Binding constants of NZB myeloma antidextrans for dextrans and isomaltose oligosaccharides determined by affinity electrophoresis.

Association constants of dextrans (Ka) and oligosaccharides (Kia) from NZB myeloma antidextrans (PC3858 and PC3936) were studied by affinity electrophoresis. With linear dextrans or with those with a low degree of branching, Ka ranged from 2.7 X 10(3) to 5.4 X 10(4) ml/g for PC3858 and from 1.3 X 10(4) to 2.6 X 10(5) ml/g for PC3936. Completely linear alpha-(1 leads to 6)-linked dextrans, LD7 and D3, showed relatively high affinities for the two NZB antidextrans. With oligosaccharides, the Kia value increased as the number oa alpha-(1 leads to 6)-linked glycosyl residues increased. Isomaltoheptaose (IM7) showed the highest Kia (1.9 X 10(4) M-1 for PC3858 and 1.63 X 10(4) M-1 for PC3936), whereas isomaltose (IM2) had the lowest Kia (2.36 X 10(2)M-1 for PC3858 and 1.32 X 10(2)M-1 for PC3936). Pullulan and glycogen showed very weak affinity for PC3936, but they did not react at all with PC3858. These findings indicate that NZB myeloma antidextrans, PC3858 and PC3936, are specific for internal chains of alpha-(1 leads to 6)-linked dextrans. Data on the precision with which Ka and Kia can be determined are presented.     

Determination of the structure of dextran by 13C-nuclear magnetic resonance spectroscopy

The ¹³C-n.m.r. spectra have been recorded for a series of dextrans whose structures, in terms of degree and type of branching, had previously been determined by methylation analysis. The spectra established that all observable linkages in these dextrans are α-linked. Correlation of the spectra with methylation data indicated that the 75–85-p.p.m. spectral region is diagnostic for establishing the presence of α-D-(1→2)-, α-D-(1→3)-, or α-D-(1→4)-linkages. Each chemical shift has been found to be temperature-dependent (Δδ/ΔT) when referenced to either the deuterium lock or an external standard (tetramethylsilane). All carbohydrate Δδ/ΔT values are positive, and range from 0.01 to 0.03 p.p.m./°C. These values are considerably larger than analogous Δδ/ΔT values previously observed for smaller molecules. Larger than average Δδ/ΔT values are associated with the non-anomeric, sugar-linking carbon atoms.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

A galactoglucomannan from the leaf and stem tissues of red clover (Trifolium pratense)

A galactoglucomannan has been isolated by fractionation of the alkali-soluble hemicelluloses of the leaf and stem tissues of red clover (Trifolium pratense L.). The hemicellulose contains galactose, glucose, and mannose residues in the molar ratios 0.25:1.0:1.1 and accounts for ca. 25% of the mannose residues present in the clover tissues. Structural studies showed that the hemicellulose has a main chain of β-(1→4)-linked D-glucopyranosyl and D-mannopyranosyl residues, to which are attached α-(1→6)-linked D-galactopyranosyl residues.     

Calorimetric study of carbohydrate binding to Concanavalin A

Flow microcalorimetry has been used to examine the ΔH of binding of two types of saccharides, a series of simple monosaccharides and a series of α-(1 → 4)-linked glucosides, to the lectin Concanavalin A. It has been found that the ΔH decreases with any change in the stereochemistry of a hydroxyl group relative to methyl α-d-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The ΔH's of binding for the α-(1 → 4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for α-(1 → 4)-linked glucopyranosides, in contrast to that proposed for α-(1 → 2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.     

Structural studies of extracellular glucans of Streptococcus mutans by proton magnetic resonance

Proton magnetic resonance spectra at 100 MHz were obtained for water-soluble and water-insoluble glucans from 11 strains of Streptococcus mutans. The percentages of α-D-(1→6) and non-α-D-(1→6)-, namely, α-D-(1→3)-, linkages were calculated from the anomeric-proton resonances in the 4.7-4.8 and 5.0-5.1 p.p.m. range, respectively. The average content of α-D(1→6) linkages in the polymer fractions precipitating from solution during synthesis of the glucans was generally much lower than that of fractions remaining in solution. The frequent appearance of the α-D-(1→3) resonances as doublets in the spectra suggested neighboring-group effects among the possible α-D-(1→3) and α-D-(1→6) linkage-configurations. These effects were confirmed from 100-MHz spectra of products of a dextranase-degraded, water-insoluble glucan, and a 270-MHz spectrum of an undegraded glucan. It was thus possible to assign the doublet resonances to α-D-(1→3), homogeneous, heterogeneous, and branch configurations, although complete differentiation among proportions of each configuration in the glucan chains could not be achieved.     

