Non-reducing Terminal Positions Research Articles

Comparative studies of flocculation and deflocculation of Saccharomyces uvarum and Kluyveromyces bulgaricus

Flocculation of Kluyveromyces bulgaricus and Saccharomyces uvarum occurred when these yeasts were grown in a peptone glucose medium enriched with calcium ions. K. bulgaricus and S. uvarum flocculated at the beginning and at the end, respectively, of the exponential growth phase. After growth, both yeasts were washed with an EDTA solution, flocculated again in an acetate buffer, and optimum flocculation was obtained at pH 4.5 in the presence of 3.75 mM Ca++. K. bulgaricus flocculation was irreversibly suppressed by incubation at 80° C for 6 min. S. uvarum needed an incubation at 100° C for 20 min to be irreversibly deflocculated. For both yeasts, flocculation stability depended on the presence of sugars. Mannose, mannose 6P and oligosaccharides bearing a mannose in a terminal non-reducing position reversed flocculation of S. uvarum, while galactose, galactose 6P and oligosaccharides bearing a galactose in a terminal nonreducing position reversed flocculation of K. bulgaricus. It is suggested that sugars specifically reverse flocculation because cell-to-cell aggregation of these yeasts is a lectin-carbohydrate-linked mechanism; not any sugar is capable of deflocculating any yeast, but the mechanism is specific.

The occurrence of l-galactose in snail galactans

The occurrence of l-galactose ( l-Gal) was investigated in twelve preparations of snail galactans from nine different species. l-Gal (11–15%) was found not only in members of the family Helicidae, a highly developed group among the Pulmonata, but was detected also in a galactan of the oldest phylogenetic subclass of snails in the Prosobranchiata. To determine the position of the l-Gal residues, the galactans of Arianta arbustorum, Cepaea nemoralis, and Helix pomatia were methylated and hydrolysed, and the partially methylated Gal derivatives were isolated. Each derivative was O-demethylated and the ratio of d- and l-galactose was determined by g.l.c. of the trifluoroacetylated methyl glycosides using a chiral stationary phase. l-Gal (22–34%) was detected only in the tetra- O-methyl fractions, and hence l-Gal occupies exclusively terminal non-reducing positions.

Plant mucilages. XXXV. Isolation and characterization of a mucous polysaccharide, Hippeastrum-H-glucomannan, from the bulbs of Hippeastrum hybridum.

A mucous polysaccharide, named Hippeastrum-H-glucomannan, was isolated from the bulbs of Hippeastrum hybridum HORT. The final preparation was homogeneous as determined by ultracentrifugal analysis, glass-fiber electrophoresis, and gel chromatography. It was composed of D-mannose and D-glucose in the molar ratio of 5 : 2, and its molecular weight was estimated to be 331000. O-Acetyl groups were identified and their content amounted to 13.2%. They were located at positions 2, 6 of some of the D-mannose units. Methylation and partial acid hydrolysis studies showed that the glucomannan is mainly composed of β-1→4-linked aldohexopyranose residues, and that it contains about eighty-three aldohexose units per six non-reducing groups on average. Both D-mannose and D-glucose units occupy non-reducing terminal positions and branching points linked through position 3.

A sulfotransferase-sulfatase system in avian oviduct which catalyzes a conversion of UDP-N-acetylgalactosamine 4-sulfate to the 6-sulfate isomer.

Magnum from quail oviduct was subfractionated to yield epithelium and tubular glands. The in vitro enzymatic activities involved in sulfated sugar nucleotide biosynthesis were assayed in these isolated tissues. The results demonstrated that the activities necessary for a series of reactions, UDP-N-acetylgalactosamine----UDP-N-acetylgalactosamine 4-sulfate----UDP-N-acetylgalactosamine 4,6- bisulfate ----UDP-N-acetylgalactosamine 6-sulfate, are located predominantly in the tubular gland. Both time course and pulse-chase studies with [35S]sulfate gave results that were consistent with this reaction scheme. A microsomal preparation from the magnum was shown to be capable of labeling all three sulfate sugar nucleotides with [35S]sulfate upon incubation with UDP-N-acetylgalactosamine and 3'- phosphoadenylyl [35S]sulfate. Again, their relative labeling rates were in the order necessary to allow for a synthesis of sulfated sugar nucleotides in the sequence described above. Furthermore, incubation of the microsomal preparation with UDP-N-[14C]acetylgalactosamine 4-sulfate and 3'- phosphoadenylyl sulfate resulted in the formation of UDP-N-[14C]acetylgalactosamine 6-sulfate. Also shown was the existence in the microsomal preparation of a sulfatase specific for the sulfate at position 4 of UDP-N-acetylgalactosamine 4,6- bisulfate . The results, together with those obtained in previous investigations, suggest that the tubular gland of quail oviduct contains a microsomal multienzyme system which catalyzes a series of sulfation and desulfation of N-acetylgalactosamine residues at the nonreducing terminal position of either sugar nucleotides or polysaccharide chains.

