycosaminoglycan biosynthesis and demonstrate the utility of Drosophila as a model organism for studying the function and biosynthesis of glycosaminoglycans in vivo.

Amidohydrolases↗
PubMed1990

Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes.

Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, concentrations of PSGAG (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of PSGAG inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that PSGAG may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by PSGAG, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight PSGAG in the treatment of equine degenerative joint disease.

Animals↗
PubMed1976

The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) upon glycosaminoglycan synthesis in vivo.

The synthesis of total and unextractable glycosaminoglycans of rabbit epiphyseal growth plate was studied by determination of the incorporation in vivo of 35S-sulphate into various glycosaminoglycans separated by microfractionation procedures using CPC and ECTEOLA columns. In the narrow hypertrophic zone, hyaluronic acid and chondroitin sulphate of low molecular weight were found to be unextractable with 3M guanidinium chloride for 24 h, thus being firmly bound within the tissue. While the unextractable chondroitin sulphate showed a sharply reduced synthesis rate in the hypertrophic zone compared with that in the columnar and resting zones, a rapid increase in the synthesis of extractable chondroitin sulphate toward the calcification front is indicated. At microfractionation, solubility profiles indicated that the unextractable chondroitin sulphate was of predominantly lower molecular weight and/or charge density compared to that of the whole tissue, most evident in the hypertrophic zone. The distribution pattern of incorporated 35S showed a close correspondence to the distribution of the various chondroitin sulphate fractions both for extracted and unextracted tissue. In the hypertrophic zone, higher radioactivity was found for chondroitin sulphate of predominantly low molecular weight and/or charge density in extracted compared to unextracted tissue. Low doses of EHDP caused no change of distribution of the various glycosaminoglycans but generally inhibited their synthesis.

Animals↗
PubMed1975

[Preparative electrophoresis of peptidic glycosaminoglycans. Application to the fractionation of peptidic glycosaminoglycans of arterial wall (author's transl)].

A new procedure for the fractionation of glycosaminoglycans by electrophoresis on Pevikon has been described. Mixtures of glycosaminoglycans were fractionated by preparative electrophoresis on Pevikon in pyridine formate or glycine-HCl buffers. By this procedure, 200 mg of a mixture of hyaluronic acid, heparan sulphate, dermatan sulphate and chondroitin sulphate isolated from arterial wall could be successfully separated without loss of material. The purified fractions were analysed by enzymatic and chemical procedures. The molar ratios of uronic acid to hexosamine and of sulphate to hexosamine and the amino acid content of each glycosaminoglycan have been determined. The peptidic content (less than 1%) is represented by five amino acids, viz. serine, glycine, alanine, aspartic acid, and glutamic acid.

Amino Acids↗