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PubMed1997

Effect of water on lamellar structure of DPPC/sugar systems.

The ability of two monosaccharides, four disaccharides and one trisaccharide, to lower the transition temperature of L-alpha-dipalmitoyl phosphatidylcholine (DPPC) was investigated using differential scanning calorimetry (DSC) and the ability of these sugars to change the lateral packing of the acyl chains of DPPC was investigated using wide-angle X-ray diffraction. The sugars affected the gel-liquid crystal transition temperature (Tc) of DPPC when the water content of the DPPC/sugar systems was less than 20 wt.%. Specifically, Tc of the DPPC without sugar increased to approximately 106 degrees C, the Tc of the DPPC/monosaccharide system remained almost constant at 43 degrees C and of the DPPC/disaccharide or trisaccharide systems decreased to approximately 24 degrees C. In the dehydrated state, di- and trisaccharides caused looser packing of the DPPC hydrocarbon chains than the monosaccharides did, and the sugars affected the packing mode in different ways. The addition of water caused this difference in the sugars' effects on the packing mode to disappear and further addition of water caused the effect of sugar to almost disappear. Thus, the addition of water to a DPPC/sugar system weakens the interaction between the sugar and lipid and strengthens the DPPC chain packing.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Activation of heparin cofactor II by heparin oligosaccharides.

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.

Animals↗
PubMed2001

The occurrence of internal (1 --> 5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances.

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.

Arabinose↗
PubMed1986

Relationship between the structure and the properties of carbohydrates in aqueous solutions: sweetness of chlorinated sugars.

The structural basis of the sweet taste of D-galactose, D-glucose, D-mannose, sucrose, and some of their chlorinated derivatives has been derived from an interpretation of their F.t.-i.r. spectra. AH-B glucophores are proposed in the light of the observed OH vibrations, and an explanation of the differences in sweetness of the monosaccharides is proposed. The hydrophobic character of the CH2Cl, "gamma" centre in the tripartite template does not seem to play the same role in monosaccharides and 4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose. The enhancement of sweetness in the disaccharide derivative is due to the enhanced hydrophobicity of the CH2Cl groups as well as to specific interactions with water. A sharp i.r. absorption characteristic of free hydroxyl is found in the spectra of most of the very sweet polyhydroxy compounds.

Deoxyglucose↗
PubMed1994

Substrate specificities of glycosyltransferases involved in formation of heparin precursor and E. coli K5 capsular polysaccharides.

The E. coli K5 capsular polysaccharide is composed of 4)GlcpA(beta 1-4)GlcpNAc(alpha 1-disaccharide units. A partially N-deacetylated/N-sulfated heptasaccharide, derived from this polymer and having a nonreducing terminal GlcNAc unit, was used as acceptor for a mastocytoma microsomal GlcA-transferase involved in heparin biosynthesis. An octasaccharide with nonreducing-terminal GlcA similarly served as acceptor for the microsomal GlcNAc-transferase. Analysis of the labeled octa- and nona-saccharides formed by transfer of monosaccharide units from UDP-[14C]GlcA and UDP-[3H]GlcNAc, respectively, showed that both glycosyltransferases could utilize partially N-sulfated acceptors. The GlcA-transferase showed a marked preference for a terminal GlcNAc-GlcA-GlcNSO3-sequence, particularly when this sequence was followed by an additional N-sulfated disaccharide unit. Enzymes catalyzing the same GlcA and GlcNAc transfer reactions were solubilized from E. coli K5 membranes. The K5 capsular polysaccharide, like the heparin/heparan sulfate precursor polysaccharide, thus probably grows by stepwise, alternating addition of the two constituent monosaccharide units, from the corresponding UDP-sugars, to the nonreducing ends of the chains. Moreover, the bacterial glycosyltransferases utilized the same partially N-sulfated oligosaccharide substrates as the mammalian enzymes, and with similar preference for N-sulfate groups in certain positions.

Animals↗
PubMed2002

Effect of (1-->3)- and (1-->4)-linkages of fully sulfated polysaccharides on their anticoagulant activity.

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.

Alcaligenes↗
PubMed2003

Effect of molecular structure on the conductivity of amorphous carbohydrate-water-KCl mixtures in the supercooled liquid state.

