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At least 19 recordsLinked to original sources

PubMed1978

Micro determination of unsaturated disaccharide formed by the action of acidic glycosaminoglycan-endoeliminases. An application of the thiobarbituric acid method to the assay of D-gluco-4-enepyranosyluronic acid-containing disaccharides.

An improved method is described for the micro determination of acidic glycosaminoglycans after digestion with chondroitinase-ABC and -AC. The determination is based on the color production of D-gluco-4-enepyranosyluronic acid-containing disaccharides produced by the action of chondroitinase-ABC and -AC (acidic glycosaminoglycans-endoeliminase) when the periodate-thiobarbituric acid method is applied to the alpha,beta-unsaturated disaccharides. Suitable conditions for the quantitative assay are described.

Chondroitin Sulfates↗

PubMed1979

Identification of N-sulphated disaccharide units in heparin-like polysaccharides.

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.

Chemical Phenomena↗

PubMed1991

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.

Carbohydrate Sequence↗

PubMed1991

Capillary electrophoresis for the analysis of chondroitin sulfate- and dermatan sulfate-derived disaccharides.

High-voltage capillary zone electrophoresis (CZE) has been used for the first time in the analysis of non-, mono-, di-, and trisulfated disaccharides derived from chondroitin sulfate, dermatan sulfate, and hyaluronic acid. These glycosaminoglycans are first depolymerized using polysaccharide lyases. The resulting unsaturated disaccharide products can be detected by their ultraviolet absorbance at 232 nm. Different retention times were obtained for each unsaturated disaccharide analyzed by CZE. The application of a constant voltage across a 70-cm fused silica capillary using a single, simple buffer system resolved an eight-component mixture within 40 min. Quantitation of disaccharides derived from chondroitin sulfate using chondroitin ABC lyase (EC 4.2.2.4) and mixtures of unsaturated disaccharide standards was possible requiring only picogram quantities of sample. The disaccharides examined had a net charge of from -1 to -4 and were resolved primarily on the basis of net charge and secondarily on the basis of charge distribution. Two unsulfated disaccharides both containing the same unsaturated uronic acid residue were analyzed. One was from chondroitin having an N-acetylgalactosyl residue and one from hyaluronate having an N-acetylglycosyl residue. Despite the fact that they differed only by the chirality at one center, these disaccharides were resolved by CZE. CZE is a fast and simple method that represents a powerful new tool for analysis and separation of acidic disaccharide components of glycosaminoglycans.

Carbohydrate Sequence↗

PubMed1979

Gas chromatography and mass spectrometry of disaccharides from glycoproteins.

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 microgram disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples.

Animals↗

PubMed1975

Demonstration of a specific metabolic effect of dietary disaccharides in the rat.

Male Wistar rats were starved and refed diets containing either 40% carbohydrate as monosaccharides (glucose, fructose, invert sugar) or disaccharides (maltose, sucrose), or 42.2% carbohydrate as glucose. Induction of various liver enzymes and changes in total liver lipid levels by the different dietary sugars were studied. Liver enzymes measured included glucose-6-phosphate dehydrogenase (g6pd), 6-phosphogluconate dehydrogenase (6PGD), malic enzyme (ME), phosphofructokinase (PFK), L-alpha-glycerol phosphate dehydrogenase (LalphaGPD), pyruvate kinase (PK), citrate cleavage enzyme (CCE), acetyl CoA carboxylase (AcCoAC), and fatty acid synthetase (FAS). The responses in enzyme activity to diets containing glucose or invert sugar were used as the basal response. Enzyme responses to refeeding the carbohydrate diets fell into three categories: (1) enzyme activity increased both by the disaccharide configuration of the carbohydrate and by fructose (G6PD, PK, CCE, AcCoAC, FAS); (2) enzyme activity increased only by the disaccharide configuration of the carbohydrate (6PGD, ME); and (3) enzyme activity increased only by fructose (PFK, LalphaGPD). Total liver lipid level was increased both by the disaccharide configuration of the carbohydrate and by fructose. Refeeding diets containing equal molar amounts of glucose or maltose did not abolish the disaccharide effect. The data indicate that the disaccharide configuration of maltose and sucrose may have an effect at the gastrointestinal level, which causes an increased induction of certain enzymes in the liver.

ATP Citrate (pro-S)-Lyase↗

PubMed1991

Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Gal beta 1-3GlcNAc) or type 2 (Gal beta 1-4GlcNAc) disaccharide units.

