Low Molecular Weight Oligosaccharides Research Articles

Surface antigens of intact Aspergillus fumigatus mycelium: their localization using radiolabelled protein A as marker.

Isotopically-labelled Protein A from Staphylococcus aureus was used as a marker to quantify binding of IgG molecules to surface antigens of Aspergillus fumigatus mycelium. The IgG class antibodies were obtained from rabbits inoculated with mycelial fractions and from patients suffering from aspergillosis and aspergillus-related diseases. The highest incorporation of label was obtained with an antiserum from rabbits inoculated with A. fumigatus wall material. Antibodies raised to other antigenic fractions of A. fumigatus and antibodies from patients infected with aspergillus gave lower levels of incorporation of Protein A. In competitive binding experiments, pre-incubation of antibodies with partially purified aspergillus antigens depressed subsequent binding to the mycelial surface by 20-30% of that of the control values. Low molecular weight disaccharides and oligosaccharides were without effect in this system. Preincubation of A. fumigatus with lectins having specificities for defined sugar residues did not reduce subsequent antigen/antibody interaction. Treatment of the mycelial surface with certain proteolytic or polysaccharolytic enzymes led to a decrease in antibody binding, while pretreatment of A. fumigatus with the hydrolytic enzyme mixture of Trichoderma harzianum culture filtrate gave increased antibody binding. Aspergillus species showed different susceptibilities to enzyme action and their surface structures could be differentiated from A. fumigatus on this basis. These differences were not obvious in direct binding experiments where antibodies raised to A. fumigatus wall bound with equal facility to antigenic sites located on the walls of other Aspergillus species.

Action patterns of immobilized dextranase

Dextranase, isolated from Penicillium funiculosum and P. lilacinum, was immobilized on porous, silanized-silica beads and a phenol-formaldehyde resin. A commercial dextran of relatively low molecular weight (∼2 × 10 6) was degraded by immobilized dextranase, with the formation of reducing sugars, but with little decrease in viscosity. In contrast, soluble dextranase caused rapid loss of viscosity, but only a slight increase in reducing sugar. Native dextran of high molecular weight, from Leuconostoc mesenteroides NRRL B-512 (F), was attacked very slowly by immobilized dextranase, with the release of oligosaccharides of low molecular weight.

Purification and properties of glucosyltransferase produced by Streptococcus salivarius HHT.

Glucosyltransferase produced by Streptococcus salivarius HHT was purified from the culture filtrate, and its enzymic properties were examined. The enzyme was specific to sucrose (Km 7.2mM), and formed a water-insoluble α-D-glucan consisting of a high proportion of (1 → 3)-α-D-glucosidic linkage (90%) together with small proportions of (1 → 6)- and (1 → 4)-α-D-glucosidic linkages. The optimum pH and temperature of the enzyme activity were 5.8 and 40°C, respectively. The enzyme activity and yield of the water-insoluble glucan were stimulated in the presence of dextran. The addition of low molecular weight oligosaccharides, such as maltose and isomaltose, inhibited the production of the water-insoluble glucan with the concomitant formation of several oligosaccharides, i.e., O-α-D-glucopyranosyl-(1 → 6)-O-α-D-glucopyranosyl-(1 → 4)-D-glucopyranose (panose), O-α-D-glucopyranosyl-(1 → 3)-O-α-D-glucopyranosyl-(1 → 4)-D-glucopyranose, maltotriose, and O-α-D-glucopyranosyl-(1 → 6)-O-α-D-glucopyranosyl-(1 → 6)-O-α-D-glucopyranosyl-(1 → 4)-D-glucopyranose.

Metabolism of parenteral glucose oligosaccharides in man.