The effect of carbohydrate structure on the conductivity of low water content amorphous carbohydrate-water, and carbohydrate-water-KCl mixtures, has been measured using both direct current and alternating current techniques at temperatures in the supercooled liquid and glassy range, ranging from -40 to 80 degrees C. The structures included homologous mono-, di- and trisaccharides (glucose, maltose and maltotriose), a monosaccharide with no exocyclic hydroxymethyl group (xylose) and a second trisaccharide (raffinose). The KCl-mixtures contained 9.3% w/w water and 0.74% w/w KCl which resulted in calorimetric glass transition temperatures, T(g), in the range -29-19 degrees C. At this concentration conduction due to KCl dominated that due to intrinsic conductors originating from the carbohydrates and water. In the supercooled liquid region, as temperature, T, is reduced to T(g), the activation energy of the molar conductivity of KCl, Lambda(m), increased as described by a Vogel-Tamman-Fulcher-type equation, Lambda(m)=Lambda(m0)exp[B/(T-T(0))], where Lambda(m0), B and T(0) are constants. Comparison of the molar conductivity of KCl in the carbohydrate mixtures at T(g) with that in aqueous solutions showed that conductivity is, to varying extents, uncoupled from viscosity. The uncoupling increased in the order D-xylose<D-glucose<maltose<maltotriose and raffinose. The results suggest that the primary structural characteristic determining conductivity is molecular weight, though the presence of the exocyclic hydroxymethyl group in the monosaccharide also has an effect. Whilst at T(g) the D-xylose mixture had the lowest conductivity, at a particular temperature the trisaccharide mixtures of maltotriose and raffinose had the lowest conductivities.

Carbohydrates↗
PubMed2000

An efficient access to protected disialylated glycohexaosyl threonine present on the leukosialin of activated T-lymphocytes.

The total synthesis of the threonine-linked core 2 class disialylated hexasaccharide in a completely protected form was accomplished for the first time. The L-threonine conjugate, N-(9-fluorenylmethoxycarbonyl)-O-[(5-acetamido-4,7,8,9-tetra-O-ben zyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2-acetam ido-2-deoxy-3,6-di-O-benzyl-beta-D-glucopyranosyl-(1-->6)-[(5-acetamido- 4,7,8,9-tetra-O-benzyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo pyranosylonic acid)-(2-->3)-2,6-di-O-benzyl-beta-D-galactopyranosyl-(1-->3)]-2-acetami do-2-deoxy-alpha-D-galactopyranosyl-(1d-->4c:1f-->4e)-dilactone ]-L-threonine allyl ester was synthesized via stereocontrolled glycosylations employing readily accessible monosaccharidic blocks; t-butyl-diphenylsilyl-2-azido-2-deoxy-3,6-di-O-benzyl-beta-D-gluco pyranose, N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-6-O-t-butyldimethylsilyl -2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester, 8, 9 and N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzylidene-3-O-ch loroacetyl-2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester. For the introduction of the amino acid, the azide group was used to temporarily mask the amino group of GalNAc so as to obtain an alpha-glycosidic linkage without participation from the C-2 substituent. The threonine was attached to the sugar unit at the monosaccharide stage to avoid loss of oligosaccharide at a later stage. The Fmoc and allyl ester protected amino acid at the reducing end facilitates efficient glycopeptide synthesis on solid-phase support.

Antigens, CD↗
PubMed1997

The effect of delta-gluconolactone, an oxidised analogue of glucose, on the nonenzymatic glycation of human and rat haemoglobin.

Nonenzymatic glycation of proteins and oxidative stress are considered independent factors important in the development of the complications of diabetes but may be interrelated by the process of autoxidative glycation. This pathway involves monosaccharide autoxidation to a reactive ketoaldehyde analogue and subsequent reaction with protein to form a ketoimine adduct. This study demonstrates that delta-gluconolactone (delta-GL), an oxidised analogue of glucose, is a potent glycating agent in vitro of haemoglobin present in blood samples from insulin-dependent diabetic and non-diabetic human subjects and from spontaneously diabetic, insulin-dependent BB/Edinburgh (BB/E) rats. The percentage glycated haemoglobin after incubation (37 degrees C, 5 h) with delta-GL (25 mmol/l) was significantly (P < 0.002) higher than that observed using an equimolar concentration of glucose. Intravenous administration of delta-GL (1 g/kg) to non-diabetic BB/E rats also significantly increased glycation of haemoglobin (6.0 +/- 0.1% vs 4.9 +/- 0.1%, P < 0.01) whereas intravenous injection of an identical dose of glucose had no significant effect (5.1 +/- 0.1% vs 5.0 +/- 0.2%). These results support the hypothesis that nonenzymatic glycation of proteins involves attachment by both native and oxidised monosaccharides. Further investigation of the interactions between diabetes-associated increases in oxidative stress and glycation on the development and progression of the vascular complications of diabetes is necessary.

Animals↗
PubMed2001

Routine o-glycan characterization in nutritional supplements--a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation.