The adhesion of Pseudomonas aeruginosa to type 1 (Gal beta 1-3GlcNAc) and type 2 (Gal beta 1-4GlcNAc) disaccharide determinants was studied in a microtiter adhesion assay and a thin-layer chromatography bacterial overlay assay. The oligosaccharides were prepared from human breast milk and human urine and were conjugated to hexadecylaniline to form neoglycolipids that were used in were used in the assays. Both the mucoid and the nonmucoid strains that were studied recognized the disaccharide determinants Sialylation of the oligosaccharides did not suppress binding in the thin-layer chromatography assay, but alpha 2-6-linked sialic acid blocked binding in the microtiter assay. The use of bovine serum albumin instead of gelatin as a blocking agent against nonspecific binding completely suppressed binding in the thin-layer chromatography assay. Isogenic nonpiliated mutants of nonmucoid strains constructed by interrupting the pilin gene retained their adhesive capacity for the disaccharide units, indicating that binding to the disaccharides was mediated by a nonpilus adhesin(s). Furthermore, two monoclonal antibodies that recognize the type 2 disaccharide determinant (Gal beta 1-4GlcNAc) partially inhibited adhesion of a pair of piliated and nonpiliated isogenic strains to mucin. This study suggests that P. aeruginosa utilizes a nonpilus adhesin(s) to bind to disaccharide units commonly found in mucins, in addition to pili and alginate, two previously described adhesins.

Amino Sugars↗

PubMed1977

The micro-assay of the disaccharide isomers of chondroitin sulphate.

Connective tissue extracts containing the 4- and 6-sulphated isomers of chondroitin sulphate can be measured at the microgram level (approximately 10 microgram) using colorimetric assays. The chondroitin sulphates are depolymerised to disaccharides using chondroitin ABC lyase (EC 4.2.2.4). The 4- and 6-sulphated disaccharides after treatment with acid periodate are determined using a thiobarbituric acid procedure. An alkaline borate p-dimethylaminobenzaldehyde reaction is used to measure 6-sulphated disaccharide, the 4-sulphated disaccharide not forming a chromogen. Unsulphated disaccharide causes interference in both assays and if present is separated prior to assay using paper electrophoresis fractionation.

Chondroitin↗

PubMed1992

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.

Animals↗

PubMed1979

Structure of the disaccharide chain of galactosyl-N-acetylgalactosaminyl-protein synthesized in vitro.

In order to obtain a [14C]galactosyl-N-acetylgalactosaminyl-protein which would be useful as an acceptor in studies on the specificity of glycosyltransferases, a porcine submaxillary gland microsomal galactosyltransferase preparation was used for the galactosylation in vitro of N-acetylgalactosaminyl-protein (desialylated ovine submaxillary mucin). The newly formed oligosaccharide unit was obtained as a reduced disaccharide after alkaline borohydride treatment of the [14C]galactosyl-N-acetylgalactosaminyl-protein product and as glycopeptides by proteolytic digestion of the glycoprotein. The reduced disaccharide consisted of equimolar amounts of galactose and N-acetylgalactosaminitol and was characterized by thin-layer chromatography, high-voltage electrophoresis and gas-liquid chromatography. Periodate oxidation studies on the reduced disaccharide revealed that [14C]galactose was linked to position C-3 on the N-acetylgalactosaminyl residue. Digestion of the reduced disaccharide and the glycopeptides with galactosidases gave equivocal results as to the anomeric configuration of the [14C]galactose residue. Nuclear magnetic resonance of the reduced disaccharide, however, definitely indicated that the configuration was beta. The specificity of the porcine submaxillary gland galactosyltransferase thus can be defined as a uridine diphosphogalactose: alpha-D-N-acetylgalactosaminyl-protein beta 1 leads to 3 transferase activity.

Acetylgalactosamine↗

PubMed1977

Specificity of the disaccharide effect in the rat.

Male rats were starved and refed diets containing 40% disaccharides (maltose, trehalose, sucrose, turanose), trisaccharide (melezitose), starch, or the monosaccharide equivalents. Responses of hepatic glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), fatty acid synthetase (FAS), and total liver lipid (TLL) or changes in concentration of portal blood total carbohydrate and fructose were determined following refeeding of the different carbohydrate diets. Maltose, trehalose, sucrose, and turanose refeeding resulted in G6PD and ME responses which were higher than the responses to their component monosaccharides (disaccharide effect). Starch refeeding decreased the responses of G6PD, ME, and FAS when compared to refeeding of glucose. Refeeding diets containing fructose (sucrose, turanose, melezitose, and monosaccharide equivalents containing fructose) increased the responses of G6PD, ME, FAS, and TLL. No correlation between portal blood carbohydrate concentration and hepatic enzyme levels could be demonstrated. It is concluded that readily digestible disaccharides produce an effect which is greater than the effect produced by their monosaccharide equivalents. If these disaccharide configurations are fed as part of a trisaccharide or polysaccharide, the disaccharide effect is no longer discernible.

Animals↗

PubMed1976

Interpretation of disaccharide-dependent electrical potential differences in the small intestine.