Low molecular weight oligosaccharides of glucose may be utilized as a calorie source when given intravenously. Preliminary safety studies included the parenteral infusion of four healthy subjects with a solution of glucose oligosaccharides at a rate of 0.5 mg/kg/min for 1, 2, or 3 hours. Approximately 100% of the oligosaccharides was retained with only trace amounts of the glucose oligosaccharides recovered in the urine. In further studies, 12 healthy males, ages 22–31, were infused with a 10% solution of glucose oligosaccharides containing 25 μCi [U‐14C]glucose oligosaccharides/liter at a rate of 2.5 or 5.0 mg/kg/min for 12 hours with or without insulin (0.12 unit/g oligosaccharides) given subcutaneously in 3 equi‐divided doses. Four additional subjects received cold oligosaccharides at a rate of 1.75 mg/kg/min without insulin. Eighty percent of the glucose oligosaccharides consisted of 2 to 8 glucose chain lengths. Blood glucose and oligosaccharide glucose concentrations and 14CO2 were measured hourly. Paper chromatography of urine sugars was also performed to determine the sugars excreted. Serum glucose and insulin concentration did not change during the oligosaccharide infusion; insulin injections significantly depressed serum glucose and produced elevations in serum insulin concentration. Plasma FFA decreased in response to oligosaccharide infusion at both 2.5 and 5.0 mg/kg/ min rates, with and without insulin. At the 1.75, 2.5, and 5.0 mg/kg/min rates, 21, 43, and 62% of the infused oligosaccharides, respectively, was excreted into the urine. Only 7–15% of the infused oligosaccharide 14C was recovered as 14CO2. Insulin did not influence these parameters. Urine chromatography showed that approximately 20, 30, and 40% of excreted sugar was maltotriose, maltose, and higher oligosaccharide chains, respectively. These studies indicate that partial, but significant quantities of glucose oligosaccharides may be metabolized after intravenous infusion, particularly at low infusion rates.

Isolation, purification, and properties of neuraminidase from Propionibacterium acnes.

Neuraminidase activity was discovered in 32 of 38 strains of Propionibacterium acnes. Enzyme production was studied in yeast extract bouillon of different pH containing various amounts of human milk as neuraminidase inductor. Enzyme activity was found in the bacterial sediments as well as in the culture filtrates. Since neither ultrasonic treatment nor lysozyme incubation of bacterial sediments did release reasonable amounts of enzyme, culture filtrates were used for enzyme preparation. Neuraminidase was isolated by 40% ammonium sulfate precipitation, dialysis, concentration and repeated gel chromatography on Sephadex G-100. The enzyme posesses a molecular weight of about 33 000 and a pH-optimum around 5.0. The Michaelis constants are 1.8 x 10(-3) M for alpha 2 leads to 3 linked N-acetylneuraminic acid (NeuAc) in II3NeuAc-Lac, 3.7 x 10(-3) M for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 2.1 x 10(-3) M for the alpha 2 leads to 8 linkage of II3 (comes from 2 alpha NeuAc8)2-Lac, respectively. Among the different groups of naturally occurring NeuAc-containing substrates, i.e. oligosaccharides, glycolipids and glycoproteins, the enzyme exhibits its highest activity towards low molecular weight oligosaccharides. Activity is considerably lower on glycoproteins. Glycolipids (gangliosides) are only little attacked under conditions used in the test. However, there is no remarkable specificity towards one of the different linkage types of N-acetylneuraminic acid. In general, the enzyme reveals a specificity pattern similar to that found in other bacteria of low pathogenicity towards man.

Use of some new poly-phenolic resins for fractionation of carbohydrates and immobilisation of carbohydrate hydrolases and isomerases

New matrices, exhibiting selective adsorption of polymeric carbohydrates, have been synthesised by copolymerisation of hydroquinone and other phenolic compounds with formaldehyde or glutaraldehyde. These gel-type resins are capable of separating high molecular weight polysaccharides from low molecular weight oligosaccharides and monosaccharides, and branched from unbranched polysaccharides. The formaldehyde–hydroquinone resin is also an effective matrix for the simple and rapid immobilisation of various enzymes and (other) proteins. It is therefore additionally suitable for the separation of proteins from low molecular weight peptides and amino-acids. α-Amylase, glucoamylase, and D-glucose isomerase immobilised on the resins retain their activity incontinuous use. Since the resin matrices are highly porous and hydrophilic, they allow high flow rates to be achieved when used in packed-bed reactors. Both the formaldehyde and glutaraldehyde types of resin are cheap and easy to produce, relatively non-toxic in preparation and use, porous, hydrophilic, and extremely stable to changes in temperature and pH.

Properties and substrate specificities of two neuraminidases from Trichomonas fetus.