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Specificities and reactivities of the antibodies formed in rabbits to stearyl-isomaltosyl oligosaccharides

The specificities and the sizes and shapes of the antibody combining sites of the 15 antisera raised against various stearyl-isomaltosyl oligosaccharides were studied by quantitative precipitin and precipitin inhibition. The antibodies precipitated well with dextrans B512 and B1424 but less well with B1299S and B1355S. Only 3 of the 15 antisera reacted with linear dextrans; however, with about 50% of the added antibodies being precipitated, showing that most of the antibodies cannot bind to internal determinants along the dextran molecules and are similar to myeloma protein W3129 in having cavity-type sites which bind only to terminal nonreducing ends of α1 → 6 dextran. Antibodies differing in the sizes of their antibody combining sites were elicited in different rabbits by the same antigen. Of the 15 antisera studied, four have antibody combining sites as large as IM3, five as large as IM4, three as large as IM5 and three as large as IM6. The association constants for various isomaltose oligosaccharides of an antiserum (R-862) showing fewest bands in isoelectric focusing gel were determined by affinity electrophoresis and were comparable to W3129.     

Purification,structural characterization and antioxidant property of an extracellular polysaccharide from Aspergillus terreus

The marine fungus Aspergillus terreus produces an extracellular polysaccharide, YSS, when grown in potato dextrose-agar medium. YSS was isolated from the fermented liquids using ethanol precipitation, anion-exchange and size-exclusion chromatography. YSS was mainly composed of mannose and galactose in a molar ratio of 7.68:1.00, its average molecular weight was estimated to be about 18.6 kDa. On the basis of chemical and spectroscopic analyses, including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopy, structure of YSS may be represented, at an average, as a backbone of mannan with two types of branches. The mannan backbone is mainly composed of (1→2)-linked α-mannopyranose with small amounts of (1→6)-linked α-mannopyranose residues. The branches consist of terminal β-galactofuranose residues, and disaccharide units of (1→6)-linked α-mannopyranose. The branches are linked to C-6 of (1→2)-linked α-mannopyranose residues of backbone. The antioxidant activity of YSS was evaluated with the scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and hydroxyl radicals in vitro, and the results indicated that YSS had good antioxidant activity, especially scavenging ability on DPPH radicals. The investigation demonstrated that YSS is a novel branched galactomannan with antioxidant activity, and differs from previously described extracellular polysaccharides.     

Stepwise synthesis of methyl 4-O-[3-O-(β-d-xylopyranosyl)-β-d-xylopyranosyl]-β-d-xylopyranoside

The general properties and specificity of a dextran α-(1→2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5–6.5, and 45°, respectively. The enzyme was stable up to 40° on heating for 10 min, and over a pH range of 6.5–9.0 on incubation at 4° for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1→2)-α-d-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces d-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the K_m value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. The debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran α-(1→2)-debranching enzyme, and its systematic name is also discussed.     

Aufbau von oligosacchariden mit glycosylfluoriden unter lewissäure-katalyse

Glycosylation of 1,2:3,4-di-O-isopropylidene-α-d-galactopyranose (6), as well as its 6-trimethylsilyl ether 7 with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl fluoride (5) was achieved stereospecifically in a mild and fast manner in the presence of Lewis acids like, e.g., titanium tetrafluoride, to give the β-(1→6)-linked disaccharide derivative 1. By use of 2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl fluoride (8) or its α anomer 10 and titanium tetrafluoride in acetonitrile with 6 or 7, a fast reaction proceeds preponderantly to yield 1,2:3,4-di-O-isopropylidene 6-O-(2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl)-α-d-galactopyranose (2). In ether, however, mainly the α-(1→6) anomer was formed. These model systems were used to elucidate the limiting conditions for this procedure, and mechanistic conceptions are discussed. By glycosylation at O-4 of 1,6:2,3-dianhydro-β-d-mannopyranose (11) with the perbenzylated α-fluoride 10 both the α- and the β-d-(1→4) disaccharide derivatives 12 and 14 were obtained, but 5 gave exclusively the β-d-(1→4) compound 16. Opening of the anhydro rings of 12 led to the synthesis of N-acetyl-maltosamine (22). 1,6-Anhydro-2-azido-4-O-benzyl-2-deoxy-β-d-glucopyranose was glycosylated with methyl (2,3,4-tri-O-acetyl-β-d-galactopyranosyl fluoride)uronate under titanium tetrafluoride catalysis to give the β-d-(1→3)-linked disaccharide 16, subsequently transformed into 29.     