The nature of maltose- and trehalose-induced electrical potential increments across everted small intestines of toads were investigated. A Michaelis-Menten-like relation was seen between the amplitude of PD increments (deltaPD) and the mucosal concentration of disaccharides over a wide range of concentration, but, at a higher concentration range, Lineweaver-Burk type plot of data always deviated from the linear line for the low and moderate concentration range. The extrapolation of the linear segment of the plots intercepted the ordinate at the same point as that of the line for the glucose-induced potential increments. Both the disaccharide- and the glucose-evoked potentials were not additive and were equally sensitive to phlorizin. Tris depressed the disaccharide-evoked potentials to about the same extent as that of inhibition of enzyme activities. The amplitude and time course of the disaccharide-induced potentials, however, could not be accounted for by the mucosal concentration of liberated glucose. These qualitative and quantitative characteristics were explained properly on the basis of a simple well-type local pool for liberated glucose assumed to exist at the brush border. In conclusion, a close functional linkage between brush border membrane disaccharidase activities and the electrogenic hexose transport is well reflected in the disaccharide-evoked potentials in the small intestine.

Animals↗

PubMed1992

Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1.

Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various linkage region oligosaccharide alditols were derived from these chains using sequential chondroitinase digestions (ABC lyase followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed the 4,5-unsaturated uronosyl residue from the nonreducing end of the linkage, and then beta-galactosidase digestion which liberated the 2 galactose residues from the xylitol reducing terminus. Alkaline phosphatase digestions were performed to verify the presence of phosphate esters. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1 chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from aggrecan synthesized by rat chondrosarcoma cells in vitro have the following properties: 1) three out of every four of their linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharide region with an unsulfated first disaccharide unit, 3) nearly twice as many nonphosphorylated chains have a sulfated first disaccharide than their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose in their linkage regions. This report also describes a borohydride reduction procedure to confer alkali stability to the 3-substituted, unsaturated disaccharides derived from chondroitinase digests of chondroitin sulfate. Furthermore, a CarboPac PA1 method is demonstrated that separates these reduced disaccharides with exceptional resolution.

Animals↗

PubMed1976

Stimulation of the gerbil's gustatory receptors by disaccharides.

The gustatory responses from the chorda tympani nerve of the Mongolian gerbil, Meriones unguiculatus, were treated with 13 disaccharides. Sucrose was the most stimulatory sugar. The ability of fructosyl glycosides to stimulate may depend upon the linkage between fructose and the glycoside. Disaccharides possessing 1 leads to 3, 1 leads to 4, or 1 leads to 6 linkages were poor stimuli compared to sucrose which has a 1 leads to 2 linkage. Glucopyranosyl disaccharides with an alpha-linkage were better stimuli than the beta-anomers, while galactopyranosyl disaccharides possessing a beta-linkage were better than their alpha-amoners.

Animals↗

PubMed1992

Preparation of unsaturated disaccharides by eliminative cleavage of heparin and heparan sulfate with heparitinases.

1. Six kinds of unsaturated disaccharides were prepared by enzymatic digestion of heparin and heparan sulfate with heparitinases I0 and IV, and subsequent column chromatography. They were identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction was determined colorimetrically, and found to range from 130.7 to 722.3 mumol. 3. Molecular extinction coefficient of each unsaturated disaccharide was calculated from absorbance at a wavelength of around 230 nm where a peak appeared on the ultraviolet spectrum of each disaccharide solution at pH 2. The values varied from 6000 to 6600.

Animals↗

PubMed1977

Determination of the linkages of disaccharides containing a 2-acetamido-2-deoxy sugar unit by solvent effects in circular dichroism.

The circular dichroism spectra of 2-acetamido-2-deoxy sugars in 1,1,1,3,3,3-hexafluoro-2-propanol (F6Pr-2-ol) solutions show a positive band in the n-pi region (209 nm) in contrast to a negative c.d. band in water solution. This difference is interpreted as an indication of a change in the average orientation of the hydroxyl groups adjacent to the amide group. C.d. spectra of 2-acetamido-2-deoxy sugars having a methyl group at O-1 and O-3 confirm this interpretation and suggest that the c.d. spectrum of a disaccharide in F6Pr-2-ol reflects strongly the disaccharide linkage. Large differences in the c.d. spectra of (1 leads to 4) and (1 leads to 6)-linked disaccharides in this solvent lead to rules for distinguishing the linkages of the disaccharides.

Acetylgalactosamine↗

PubMed1992

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.

Carbohydrate Conformation↗

PubMed1992

Disaccharide analysis of the skin glycosaminoglycans in systemic sclerosis.

The disaccharides constituting chondroitinase-digestible glycosaminoglycan (GAG) in the skin lesions of patients with systemic sclerosis were determined using high-performance liquid chromatography (HPLC). In scleroderma there was an increase in the amount of delta Di-4S(DS), the main disaccharide unit of dermatan sulphate, and a decrease in delta Di-HA, the disaccharide unit of hyaluronic acid, as compared with normal skin from a similar site. The distribution pattern of the main disaccharides constituting chondroitin sulphate and dermatan sulphate in scleroderma differed from that in scars or scleredema.

Adult↗