Structure of the Arabinogalactan from Zea Shoots

The structure of the arabinogalactan obtained from the buffer-homogenate of Zea mays L. (hybrid B73 x Mo17) shoots has been studied. The purified polysaccharide was investigated by methylation analysis before and after controlled acid hydrolysis. Arabinogalactan-1 consists of arabinose, galactose, xylose, uronic acid, and glucose in the molar ratio of 37.1:55.8:3.0:4.1:trace, and arabinogalactan-2 consists of the same sugars in the ratio of 35.4:53.9:1.6:9.2:trace. A trace of protein was detected in arabinogalactan-1 and about 0.2% was present in 2. About 20% of the galactose residues in arabinogalactan-1 constitute a (1 --> 3)-linked galactan chain and approximately 60% constitute a (1 --> 6)-linked galactan sequence. About 15% of the galactose residues in arabinogalactan-1 are substituted by galactose in the 3- and 6-positions, thereby constituting branch points of the galactan framework. The remainder (5%) of the galactose residues in arabinogalactan-1 are located at nonreducing terminal positions. About 85% of the (1 --> 6)-galactosyl sequence is substituted, mostly by single arabinose residues. Nonreducing terminal glucuronic acid is attached to C-6 of galactose residues. The basic structure of arabinogalactan-2 is similar to that of arabinogalactan-1.

Plant mucilages. XXXIII. An acetyl-rich mucilage, "lycoris-S-glucomannan," from the bulbs of Lycoris squamigera.

A mucilage, named Lycoris-S-glucomannan, was isolated from the bulbs of Lycoris squamigera MAXIM. The final preparation was homogeneous as determined by ultracentrifugal analysis, glass-fiber electrophoresis, and gel chromatography. It was mainly composed of D-mannose and D-glucose in the molar ratio of 7 : 2, and its molecular weight was estimated to be about 1800000. O-Acetyl groups were identified in the glucomannan and their content amounted to 16.7%. They were located at positions 2, 6 of about half of the D-mannose units. Methylation, periodate oxidation, and partial acid hydrolysis studies showed that the glucomannan is mainly composed of β-1→4 linked aldohexopyranose residues, and that it contains about twenty aldohexose units per three non-reducing groups on average. D-Mannose units occupy all branching points linked through position 3, while both D-mannose and D-glucose units occupy non-reducing terminal positions.

Structure of carbohydrate units of ovine luteinizing hormone.

The detailed structures of the three asparagine-linked carbohydrate units of ovine lutropin subunits alpha and beta (oLH-alpha and oLH-beta) have been determined by carrying out the structural analysis on the three glycopeptides alpha GP-1, alpha GP-2, and beta GP-3, and the oligosaccharide obtained by alkaline sodium borohydride treatment of oLH and the individual subunits. Based on the results of methylation, periodate oxidation, deamination, acetolysis, and enzymatic studies with exoglycosidases, the following structure is proposed for the carbohydrate units of oLH. Methylation studies indicated that the 3 N-acetylglucosaminyl residues were substituted at 1-, 1,4-, and 1,4,6-positions. Two of the three mannosyl residues were 1,2-linked and 3rd mannosyl residue was 1,3,6-linked. Fucose and galactose were terminally located and N-acetylgalactosamine was substituted at C-4. Periodate oxidation studies were consistent with the methylation data. The isolation of (formula, see text) 2,5-anhydromannose by the deamination of the oLH oligosaccharide established the branched structure for the mannosyl residues as well as the linkage of the trimannose unit to N-acetylglucosamine. The nonreducing terminal position of N-acetylgalactosamine was indicated by the formation of N-acetylgalactosaminyl glyceraldehyde on Smith degradation of oLH. The presence of a substituent "X" on N-acetylgalactosamine was established by the isolation of X-2,5-anhydrotalitol from the deamination products of oLH oligosaccharide. X was found to be an acid-labile substituent and was identified as a sulfate ester. Last but not least, since oLH oligosaccharide, obtained by hydrolysis of the hormone with NaOH + NaBH4, contained N-acetylglucosaminitol at the reducing end, the carbohydrate must be linked to the protein through N-acetylglucosamine.