Analytical procedures, including capillary isoelectric focusing (CIEF), high-performance anion-exchange chromatography coupled to amperometric detection (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinomacropeptide (CGMP) and the detection of subtle glycosylation differences between CGMP Batches obtained with two different preparation procedures. Modified two-step CIEF allowed monitoring of glycopeptide heterogeneity and determination of the isoelectric points of acidic glycoforms. The mixture of wide and narrow pH range ampholytes was optimized to improve glycoform resolution. The pI of the different CGMP glycoforms was evaluated with pI internal standards and found to range between 3.08 and 3.58, which indicates a very acidic glycopeptide. Moreover, the monosaccharide composition was determined with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Batches I and II, but the monosaccharide percentages were 3-4 fold higher in Batch I, particularly for galactose and glucose. This likely reflects a higher content in lactose in the case of Batch I. Finally, O-linked oligosaccharides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then analyzed by normal-phase HPLC coupled with fluorescence detection, and separated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylated than Batch II. Additionally, the sizes of the separated glycans, expressed as the number of glucose units, were tentatively assigned using calibration with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studying glycoprotein heterogeneity and assessing batch-to-batch consistency.

Animals↗
PubMed1991

Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 9-13) separates aminosaccharides, neutral saccharides and glycuronic acids based upon their molecular size, saccharide composition and glycosidic linkages. Carbohydrates were extracted by utilizing 0.5 M H2SO4 (neutral monosaccharides), 0.25 M H2SO4 coupled with enzyme catalysis (glycuronic acids) and 3 M H2SO4 (aminosaccharides). Solid-phase extraction with strong cation and strong anion resins was used to partition the cationic aminosaccharides and anionic glycuronic acids and to deionize acid extracts for neutral saccharides. Separation was conducted on a medium-capacity anion-exchange column (36 mequiv.) utilizing sodium hydroxide (5-200 mM and sodium acetate (0-250 mM) as the mobile phase. The saccharides were detected by oxidation at a gold working electrode with triple-pulsed amperometry. HPAEC-PAD was found superior to high-performance liquid chromatography with refractive index (RI) detection for neutral monosaccharides and aminosaccharides and to low-wavelength UV detection for glycuronic acids in terms of resolution and sensitivity. HPAEC-PAD was not subject to interferences as was the case for low UV detection (210 nm) or RI analyses and was highly selective for mono- and aminosaccharides and glycuronic acids. The use of HPAEC-PAD was applied for the determination of the saccharide composition of organic materials (plant residues, animal wastes and sewage sludge), microbial polymers and soil.

Animals↗
PubMed1990

Sensitive blotting assay for the detection of glycopeptides in peptide maps.

A dot blotting assay using digoxigenin hydrazide (Glycan detection kit, Boehringer Mannheim Biochemicals) was used to screen an endoproteinase Lys-C peptide map of ribonuclease B for the presence of glycopeptides. The carbohydrate content of the identified glycopeptide fraction was then further characterized by monosaccharide analysis using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). The tandem use of a hydrazide dot blotting technique to screen peptide maps for glycopeptides and subsequent use of HPAE-PAD to identify the monosaccharide composition of glycopeptide hydrolyzates proved to be a quick, sensitive and reliable method for identifying glycopeptides and analyzing their glycan composition without derivatization of the carbohydrate.

Animals↗
PubMed2002

Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides.

A high-throughput method is described by which Arabidopsis thaliana stems can be screened for variation in cell wall composition after hydrolysis with Driselase or trifluoroacetic acid (TFA). Driselase, a mixture of fungal enzymes, hydrolyses cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides); TFA hydrolyses the matrix polysaccharides, but not cellulose, to monosaccharides. Two different wild-type ecotypes, Columbia and Wassilewskija, showed only minor differences in wall carbohydrate composition. A small number of T-DNA-tagged populations that were screened contained individuals in which the proportion of cellulose, xyloglucan or xylan differed quantitatively from the wild-type. Differences from the wild-type were also observed in the susceptibility of the hemicelluloses to hydrolysis by Driselase, probably reflecting differences in wall architecture.

Acids↗
PubMed2005

Methods in enzymology: O-glycosylation of proteins.