Trichomonas fetus, a protozoon belonging to the class of flagellates causes vaginal infections in cows, leading to sterility or abortion in early stage of pregnancy. Two neuraminidases were isolated from the culture medium and purified by various procedures of gel chromatography, ion exchange chromatography, and by affinity chromatography on N-(4-nitrophenyl)-oxamic acid-Sepharose 4B. The molecular weights of the two neuraminidases were determined as 320 000 (enzyme I) and 38 000 (enzyme II) respectively. However, enzyme I seems to consist of two isoenzymes containing four subunits of almost equal molecular weight. The pH optima of both enzymes depend on the substrates and range from pH 4.7 to 5.5. Due to the type of substrate, the Michaelis constants (Km) vary between 5.0 x 10(-2)M and 6.6 x 10(-3)M for enzyme I and between 1.4 x 10(-2)M and 4.9 x 10(-3)M for enzyme II. Among the different groups of NeuAc-containing substrates, i.e. glycoproteins, glycolipids, oligosaccharides and synthetic ketosides, enzyme I preferably cleaves high molecular weight glycoprotein type substrates whereas enzyme II shows higher affinities to low-molecular weight oligosaccharides. The ganglioside II3NeuAcGgOse4Cer is susceptible to both enzymes only after removal of the lipophilic ceramide residue. Both enzymes show differences in the specificity towards alpha 2 leads 3 to 3, alpha 2 leads to 6, and alpha 2 leads to 8 glycosidic linkages of NeuAc. Taking the rate of cleavage of the alpha 2 leads to linkage in II3NeuAc-Lac as 100, enzyme I reveals 65 for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 15 for the alpha 2 leads to 8 linkage in II3(comes from 2 alpha NeuAc8)2-Lac, whereas enzyme II exhibits values around 50 for both the alpha 2 leads to 6- and the alpha 2 leads to 8-linked substrates. The activity of neuraminidase I and II is not influenced by Ca2 but is inhibited by Cu2, Hg2, ann 4-hydroxymercurisulfonic acid. The inhibition by Hg2 and by the latter is reversible with enzyme I by addition of dithioerythritol.

Exoenzymic activity of alpha-amylase immobilized on a phenol-formaldehyde resin

Amylose and amylopectin from two starch sources were partially degraded by alpha-amylase immobilized on a phenol-formaldehyde resin. The degradation products were fractionated by gel-permeation chromatography and high-pressure, liquid chromatography. Two distinct fractions were obtained from tapioca amylose. One is a fragment having a molecular weight exceeding 200,000, and the other consists of oligosaccharides of low molecular weight with a degree of polymerization of 1–8. In contrast, treatment of tapioca amylose with soluble alpha-amylase produces a single fraction, nearly all of which has a molecular weight of <35,000, with only traces of small oligosaccharides detectable by high-pressure, liquid chromatography. Even wider differences were observed in degradation products from tapioca amylopectin. Similar activity-patterns were obtained with immobilized and soluble enzymes, using corn amylose and corn amylopectin as substrates. Immobilization of alpha-amylase on the resin apparently restricts the activity of the enzyme to the ends of the starch molecules, making it appear to be limited to exoenzymic activity.

Alkali-labile oligosaccharides of brain glycoproteins

Treatment of glycopeptides prepared from rabbit brain glycoproteins with NaOH or NaOH-NaBH 4 released approximately 10% of the carbohydrate as low molecular weight oligosaccharides. After treatment with alkali alone, a disaccharide and a trisaccharide were isolated by gel filtration and preparative paper chromatography, and have been identified as N- acetylneuraminyl-(2→6)-O-2- acetamido-2- deoxy- D- galactose (Component B-I) and di-N- acetylneuraminyl- D- galactose (Component B-III). The N-acetylgalactosamine in Component B-I was obtained as preformed Morgan-Elson chromogen, and Components B-I and B-III were not obtained when the glycopeptides were treated with alkaline NaBH 4 under conditions which prevent peeling. These results indicate that the trisaccharide (Component B-III) was originally present as a substituent at C-3 of the N-acetylgalactosamine involved in the carbohydrate-peptide linkage, and was eliminated as a result of alkali treatment. A disaccharide (Component M) was isolated after alkaline NaBH 4 treatment of the desialylated glycopeptides, and yielded equimolar amounts of galactose and galactosaminitol after hydrolysis. Smith degradation of Component M gave galactose and threosaminitol, indicating that the disaccharide was galactopyranosyl-(1→3)-O-2- acetamido-2- deoxy- D- galactitol . From the isolation of Components B-I, B-III and M after NaOH and NaOH-NaBH 4 treatment of the glycopeptides, it is concluded that the major alkali-labile oligosaccharide of brain glycoproteins is the pentasaccharide di-N- acetylneuraminyl- D- galactopyranosyl-(1→3) [N- acetylneuraminyl-(2→6)]-O-2- acetamido-2- deoxy- D- galactose .