Plant mucilages. XXXI. An acety-rich mucous polysaccharide, "lycoris-R-glucomannan," from the bulbs of Lycoris radiata.

A mucous polysaccharide, named Lycoris-R-glucomannan, was isolated from the bulbs of Lycoris radiata HERBERT. The final preparation was homogeneous as determined by ultracentrifugal analysis, glass-fiber electrophoresis, and gel chromatography. It was composed of D-mannose and D-glucose in the molar ratio of 12 : 1, and its molecular weight was estimated to be 468000. O-Acetyl groups were identified in the glucomannan and their content amounted to 15.5%. They were located at positions 2, 6 of some of the D-mannose units. Methylation, periodate oxidation, and partial acid hydrolysis studies showed that the glucomannan is mainly composed of β-1→4 linked aldohexopyranose residues, and that it contains about thirteen aldohexose units per three non-reducing groups on average. D-Mannose units occupy non-reducing terminal positions and branching points linked through both positions 3 and 6.

A mitogenic lactose-binding lectin from the sponge Geodia cydonium.

Two lectins with affinity for hog A + H blood group substance were extracted from the Mediterranean sponge Geodia cydonium. One, termed Geodia lectin I, was purified to near homogeneity by lactose elution from a column of hog A + H coupled to Sepharose 4B. Geodia lectin I was a glycoprotein (carbohydrate content about 14%) with m.w. of about 60,000 and an isoelectric point of pH 4.4. Subunits of about 15,000 daltons were linked by disulfide bonds. The lectin precipitated with various snail galactans composed entirely of DGal and LGal, to a slight degree with guaran, which has DGal alpha 1 leads to 6 in a terminal nonreducing position, and with blood group substances. Of 42 sugars tested, only lactose, dGal beta 1 leads to 4 DGlcNAc, DGal beta 1 leads to 3DGlcNAc, DGalNAc, inhibited precipitation. In view of the relatively high concentration of those disaccharides that inhibited relative to other lectins, the specificity of interaction is thought to be via beta-linked DGal residues that are part of a more complex combining site. Geodia lectin I was mitogenic for human peripheral blood lymphocytes with an optimal concentration of about 5.6 micrograms/ml if serum was omitted for the first 24 hr of culture to allow "triggering." Fetal calf serum apparently contained a high concentration of substances that inhibited mitogenicity and hemagglutination by Geodia lectin I, presumably by specifically interacting with the binding site.

A novel glycosaminoglycan from the fungus Omphalia lapidescence

The major, water-soluble polysaccharide isolated from the sclerotia of Omphalia lapidescence, and purified by chromatography on DEAE-cellulose and zone electrophoresis, is a heteroglycan (OL-1), ▪ ( c 1, water), which is composed of d-glucose, 2-acetamido-2-deoxy- d-glucose, and d-glucuronic acid in the molar ratios 1.0:1.95:2.0. The results of periodate oxidation, Smith degradation, and methylation analysis showed that the polysaccharide has a highly branched structure involving 1,3-, 1,4-, 1,6-, and 1,3,6-linked d-glucopyranosyl, 1,3,4-linked 2-acetamido-2-deoxy- d-glucopyranosyl, and (1,3 ?)- and 1,4-linked d-glucopyranosyluronic acid residues. The non-reducing terminal positions are occupied by the three component sugars. The branched glycosaminoglycan OL-1 is the first to be found in the fungi.

