NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

Structure of a fucoidan from the brown seaweed Fucus serratus L

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1:0.1 and small amounts of xylose and galactose were isolated from the brown seaweed Fucus serratus L. The fucoidan structure was investigated by 1D and 2D 1H and 13C NMR spectroscopy of its desulfated and de-O-acetylated derivatives as well as by methylation analysis of the native and desulfated polysaccharides. A branched structure was suggested for the fucoidan with a backbone of alternating 3- and 4-linked alpha-L-fucopyranose residues, -->3)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->, about half of the 3-linked residues being substituted at C-4 by trifucoside units alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-alpha-L-Fucp-(1-->. Minor chains built up of 4-linked alpha-fucopyranose and beta-xylose residues were also detected, but their location, as well as the position of galactose residues, remained unknown. Sulfate groups were shown to occupy mainly C-2 and sometimes C-4, although 3,4-diglycosylated and some terminal fucose residues may be nonsulfated. Acetate was found to occupy C-4 of 3-linked Fuc and C-3 of 4-linked Fuc in a ratio of about 7:3.     

Substrate specificity of the alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

The alpha-L-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1-->5)-alpha-L-arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [alpha-(1-->5)-linked] of the arabinan backbone rather than the arabinosyl side chain [alpha-(1-->3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1-->5)-Araf, T-Araf, (1-->3, 5)-Araf and (1-->3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1-->5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [alpha-(1-->5)-linked residues]. Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and 13C NMR spectra are dominated by signals of the alpha-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this alpha-L-arabinofuranosidase can only hydrolyse alpha-L-arabinofuranosyl residues of arabinan.     

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.     

Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1.23:0.36 was isolated from the Pacific brown seaweed Fucus evanescens. The structures of its desulfated and de-O-acetylated derivatives were investigated by 1D and 2D (1)H and (13)C NMR spectroscopy, and the data obtained were confirmed by methylation analysis of the native and desulfated polysaccharides. The fucoidan was shown to contain a linear backbone of alternating 3- and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp(2SO(3)(-))-(1-->4)-alpha-L-Fucp(2SO(3)(-))-(1-->. Additional sulfate occupies position 4 in a part of 3-linked fucose residues, whereas a part of the remaining hydroxyl groups is randomly acetylated.     

Synthesis of beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3) linked bond with antitumor activity

A beta-(1-->6)-branched beta-(1-->3)-glucohexaose, present in many biologically active polysaccharides from traditionally herbal medicines such as Ganoderma lucidum, Schizophyllum commune and Lentinus edodes, was synthesized as its lauryl glycoside 32, and its analogues 18, 20 and 33 containing an alpha-(1-->3) linked bond were synthesized. It is interesting to find that coupling of a 3,6-branched acylated trisaccharide trichloroacetimidate donor 9 with 3,6-branched acceptors 13 and 16 with 3'-OH gave the alpha-(1--> 3)-linked hexasaccharides 17 and 19, respectively, in spite of the presence of C-2 ester capable of neighboring group participation. However, coupling of 9 with 4-methoxyphenyl 4,6-O-benzylidene-beta-D-glucopyranoside (27) selectively gave beta-(1-->3)-linked tetrasaccharide 28. Simple chemical transformation of the tetrasaccharide 28 gave acylated tetrasaccharide trichloroacetimidate 29. Coupling of 29 with lauryl (1-->6)-linked disaccharide 26 with 3-OH gave beta-(1-->3)-linked hexasaccharide 30 as the major product. Bioassay showed that in combination with the chemotherapeutic agent cyclophospamide (CPA), the hexaose 18 at a dose of 0.5-1mg/kg substantially increased the inhibition of S(180) for CPA, but decreased the toxicity caused by CPA. Some of these oligosaccharides also inhibited U(14) noumenal tumor in mice effectively.     