Polysaccharide synthesis in mung bean roots — An X-ray investigation

The early intracellular localisation experiments on “cellulose synthesis” from mung bean roots have been repeated and two active fractions, one of which resembles plasmalemma elements, have been separated. Attempts to characterise physically the synthesized product from the reaction between the active fractions and UDPGlc, GDPGlc, necessitated using large amoung of both fraction and nucleotide sugar. Hydrolysis and chromatography revealed that under the experimental conditions used only a glucan was formed and X-ray diffraction analysis of the product showed that cellulose I, i.e. natural cellulose was not synthesized but only low molecular weight oligosaccharides.

Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of glycogen synthetase

The presence of glycogen synthetase has been demonstrated in cell extracts of five strains of S. mitis. The purified enzyme was free from transglucosylase, α-(1→6)- d-glucosidase, pullulanase, phosphorylase, and 5′-adenylate kinase, but contained a trace of branching enzyme. The enzyme transferred d-glucosyl residues from ADPG to the acceptor with the formation of α-(1 → 4)- d-glucosidic linkages. The preferred acceptors were highly branched polysaccharides. The efficiency (relative to S. mitis glycogen) of various acceptors, present in excess in activity digests, was as follows: glycogen phosphorylase limit-dextrin (107), glycogen (100), amylopectin phosphorylase limit-dextrin (78), amylopectin (65), amylopectin β-amylase limit-dextrin (30), and amylose (23). For branched oligosaccharide and polysaccharide acceptors, the side chain was the favoured site of d-glucosyl transfer. The enzyme displayed an absolute requirement for three or more d-glucosyl residues in the side chain of branched oligosaccharides of low molecular weight.

Structural Studies on Human Erythrocyte Glycoproteins: ALKALI-LABILE OLIGOSACCHARIDES

Abstract Treatment of M- or N-active sialoglycopeptides, derived from human erythrocytes, with alkaline borohydride (0.1 m NaOH-0.4 m NaBH4, room temperature, 26 hours) released 25 to 30% of the carbohydrate as low molecular weight oligosaccharides. No difference was found in the composition of oligosaccharides obtained from M- or N-active erythrocytes. The most complex oligosaccharide (Component I) was a tetrasaccharide, N-acetylneuraminyl-(2 → 3)-β-d-galactopyranosyl-(1 → 3) [N-acetylneuraminyl-(2 → 6)]-d-N-acetylgalactosaminitol. In addition, the following compounds were identified: N-acetylneuraminyl-(2 → 3)-β-d-galactopyranosyl-(1 → 3)-d-N-acetylgalactosaminitol (Component II); N-acetylneuraminyl-(2 → 3)-β-d-galactitol (Component III); β-d-galactopyranosyl-(1 → 3)-d-N-acetylgalactosaminitol, together with free sialic acid, galactitol, N-acetylgalactosaminitol, 3-deoxy-d-glucitol, and the reduced Kuhn chromogen. It is concluded that the major, alkali-labile oligosaccharide of sialoglycopeptides is a tetrasaccharide and that other components are degradation products thereof.

Oligosaccharides from M-active sialoglycopeptides of human erythrocytes

A sialoglycopeptide released from human type M erythrocytes by incubation with trypsin was treated with 0.1 M NaOH containing 0.4 M NaBH 4. Nine low molecular weight sugars and oligosaccharides were released, including four which had N-acetylgalactosaminitol at the “non-reducing” terminus. All of these appear to be derived or derivable from a tetrasaccharide containing two sialic acids, one galactose and one galactosamine with the latter linked O-glycosidically to serine or threonine. Sialoglycopeptides with M or N bood group activity are liberated from the surface of human erythrocytes on incubation with trypsin ( Mäkela et al , 1960 ; Cook et al , 1960 ; Eylar et al , 1962 . A previous report ( Winzler et al , 1967 ) has established that such glycopeptides have a portion of their carbohydrate linked to the peptide chain by alkali-labile, O-glycosidic bonds. In the present paper the nature of the oligosaccharides released by treating sialoglycopeptides with alkaline borohydride will be considered.