Investigations on the occurrence of lectins in marine sponges with special regard to some species of the family axinellidae

1. 1. Thirty-two species of eighteen families of sponges were screnned for their content of lectins. Twenty-one produced agglutinins reacting with human and animal red blood cells. Hemagglutiation inhibition tests were carried out with six species of different families to look for possible purification procedures. 2. 2. All species available of the family Axinellidae were compared by Ouchterlony tests, as well as by immuno- and disc electrophoresis. Axinella polypoides and Axinella dissimilis showed close structural relationships, whereas Axinella flustra had only restricted immunological similarities. The other three species displayed no actions with anti- Axinella polypoides antisera. 3. 3. Within the family Axinellidae lectins were detected only in Axinella polypoides and Axinella dissimilis. They were purified and their carbohydrate specificities studied, which are directed against dGal moieties in terminal non-reducing position. dGal β linked is of higher inhibitory potency than g linked dGal residues. A major difference between the lectins of both species is seen in inhibition studies with d GalN Ac , which is 250 times more active against Axinella dissimilis than Axinella polypoides.

Blood group A and B activity associated with factor VIII - von Willebrand factor

This report presents further progress on the characterization of blood group activities associated with F VIII/vWf. Evidence is provided that a relationship exists between the nature of the soluble blood group substance of plasma and of F VIII/vWf isolated from this plasma. A mixture of F VIII/vWf from O blood group plasma with A and B substances leads to a purified F VIII/vWf with AB blood group activity. Furthermore, chemical analysis of glycans released from F VIII/vWf by alkaline-borohydride treatment or hydrazinolysis shows the absence of N-acetylgalactosamine residues in non-reducing terminal positions. These results suggest that the blood group activity of F VIII/vWf preparations may be related to minor contamination of this molecule by glycolipidic or glycoproteinic plasma components with A or B oligosaccharide structures.

Interaction between heparan sulphate chains. II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains.

Abstract A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. 13 C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N -acetyl-glucosamine than N -sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N -acetyl-to- N -sulphate ratio was approx. 2 : 1, the N -sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species. Both preparations were subjected to deaminative cleavage which produces fragments like uronate-( N -acetylglucosamine-uronate) n -anhydromannose. Tetrasaccharides ( n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate- N -acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate- N -acetylglucosamine-glucuronate-anhydromannose was the major sequence. In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-A., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N -sulphated block regions and in regionsthat carry alternating N -acetyl- and N -sulphated glucosamine.

The biosynthesis of oligosaccharide-lipids. Formation of an alpha-1,2-mannosyl-mannose linkage.

An enzyme present in rabbit liver microsomes has been found to catalyze mannosyltransfer from GDP-mannose to exogenously added oligosaccharide-lipid acceptor resulting in the formation of an alpha-1,2-mannosyl-mannose linkage. Several lines of evidence suggest that the product is a heptasaccharide containing a single 1,6-linked branched glycosyl unit with 65% of the newly added mannosyl units in a terminal nonreducing position. The enzyme has been solubilized with non-ionic detergents and was partially purified on DEAE-cellulose and hydroxylapatite. The partially purified enzyme no longer catalyzed the formation of dolichol-P-mannose indicating a direct transfer from GDP-mannose to oligosaccharide-lipid acceptor.

Plant mucilages XXVII. Isolation and characterization of a mucous polysaccharide,"Narcissus-T-glucomannan," from the bulbs of Narcissus tazetta var. chinensis.

A mucous polysaccharide, named Narcissus-T-glucomannan, was isolated from the bulbs of Narcissus tazetta L. var. chinensis ROEMER. The final preparation was homogeneous as determined by ultracentrifugal analysis, glass-fiber electrophoresis, and gel chromatography. It was composed of D-mannose and D-glucose in the molar ratio of 5 : 1, and its molecular weight was estimated to be 119000. The O-acetyl groups in the glucomannan was identified and the content amounted to 22.7%. They were located at positions 6 and 2, 6 of most of the D-mannose units. Methylation, periodate oxidation, partial acetolysis, and enzymatic degradation studies showed that the glucomannan is mainly composed of β-1→4 linked aldohexopyranose residues, and that it contains about 42 aldohexose units per non-reducing group on average. D-Mannose units occupy non-reducing terminal positions and branching points linked through position 3.