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

Mammalian-lung galactan

Exopolysaccharides of Agrobacterium tumefaciens and Rhizobium meliloti, containing d-glucose, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 6:1:1:1.5, were analysed by methylation. They were found to contain the following main structural units (all β-glycosidic): chain residues of (1→3)-linked d-glucose (24%), (1→3)-linked d-galactose (15%), (1→4)-linked d-glucose (20%), and (1→6)-linked d-glucose (18%); (1→4,1→6)-linked branching residues of d-glucose (12%), and terminal d-glucose residues substituted at positions 4 and 6 by pyruvate (11%). Uronic acid-containing exopolysaccharides of Rhizobium leguminosarum, R. phaseoli, and R. trifolii contained d-glucose, d-glucuronic acid, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 5:2:1:2:3. Methylation gave identical patterns of methylated sugar components, from which the following structural elements were deduced: chain residues of (1→3)-linked d-glucose substituted at positions 4 and 6 by pyruvate (13%), (1→4)-linked d-glucose (32%), and (1→4)-linked d-glucuronic acid (20%); (1→4,1→6)-linked branching residues of d-galactose and/or d-glucose (13%), and terminal d-glucose and/or d-galactose residues substituted at positions 4 and 6 by pyruvate (13%).     

Growth-inhibitory activity of the d-mannan of saccharomyces cerevisiae X2180-1A-5 mutant strain against mouse-implanted sarcoma 180 and ehrlich-carcinoma solid tumor

The d-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain, which possesses a main chain composed of α-(1→6) linked d-mannopyranosyl residues and a small proportion of branches composed of α-(1→2)- and α-(1→3)-linked d-mannopyranosyl residues, showed strong growth-inhibitory activity against mouse-implanted Sarcoma 180 and Ehrlich-carcinoma solid tumor. The observation that the level of this activity was nearly identical with that of the d-mannan of a wild-type strain of bakers' yeast, which possesses a high proportion of branches composed of α-(1→2)- and α-(1→3)-linked d-mannopyranosyl residues, suggests that the branches are not essential for antitumor activity. The partial acid-degradation products of both d-mannans, the molecular weight of which was one-third of that of each parent d-mannan, had only one half of the antitumor activity of the parent d-mannans. This suggests that molecular size is the most important factor for the differences in activity of the polysaccharides of wild and mutant strains.     

Characterization of anti-complementary acidic heteroglycans from the seed of Coix lacryma-jobi var. ma-yuen

The anti-complementary polysaccharides, CA-1 and CA-2, were purified from the seed of Coix lacryma-jobi L. var. ma-yuen. CA-1 consists of rhamnose, arabinose, xylose, galactose, galacturonic acid and glucuronic acid in molar ratios of 1.8:43.8:10.8:33.2:3.2:7.2, and CA-2 consists of rhamnose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid and glucuronic acid in molar ratios of 2.4:37.0:11.8:1.7:35.6:2.9:2.6:6.0. CA-1 and CA-2 contained 8 ∼ 11% protein. Their M_rs were estimated to be 160 000 in CA-1 and 70 000 in CA-2 by gel filtration. CA-2 showed more potent anti-complementary activity than CA-1 in low dose.Methylation analysis of CA-1, its carboxyl-reduced products (reduced CA-1a and CA-1b) and CA-2 were carried out by the use of GC/MS and the results suggested that CA-1 has a very complicated and highly branched structure, and CA-2 is also composed of the same glycosidic linkages as CA-1 in different molar proportions. The results of exo α-L-arabinofuranosidase treatment and partial acid hydrolysis suggested that CA-1 and CA-2 contained arabino 3,6-galactan moiety and most of the arabinose was present as an α-L-furanosyl residues in the non-reducing terminals and (1 → 5)-linked side chains which mostly attached to the O-3 of (1 → 6)-linked galactopyranosyl residues. The results also suggested that CA-1 and CA-2 contained rhamnogalacturonan moiety which has a main chain consisting of (1 → 4)-linked galacturonic acid and (1 → 2)-linked rhamnose, and arabino 3,6- and 4-galactan might be attached to the rhamnosyl residue at the O-4. All glucuronic acid residues were present at the non-reducing terminals.     

