scholarly journals Detection of secondary structure in glycosaminoglycans via the1H n.m.r. signal of the acetamido NH group

1982 ◽  
Vol 207 (1) ◽  
pp. 139-144 ◽  
Author(s):  
J E Scott ◽  
F Heatley
Keyword(s):  
Secondary Structure ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Periodate Oxidation ◽  
Close Approach ◽  
Dimethyl Sulphoxide ◽  
Space Filling ◽  
Keratan Sulphate ◽  
Inorganic Cations ◽  
Acid Glycosaminoglycans

Two simple methods for dissolving salts of acid glycosaminoglycans with inorganic cations (e.g. Li+ and Na+) in dry dimethyl sulphoxide are described. Complete n.m.r. spectra of, e.g., Na+ and Li+ salts of chondroitin sulphate and keratan sulphate were obtained on these solutions. In [2H6]dimethyl sulphoxide the NH resonance of 2-acetamido-2-deoxy hexosides is in the range 7.2-8.0 delta, but is downfield (8.3-9.3 delta) when the NH is H-bonded to -CO2-. Heparan sulphate shows two NH resonances, of which one (at 8.3 delta) is probably indicative of H-bonding. Space-filling models show that a very close approach of NH to -CO2- across the alpha-glucosaminidic bond is possible, and a solution configuration for heparan sulphate is proposed. The n.m.r. results are entirely compatible with interpretations of periodate-oxidation kinetics, based on H-bonded secondary structures present in hyaluronate and chondroitin sulphates, but not in dermatan (or keratan) sulphate.

scholarly journals Secondary structure of hyaluronate in solution. A 1H-n.m.r. investigation at 300 and 500 MHz in [2H6]dimethyl sulphoxide solution

1984 ◽  
Vol 220 (1) ◽  
pp. 197-205 ◽  
Author(s):  
J E Scott ◽  
F Heatley ◽  
W E Hull
Keyword(s):  
Secondary Structure ◽  
Hydrogen Bonds ◽  
Oxygen Atom ◽  
Glucuronic Acid ◽  
Chondroitin Sulphate ◽  
Chemical Shifts ◽  
Heparan Sulphate ◽  
Carbonyl Oxygen ◽  
Dimethyl Sulphoxide ◽  
Coupling Constants

The 1H-n.m.r. spectra of solutions in [2H6]dimethyl sulphoxide of the sodium salts of tetra-, hexa- and octa-saccharides prepared from hyaluronate by testicular-hyaluronidase digestion were examined at 300 and 500 MHz. The signals from hydroxy groups at positions 2 and 3 in the glucuronic acid moiety were assigned. Their chemical shifts and associated temperature-dependencies, as well as their coupling constants, depended on whether or not the uronic acid was at the non-reducing end. Deviations from the ‘normal’ pattern of hydroxy-group proton n.m.r. behaviour were attributable to participation in hydrogen bonds, either to the acetamido carbonyl oxygen atom or the pyranose ring oxygen atom of neighbouring N-acetylhexosamine moieties. A secondary structure, containing four different hydrogen bonds per trisaccharide unit of glucuronsyl-hexosaminyl-glucuronic acid, was demonstrated. This is the first complete and detailed secondary structure to be established for hyaluronate in any solvent. Hyaluronate is compared with chondroitin sulphate, dermatan sulphate, heparan sulphate and keratan sulphate in their potential to form secondary structures with features in common. The significance of the details of the structure to its overall stability, and the probability of their persistence into aqueous environments, are discussed. The presence of all or most of the secondary structure in glycosaminoglycuronans is correlated with a space-filling function in the tissue, and with a high carbohydrate content in the parent proteoglycan in the case of the chondroitin sulphates.

scholarly journals 1H nuclear-magnetic-resonance spectra of the methyl group of the acetamido moiety and the structure of acid glycosaminoglycans in solution