The 6-O-Methylglucose-containing Lipopolysaccharide of Mycobacterium phlei: Identification of d-Glyceric Acid and 3-O-Methyl-d-Glucose in the Polysaccharide

Abstract The polysaccharide containing 6-O-methylglucose, from Mycobacterium phlei, is an acidic molecule with at least seven 6-O-methyl-d-glucopyranosyl residues linked α-1,4', and with a side chain of at least three α-1,4'-d-glucopyranosyl units. A simplified procedure is described for isolating the polysaccharide (MGP) from acetone-dried M. phlei cells with improved yield. Exhaustive α-amylase digestion of the polysaccharide released glucose, maltose, and a disaccharide. The last yielded d-glucose and 3-O-methyl-d-glucose after acid hydrolysis; while hydrolysis, after reduction with sodium borohydride, gave glucitol and 3-O-methylglucose. Methylation analysis established that the disaccharide was 3-O-methyl-α-d-glucopyranosyl-(1 → 4)-d-glucose. Limited α-amylolysis of MGP yielded a trisaccharide and a tetrasaccharide, each containing 1 mole of 3-O-methylglucose at the nonreducing end. The trisaccharide was digested by α-amylase to the disaccharide 3-O-methylglucosyl-(1 → 4)-glucose and glucose, while α-amylolysis of the tetrasaccharide gave these products plus maltose. A glucoamylase preparation converted the tetrasaccharide to the trisaccharide and glucose. That 3-O-methylglucose was at the nonreducing terminus of each saccharide was shown by partial acid hydrolysis studies and by the products of exhaustive propylation, which yielded 3-O-methyl-2,4,6-tri-O-propylglucose. Propylation of the intact polysaccharide and analysis of the fully alkylated methylglucosides by gas-liquid chromatography established that 3-O-methylglucose and glucose were in the nonreducing terminal positions. After α-amylase digestion, glucose was the only terminal residue. Glucoamylase removed an additional glucosyl residue, which had been linked α-1,4' to a 6-O-methylglucosyl residue. An organic acid having the chromatographic and electrophoretic properties of glyceric acid was isolated from acid hydrolysates of MGP. The glyceric acid was oxidized by 1 mole of sodium periodate, with the release of 1 mole of formaldehyde and 1 of glyoxalate. It was shown to have the d configuration. 3-O-Methylglyceric acid was obtained by hydrolysis of methylated MGP, showing that the linkage to the polysaccharide involved the 2-hydroxyl group. MGP was converted to the hydroxamate, and a Lossen rearrangement was performed in water at neutral pH. The reducing sugar thus formed was reduced with sodium borotritide, and the radioactive polyol obtained by acid hydrolysis was identified as glucitol. Thus, the glyceric acid must be attached glycosidically to a glucose residue. The intact lipopolysaccharide, MGLP, was not associated with purified M. phlei cell wall or particulate preparations. The material was recovered quantitatively from the high speed supernatant, apparently in free form. An attempt to degrade the main chain enzymatically with extracts from M. phlei was unsuccessful, and a search both for low molecular weight oligosaccharides containing 6-O-methylglucose and for large polysaccharides into which MGLP might have been incorporated was unproductive. Thus, our investigations have failed to suggest a structural or metabolic function for the lipopolysaccharide. MGLP, isolated from the cultural filtrate of M. phlei cells in the stationary phase, was indistinguishable from the intracellular molecule. It was released into the medium during all stages of the growth cycle, the kinetics following those for the appearance of low molecular weight carbohydrates and of mannose- and arabinose-containing polysaccharides.

Intrinsic viscosity‐molecular weight relationship for low molecular weight cellulose and oligosaccharides

AbstractThe viscosity behaviour of low molecular weight cellulose and oligosaccharides has been investigated. In the high molecular weight region the double‐logarithmic plot of intrinsic viscosity vs molecular weight is linear down to approximately DP = 150. On passing to lower molecular weights the slope of the curve at first increases, passes through a maximum and finally decreases to very low values in the oligomere region.

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light‐grown soybean cotyledons increased approximately 5‐fold during the first 9 days of germination, whereas that of dark‐grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.

Studies on the Soluble Carbohydrates and Carbohydrate Precursors in Germinating Soybean Seed

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.