Comparison of Structure and Antitumor Activity of Polysaccharides Isolated from Fukurotake,the Fruiting Body of Volvariella volvacea

An α-linked d-manno-d-galactan, glycogen, and three kinds of branched (1→3)-γ-d-glucans were isolated from Fukurotake, the fruiting body of Volvariella volvacea by successive extractions with cold and hot water, cold and hot alkali, and dimethyl sulfoxide. The mannogalactan, purified from the cold water extract, had a MW of 4x 10⁵, and consisted of an α-(1→6) linked d-galactose backbone, one out of every three d-galactose residues being substituted with a single α-d-mannosyl group. The glycogen, isolated from the hot water extract, had a MW of 12 × 10⁵, and 14 ~ 15 d-glucose residues as an average chain length, as revealed by methylation analysis. The α-(1→4)-linked unit chains of this glycogen were distributed from DP 6 to 13, approximately in equal numbers.

There were three kinds of branched (1→3)-γ-d-glucans, isolated from alkali and dimethyl sulfoxide extractions. They contain a backbone of (1→3)-linked d-glucose residues with side chains of single d-glucosyl groups, but having different degrees of branching. In addition, alkali-extracted glucans contain small but significant proportions of (1→6)-linked sugar units.

Among these polysaccharides, the cold alkali-extracted glucan (degree of branching, 1:5) showed a potent antitumor activity against Sarcoma 180 solid tumors implanted in mice, and chemical modifications changed its original activity, confirming our previous results. Other polysaccharides, such as the mannogalactan and other (1→3)-γ-d-glucans, showed no or lower antitumor activity.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

Structure of Hemicellulose Isolated from Rice Endosperm Cell Wall : Mode of Linkages and Sequences in Xyloglucan, β-Glucan and Arabinoxylan

A hemicellulosic polysaccharide, which was homogeneous on sedimentation analysis and also on electrophoresis, was isolated from the rice endosperm cell walls by the combination of alkaline extraction, ion exchange chromatography and iodine complex formation. It is composed of arabinose, xylose and glucose (molar ratio, 1.0: 2.0: 5.7) together with a small amount of galactose and rhamnose. Methylation analysis, Smith degradation and fragmentation with cellulase showed that this polysaccharide is composed of three distinct polysaccharide moieties i.e., xyloglucan, β-glucan and arabinoxylan. The xyloglucan consists of β-(1→4)-linked glucan back bone and short side chains of single xylose units or galactosylxylose both attached to C-6 of the glucose residues. The β-glucan contains both (1 →3)-and (1→4)-linkages similarly to the other cereal β-glucans, but differ from them in containing the blocks of (1→3)-linked glucose residues in the chain. The arabinoxylan has a highly branched structure, in which 78% of (1→4)-linked xylose residues have short side chains of arabinose at C-3 position.

On the basis of these findings, the interconnection of these polysaccharide moieties is discussed.     

High-temperature enhancement of 13C-N.M.R. chemical-shifts of unusual dextrans,and correlation with methylation structural analysis

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and Streptobacterium dextranicum B-1254 were examined by ¹³C-n.m.r. spectroscopy at 34 and 90°, and by methylation structural analysis. The native, structurally homogeneous dextran from L. mesenteroides NRRL B-1402 was also examined. The data allow correlations to be made between the structure and physical properties of the S (soluble) and L (less-soluble) fraction pairs of dextrans B-742, B-1254, B-1299, and B-1355. For the dextrans under consideration here, increasing solubility of the dextran (both in water and in aqueous ethanol) was found to correlate with decreasing percentages of α-d-(1→6)-linked d-glucopyranosyl residues. Both the diagnostic nature of the 70–75-p.p.m. spectral region with regard to type of dextran branching, and the increase in resolution of the polysaccharide spectra at higher temperatures, have been further confirmed.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.