1979 ◽  
Vol 181 (2) ◽  
pp. 445-449 ◽  
Author(s):  
J E Scott ◽  
F Heatley
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Heparan Sulphate ◽  
Periodate Oxidation ◽  
Evidence Based ◽  
Keratan Sulphate ◽  
Methyl Group ◽  
Nuclear Magnetic Resonance Spectra ◽  
1H Nuclear Magnetic Resonance ◽  
Acid Glycosaminoglycans ◽  
Chemical Evidence

The 1H resonances of the methyl group in the acetamido moiety of several types of glycosaminoglycans are reported at 300 MHz in 2H2O. Dermatan sulphates with various L-iduronate/D-glucuronate ratios are compared with chrondroitin sulphates with various contents and positions of substitution of sulphate esters. Hyaluronate oligomers are compared with 2-acetamido-2-deoxy-D-glucose, and with heparan sulphate and keratan sulphate. The major determinant of the chemical shift of the acetamido methyl resonance is the closeness of approach between carboxy groups and the acetamido group, in agreement with chemical evidence based on periodate-oxidation kinetics.

scholarly journals Secondary Structure of Chondroitin Sulphate in Dimethyl Sulphoxide

1983 ◽  
Vol 130 (3) ◽  
pp. 491-495 ◽  
Author(s):  
John E. SCOTT ◽  
Frank HEATLEY ◽  
Malcolm N. JONES ◽  
Allan WILKINSON ◽  
Anthony H. OLAVESEN
Keyword(s):  
Secondary Structure ◽  
Chondroitin Sulphate ◽  
Dimethyl Sulphoxide

scholarly journals A water molecule participates in the secondary structure of hyaluronan

1988 ◽  
Vol 254 (2) ◽  
pp. 489-493 ◽  
Author(s):  
F Heatley ◽  
J E Scott
Keyword(s):  
Secondary Structure ◽  
Water Molecule ◽  
High Field ◽  
Cell Membranes ◽  
Proton Exchange ◽  
Dimethyl Sulphoxide ◽  
Molecular Models ◽  
Saturation Transfer ◽  
Water Bridge ◽  
Space Filling

The structure of hyaluronan was investigated in water/dimethyl sulphoxide mixtures by using high-field n.m.r. and space-filling molecular models. The secondary structure previously established in detail in ‘dry’ dimethyl sulphoxide [Heatley, Scott & Hull (1984) Biochem. J. 220, 197-205] undergoes changes on addition of water, compatible with the incorporation of a water bridge between the uronate carboxylate and acetamido NH groups. Molecular models show that such a configuration is highly probable, and saturation-transfer experiments yield rates of NH proton exchange that support this proposed structure. The existence of two distinct stable configurations for hyaluronan, in water-rich and water-poor conditions respectively, may have biological implications, e.g. during its biosynthesis in cell membranes. There are extensive hydrophobic regions in both forms, which may be important for interactions with e.g., membranes, proteins and itself.

scholarly journals Synthesis of glycosaminoglycans by human embryonic lung fibroblasts. Different distribution of heparan sulphate, chondroitin sulphate and dermatan sulphate in various fractions of the cell culture

1977 ◽  
Vol 167 (2) ◽  
pp. 383-392 ◽  
Author(s):  
Ingrid Sjöberg ◽  
Lars-Åke Fransson
Keyword(s):  
Glucuronic Acid ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Periodate Oxidation ◽  
Exchange Chromatography ◽  
Lung Fibroblasts ◽  
Relative Distribution ◽  
Dermatan Sulphate ◽  
Human Embryonic Lung ◽  
Iduronic Acid

Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate 35SO42− for various periods of time. 35S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO2 and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various 35S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The 35S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the 35S-labelled galactosaminoglycans were extensively degraded by periodate oxidation–alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Åberg & Fransson (1975) Biochem. J.151, 477–489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected.

scholarly journals The nature of the non-ultrafilterable glycosaminoglycans of normal human urine

1971 ◽  
Vol 122 (3) ◽  
pp. 373-384 ◽  
Author(s):  
E. Wessler
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Molar Ratios ◽  
Sodium Barbital ◽  
Normal Human ◽  
Normal Men ◽  
Barbital Buffer ◽  
Acidic Glycosaminoglycans