Remote control of alpha- or beta-stereoselectivity in (1-->3)-glucosylations in the presence of a C-2 ester capable of neighboring-group participation

In (1-->3)-glucosylation the glycosyl bond originally present in either donor or acceptor is shown to control the stereoselectivity of the forthcoming bond, i.e., the newly formed glycosidic linkage has the opposite anomeric configuration of that of either the donor or acceptor. Therefore, with alpha-(1-->3)-linked disaccharides with nonreducing ends that have the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with an alpha-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with 3-OH free as the acceptor, beta-linked trisaccharides were obtained. Meanwhile, with beta-(1-->3)-linked disaccharides that have nonreducing ends with the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with a beta-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with the 3-OH free as the acceptor, alpha-linked trisaccharides were obtained in spite of the C-2 neighboring group participation.     

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6 microg/ml were much lower than the cytotoxic concentration 50% (CC50) values (1000 microg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60-100 microg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160 kDa, contained a backbone of alpha-(1-->3)-linked D-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of beta-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of alpha-(1-->3)-linked D-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.     

Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and methylation analysis

Two galactofuranomannans, Ths-4 and Ths-5, were isolated from the lichen, Thamnolia vermicularis var. subuliformis, using ethanol fractionation and anion-exchange and size-exclusion chromatography. The average molecular weights of Ths-4 and Ths-5 were estimated to be 19 and 200 kDa, respectively. Structural characterisation of Ths-4, Ths-5 and their partially hydrolysed derivatives was performed by methanolysis and methylation analysis. The intact and partially hydrolysed Ths-4 was further analysed using NMR spectroscopy (1D, COSY, NOESY, TOCSY, HSQC and HMBC). According to the data obtained, the heteroglycans Ths-4 and Ths-5 have similar structures, but have large differences in molecular weight. The structure is composed of 3-O-linked and 5-O-linked galactofuranosyl chains linked to a mannan core. The mannan core consists of a main chain of alpha-(1-->6)-linked mannopyranosyl residues, substituted at O-2 with either a single alpha-mannopyranosyl unit or an alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown.     

Comparative studies of the polysaccharides from species of the genus Ramalina-lichenized fungi-of three distinct habitats

Several structurally different glucans (alpha- and beta-) and galactomannans were characterized as components of four species of the genus Ramalina, namely R. dendriscoides, R. fraxinea, R. gracilis and R. peruviana. Freeze-thawing treatment of hot aqueous extracts furnished as precipitates (PW) linear alpha-D-glucans of the nigeran type, with regularly distributed (1-->3)- and (1-->4)-linkages in a 1:1 ratio. The supernatants (SW) contained alpha-D-glucans with (1-->3)- and (1-->4)-linkages in a molar ratio of 3:1. The lichen residues were then extracted with 2% aq. KOH, and the resulting extracts submitted to the freeze-thawing treatment, giving rise to precipitates (PK2) of a mixture of alpha-glucan (nigeran) and beta-glucan, which were suspended in aqueous 0.5% NaOH at 50 degrees C, dissolving preferentially the beta-glucan. These were linear with (1-->3)-linkages (laminaran). The mother liquor of the KOH extractions (2% and 10% aq. KOH) was treated with Fehling's solution to give precipitates (galactomannans). The galactomannans are related, having (1-->6)-linked alpha-D-mannopyranosyl main chains, substituted at O-4 and in a small proportion at O-2,4 by beta-D-galactopyranosyl units. Despite the different habitats of these lichenized fungi, all species studied in this investigation have a similar pool of polysaccharides.     