Hepatic storage of oligosaccharides and glycolipids in a cat affected with GM1 gangliosidosis

1. Glycopeptides and glycolipids were isolated from normal cat liver and liver from a cat affected with GM1 gangliosidosis. 2. Bio-Gel P-6 chromatography of the crude glycopeptide fractions demonstrated three major peaks of hexose-containing compounds that were greatly increased in the mutant liver sample; these peaks contained oligosaccharides that comprised over 2% of the liver wet weight. 3. Two of the major pathological oligosaccharides, GP5 and GP6, were purified by chromatography on charcoal/Celite and Sephadex G-25. Oligosaccharides GP5 and GP6 had apparent mol.wts. of 1800 +/- 200 and 1350+/-200 respectively, and contained galactose, mannose and N-acetylglucosamine in molar proportions of 2.0:3.1:4.1 (GP5) and 1.0:2.2:2.7 (GP6). Periodate oxidation studies demonstrated the presence of galactose in a non-reducing terminal position. 4. The neutral glycolipid fraction from the mutant cat liver has a 1.3-fold increase in hexose content accompanied by an increased concentration of asialo-(ganglioside GM1). 5. There was a 2-fold increase of gangliosides in the mutant cat liver compared with normal cats. Ganglioside GM1 and a compound tentatively identified as N-glycolloyl-(ganglioside GM1) were the major glycolipids accumulated.

Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Studies with beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, ribonuclease B and agalacto-orosomucoid.

Glycoproteins having mannose and/or N-acetylglucosamine in the terminal non-reducing position [Stockert, Morell & Scheinberg (1976) Biochem. Biophys. Res. Commun. 68, 988--993], and various lysosomal enzymes [Stahl, Schlesinger, Rodman & Doebber (1976) Nature (London) 264, 86--8] are rapidly cleared from plasma by the liver after intravenous administration. A liver cell-separation technique was used to determine the cellular localization of 125I-labelled beta-glucuronidase, ribonuclease B, agalacto-orosomucoid and asialo-orosomucoid. On a specific readioactivity basis, all ligands except 125I-labelled asialo-orosomucoid were enriched in the non-parenchymal cell fraction. Isolated cells, fixed and stained for beta-glucuronidase or N-acetyl-beta-D-glucosaminidase activity after intravenous injection of the enzymes, showed enrichment in the non-parenchymal cell fraction (probably Kupffer cells). After uptake by the non-parenchymal cells, liver lysosomal beta-glucuronidase and N-acetyl-beta-D-glucosaminidase showed degradation half-times of 2.2 and 0.4 days respectively.

Studies on the relationship between lectins from Axinella polypoides agglutinating bacteria and human erythrocytes

A new lectin, called Axinella IV, with bacteria agglutinating activity was detected in the crude extract of the sponge Axinella polypoides. Immunofluorescence studies and immunoelectrophoresis revealed no cross-reactivities with the already known lectins Axinella I. II, and III. d-Galactose and oligosaccharides with d-Galactose in terminal nonreducing position are inactive in agglutination inhibition tests demonstrating also different specificity of Axinella IV lectin when compared with the three aforementioned lectins.

Structural studies on glycolipid of shellfish. II. Occurrence of 3-O-methylgalactosamine in oyster glycolipid.

3-O-Methyl hexosamine was found for the first time and characterized as one of the sugar components of oyster glycolipid. The alditol acetate of this sugar was identified as 3-O-methyl-N-acetylgalactosaminitol acetate by comparing its retention time on gas chromatography and mass spectrum with those of the authentic sample synthesized in this laboratory. The new sugar, 3-O-methylgalactosamine, occupied the non-reducing terminal position of the carbohydrate moiety of the lipid and was linked to the penultimate hexose by a (1 leads to 3) bond.

A rapid semiquantitative assay that facilitates purification of endo-β- N-acetylglucosaminidase H from Streptomyces plicatus

A simple procedure for the detection of endo-β- N-acetylglucosaminidase H activity is described. The method utilizes N-[ 14C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [ 14C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β- N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di- N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β- N-acetylglucosaminidase H from Streptomyces plicatus.