1. The non-ultrafilterable acidic glycosaminoglycans from pooled urine of normal men, aged about 20, were isolated and characterized. The isolation procedure included digestion with sialidase and pronase, and fractionation by stepwise elution from an ECTEOLA-cellulose column. The glycosaminoglycans in each fraction were separated from each other by preparative electrophoresis in sodium barbital buffer and in barium acetate. 2. Approximate relative amounts of the different glycosaminoglycans were: chondroitin sulphate 60%, chondroitin 2%, hyaluronic acid 4%, dermatan sulphate 1%, heparan sulphate 15% and keratan sulphate 18%. Chondroitin sulphate–dermatan sulphate hybrids seemed to occur in trace amounts. 3. Chondroitin sulphate, heparan sulphate and keratan sulphate were heterogeneous with respect to degree of sulphation. Two distinct groups of chondroitin sulphate fractions were found, with sulphate/hexosamine molar ratios of about 0.5 and 1 respectively. The sulphate/hexosamine molar ratios in the heparan sulphate fractions varied from 0.5 to 0.9; the N-sulphate/hexosamine ratio was about 0.5 in all fractions. The sulphate/hexosamine molar ratios in the keratan sulphate fractions varied from 0.2 to 0.7.

PRODUCTION AND CHARACTERISATION OF MONOCLONAL ANTIBODIES AGAINST HEPARIN Deborah

1987 ◽  
Author(s):  
A Rathjen ◽  
Carolyn L Geczy
Keyword(s):  
Monoclonal Antibodies ◽  
Antithrombin Iii ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Tissue Level ◽  
Cross Reactivity ◽  
Keratan Sulphate ◽  
Cell Mediated Immune Response ◽  
At Iii ◽  
The Many

To complement the studies using MAbs to AT III and because of the reported ability of heparin to modulate several aspects of the cell-mediated immune response, we have prepared two mouse monoclonal antibodies (MAbs) to porcine mucosal heparin.MAb 25/15 is an IgGl and MAb 26/7 is an IgM. Both MAbs have iso-electric points between pH5.85 and 6.55. The MAbs recognise porcine and bovine mucosal heparin and rat mast cell heparin. Heparins with both high and low affinitiesfor antithrombin III (ATIII) bound both MAbs but neither MAb altered the binding of heparin to AT III. These antibodies did not recognise other proteoglycans (chondroitin sulphate types A, B and C, keratan sulphate and hyaluronic acid) with the exception of heparan sulphate, (the cellular equivalent of heparin) and Arte- paron (Luitpold-Werk, Munchen; a synthetically poly- sulphated chondriotin sulphate), in competition and solid-phasebinding assays. Dextransulphate(Pharmacia) was also recognised by these MAbs. Cross-reactivity with Arteparon and dextran sulphate indicate that charged sulphate goups on the mucopolysaccharides may be importantBfor MAb binding. The Mabs described may beuseful probes for endogenous heparin at the cellular and tissue level and may allow further investigation of the many biological activitiesof heparin

scholarly journals Absence of keratan sulphate from skeletal tissues of mouse and rat

1985 ◽  
Vol 228 (2) ◽  
pp. 443-450 ◽  
Author(s):  
G Venn ◽  
R M Mason
Keyword(s):  
Monoclonal Antibody ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Lumbar Disc ◽  
Heparan Sulphate ◽  
Costal Cartilage ◽  
Animal Experiments ◽  
Keratan Sulphate

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.

scholarly journals Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1970 ◽  
Vol 119 (5) ◽  
pp. 885-893 ◽  
Author(s):  
T. E. Hardingham ◽  
C. F. Phelps
Keyword(s):  
Uronic Acid ◽  
Heparan Sulphate ◽  
Neonatal Rat ◽  
Rat Skin ◽  
Acid Biosynthesis ◽  
Keratan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Acid Glycosaminoglycan ◽  
Acid Glycosaminoglycans

1. The incorporation of [35S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-14C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-14C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-14C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [35S]sulphate and were in agreement with those from 14C-labelling studies.

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