Polysaccharides from Gracilaria corticata: sulfation, chemical characterization and anti-HSV activities

In this study, we have analyzed water-extracted polysaccharides of Gracilaria corticata. The water extract (WE), a galactan-containing sub-fraction (F3) and their hyper sulfated derivatives (WES1, WES2, F3S1 and F3S2) had anti-HSV activity with inhibitory concentration 50% (IC50) from 1.1 to 27.4 microg/ml. Sub-fraction F3, which has a molecular mass of 30 kDa, consists of a backbone of beta-(1-->3) and alpha-(1-->4)-linked-galactopyranosyl residues. This linear galactan contained Gal2Xyl1, Gal2AnGal2, Gal4 and Me-Gal3AnGal2 as oligomeric building subunits. Sulfate group was located at C-4 of (1-->3)-linked galactopyranosyl residues of the native galactan, and appeared to be very important for the anti-herpetic activity.     

Candida albicans cell wall comprises a branched beta-D-(1-->6)-glucan with beta-D-(1-->3)-side chains

The structure of immunogenic and immunomodulatory cell wall glucans of Candida albicans is commonly interpreted in terms of a basic polysaccharide consisting of a beta-D-(1-->3)-linked glucopyranosyl backbone possessing beta-D-(1-->6)-linked side chains of varying distribution and length. This proposed molecular architecture has been re-evaluated by the present study on the products of selective enzymolysis of insoluble C. albicans glucan particles (GG). High resolution 1H (400 and 700 MHz) and 13C (100 and 175 MHz) NMR analyses were performed on a soluble beta-glucan preparation (GG-Zym) obtained by GG digestion with endo-beta-D-(1-->3)-glucanase and on its high- (Pool 1) and low-molecular weight (Pool 2) sub-fractions. The resonances typical of uniformly beta-D-(1-->6)- and beta-D-(1-->3)-linked linear glucans, together with additional multiplets assigned to short-chain oligoglucosides, were detected in GG-Zym. Pool 1 (46.3+/-6.4% of GG-Zym content) consisted of beta-D-(1-->6)-linked glucopyranosyl polymers, with short beta-D-(1-->3)-branched side chains of 2.20+/-0.02 units (branching degree (DB)=0.14+/-0.03). Pool 2 was a mixture of glucose and linear short-chain beta-D-(1-->3)-oligoglucosides. Further digestion of Pool 1 by beta-D-(1-->6)-glucanase yielded a mixture of glucose and short beta-D-(1-->6)-linked, either linear or beta-D-(1-->3,6) branched, oligomers. These endoglucanase digestion patterns were consistent with the presence in C. albicans cell wall glucans of beta-D-(1-->6)-linked glucopyranosyl backbones possessing beta-D-(1-->3)-linked side chains, a structure very close to that of beta-D-(1-->6)-glucan from Saccharomyces cerevisiae yeast. This finding may provide the grounds for further elucidation of the cell wall structure and a better understanding of the biological properties of C. albicans beta-glucans.     

Synthesis of mannopyranose disaccharides as photoaffinity probes for mannosyltransferases in Mycobacterium tuberculosis

Mannosyltransferases play a crucial role in mycobacterial cell-wall biosynthesis and are potential new drug targets for the treatment of tuberculosis. Herein, we describe the synthesis of alpha-(1-->2)- and alpha-(1-->6)-linked mannopyranosyl disaccharides possessing a 5-azidonaphthlene-1-sulfonamidoethyl group as photoaffinity probes for active-site labeling studies of mannosyltransferases in Mycobacterium tuberculosis.     

A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7. Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.     

Polysaccharide recognition by surfactant protein D: novel interactions of a C-type lectin with nonterminal glucosyl residues.