Cell surface and extracellular proteins are O-glycosylated, where the most abundant type of O-glycosylation in proteins is the GalNAc attachment to serine (Ser) or threonine (Thr) in the protein chain by an a-glycosidic linkage. Most eukaryotic nuclear and cytoplasmic proteins modified by a-linked O-GlcNAc to Ser or Thr exhibit reciprocal O-GlcNAc glycosylation and phosphorylation during the cell cycle, cell stimulation, and/or cell growth. Less-investigated types of O-glycosylation are O-fucosylation, O-mannosylation, and O-glucosylation, but they are functionally of high relevance for early stages of development and for vital physiological functions of proteins. Glycosaminoglycans are a-linked to proteoglycans via a xylose-containing tetrasaccharide, represented by linear chains of repetitive disaccharides modified by carboxylates and O- or/and N-linked sulfates. Analysis of O-glycosylation by mass spectrometry (MS) is a complex task due to the high structural diversity of glycan and protein factors. The parameters in structural analysis of O-glycans include determination of (i) O-glycosylation attachment sites in the protein sequence, (ii) the type of attached monosaccharide moiety, (iii) a core type in the case of GalNAc O-glycosylation, (iv) the type and size of the oligosaccharide portion, (v) carbohydrate branching patterns, (vi) the site of monosaccharide glycosidic linkages, (vii) the anomericity of glycosidic linkages, and (viii) covalent modifications of the sugar backbone chains by carbohydrate- and noncarbohydrate-type of substitutents. Classical and novel analytical strategies for identification and sequencing of O-glycans by MS are described. These include methods to analyze O-glycans after total or partial release from the parent protein by chemical or enzymatic approach or to analyze O-glycosylated peptides by mapping and sequencing from proteolytic mixtures. A recombination process of multiply charged glycopeptides with electrons by electron capture dissociation Fourier transform ion cyclotrone resonance (FTICR)-MS has been introduced and is instrumental for nonergodic polypeptide backbone cleavages without losses of labile glycan substituents. A method for O-glycoscreening under increased sensitivity and efficient sequencing as a combination of an on-line coupling of capillary electrophoresis separation, as well as an automated MS-tandem MS (MS/MS) switching under variable energy conditions collision-induced dissociation (CID) protocol, is beneficial for determination of O-acetylation and oversulfation (Bindila et al., 2004a; Zamfir et al., 2004a). O-glycomics by robotized chip-electrospray/ionization (ESI)-MS and MS/MS on the quadrupole time-of-flight (QTOF) and FTICR analyzers, accurate mass determination, and software for assignment of fragmentation spectra represent essentials for high-throughput (HTP) in serial screenings (Bindila et al., 2004b; Froesch et al., 2004; Vakhrushev et al., 2005). Dimerization of intact O-glycosylated proteins can be investigated by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS after blotting.

Animals↗
PubMed2000

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing.

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.

Acrosome↗
PubMed1998

Architecture of the sugar binding sites in carbohydrate binding proteins--a computer modeling study.

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.

Acetylgalactosamine↗
PubMed1999

Structure of extracellular polysaccharide produced by lignin-degrading fungus Phlebia radiata in liquid culture.

The extracellular material (EM) produced by the white rot fungus Phlebia radiata cultured in an N-limited liquid medium was studied. Carbohydrate analysis showed maximum concentration of glucose as the major monosaccharide component of EM was found on postinoculation day 9. Beyond day 9 of cultivation the proportion of glucose decreased suggesting that the glucan component of EM had been further metabolized. The analysis of EM at day 9 revealed the presence of the following monosaccharides (in relative %): glucose (62); galactose (16); mannose (13); xylose (4); and fucose (5). The carbohydrate analysis together with the presence of protein in EM corresponds to a mixture of glucan and glycoprotein. Purification by trypsin treatment yielded an enriched glucose-containing extracellular polysaccharide (EPS). Methylation analysis identified EPS as (1-3)-beta-D-glucan highly branched at C-6. The structure of the glucan was confirmed by 13C-NMR spectroscopy. The results suggest that P. radiata's EPS is entangled with a glycoprotein in a complex that makes the extracellular sheath surrounding the hyphae.

Chromatography, Gas↗
PubMed1997

The glycosylated enzyme-binding assay for the study of the interaction of free and immobilized lectins with carbohydrates.

The glycosylated enzymes (invertase and glucose oxidase) were used as the competitive markers for a simple and rapid determination of the lectin-saccharide interactions. The method, based on the formation of the conjugate of an appropriate glycoenzyme with the specific carbohydrate-binding lectins and the inhibition of the conjugate formation with a monosaccharide, was described. This method was used to estimate the relative carbohydrate specificity of Concanavalin A for monosaccharides derived from D-mannose. The inhibition effect of the saccharides on the formation of Concanavalin A-glycosylated enzyme precipitate was compared with their influence on the enzyme sorption on conjugate Concanavalin A-bead cellulose support. The amount of the interacting enzyme was estimated either indirectly from its concentration in a supernatant that was determined spectrophotometrically (Con A was in a free or immobilized form) or directly in the immobilized form linked to Con A-sorbent using the flow microcalorimetric method. The results obtained, using different methods, agreed in general.

Calorimetry↗