Surfactant protein D (SP-D), a C-type lectin, is an important pulmonary host defense molecule. Carbohydrate binding is critical to its host defense properties, but the precise polysaccharide structures recognized by the protein are unknown. SP-D binding to Aspergillus fumigatus is strongly inhibited by a soluble beta-(1-->6)-linked but not by a soluble beta-(1-->3)-linked glucosyl homopolysaccharide (pustulan and laminarin, respectively), suggesting that SP-D recognizes only certain polysaccharide configurations, likely through differential binding to nonterminal glucosyl residues. In this study we have computationally docked alpha/beta-D-glucopyranose and alpha/beta-(1-->2)-, alpha/beta-(1-->3)-, alpha/beta-(1-->4)-, and alpha/beta-(1-->6)-linked glucosyl trisaccharides into the SP-D carbohydrate recognition domain. As with the mannose-binding proteins, we found significant hydrogen bonding between the protein and the vicinal, equatorial OH groups at the 3 and 4 positions on the sugar ring. Our docking studies predict that alpha/beta-(1-->2)-, alpha-(1-->4)-, and alpha/beta-(1-->6)-linked but not alpha/beta-(1-->3)-linked glucosyl trisaccharides can be bound by their internal glucosyl residues and that binding also occurs through interactions of the protein with the 2- and 3-equatorial OH groups on the glucosyl ring. By using various soluble glucosyl homopolysaccharides as inhibitors of SP-D carbohydrate binding, we confirmed the interactions predicted by our modeling studies. Given the sequence and structural similarity between SP-D and other C-type lectins, many of the predicted interactions should be applicable to this protein family.     

Dextran acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I as revealed by using uniformly 13C-labeled sucrose.

A sucrose glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus transferred the uniformly 13C-labeled glucosyl residue ([U-(13)C]Glc) from [U-(13)C]sucrose to exogenous dextran T500 at the non-reducing-end, mostly by alpha-(1-->6) linkages and partially by alpha-(1-->3) linkages, as revealed by the 13C-(13)C NMR coupling pattern. With increasing amounts of [U-(13)C]sucrose, transfer of [U-(13)C]Glc to the alpha-(1-->3)-linked chain became predominant without increase in the number of chains. The transfer of [U-(13)C]Glc to an isomaltopentaose acceptor occurred similarly to its transfer to T500. alpha-(1-->3)-branches in the [U-(13)C]dextran, specifically synthesized from [U-(13)C]sucrose by a Streptococcus bovis dextransucrase, were not formed by GTF-I, as judged by the observation that a newly-formed alpha-1,3,6-branched [U-(13)C]Glc was not detected, which could have been formed by transferring the unlabeled Glc from sucrose to the internal alpha-(1-->6)-linked [U-(13)C]Glc at C-3. The 13C-(13)C one-bond coupling constants (1J) were also recorded for the C-1--C-6 bond of the internal alpha-(1-->6)-linked [U-(13)C]Glc and of the non-reducing-end [U-(13)C]Glc.     

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases

Glucanohydrolases, especially mutanase [alpha-(1-->3) glucanase; EC 3.2.1.59] and dextranase [alpha-(1-->6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C [synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1-->6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.     

On the stringent requirement of mannosyl substitution in mannooligosaccharides for the recognition by garlic (Allium sativum) lectin. A Surface Plasmon Resonance Study

The kinetics of the binding of mannooligosaccharides to the heterodimeric lectin from garlic bulbs was studied using surface plasmon resonance. The interaction of the bound lectin immobilized on the sensor chip with a selected group of high mannose oligosaccharides was monitored in real time with the change in response units. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal alpha-1,2-linked mannose residues. An increase in binding propensity can be directly correlated to the addition of alpha-1,2-linked mannose to the mannooligosaccharide at its nonreducing end. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied, exhibited the greatest binding affinity (Ka = 1.2 x 10(6) m(-1) at 25 degrees C). An analysis of these data reveals that the alpha-1,2-linked terminal mannose on the alpha-1,6 arm is the critical determinant in the recognition of mannooligosaccharides by the lectin. The association (k1) and dissociation rate constants (k(-1)) for the binding of Man9GlcNAc2Asn to Allium sativum agglutinin I are 6.1 x 10(4) m(-1) s(-1) and 4.9 x 10(-2) s(-1), respectively, at 25 degrees C. Whereas k1 increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k(-1) decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with Allium sativum agglutinin I with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition.     

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) seedlings

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.