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.     

Isolation, characterization, and antitumor activities of the cell wall polysaccharides from Elsinoe leucospila.

A cell-wall preparation from the cells of Elsinoe leucospila, which produces elsinan extracellularly when grown on sucrose or glucose-potato extract medium, was fractionated systematically. The heteropolysaccharide that was released by treatment with Actinase E digestion, comprised D-mannose, D-galactose, and D-glucose (molar ratio, 1.5:1.0:0.1). Methylation, mild acid hydrolysis, and 13C-NMR studies suggested that the polysaccharide contains a backbone of alpha-(1----6)-linked D-mannose residues having two kinds of side chains, one attached at the O-4 with single or short beta-(1----6)-linked D-galactofuranosyl residues, and the other attached at O-2 with short side chains, most probably, of alpha-(1----3)-linked D-mannopyranosyl residues. A moderately branched D-glucan fraction, obtained from the cold alkali extract, was fractionated to give an antitumor-active purified beta-(1----3)-glucan having branches of single beta-D-glucosyl groups, one out of eight D-glucose residues being substituted at the O-6.     

Isolation and structural study of polysaccharides from campion Silene vulgaris

Silenan SV, a pectic polysaccharide, was isolated from the aerial part of Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn.), widespread through the European North of Russia. The polysaccharide was found to contain residues of galacturonic acid (63%), arabinose, galactose, and rhamnose as the main constituents. The results of a partial acidic hydrolysis, pectinase digestion, and NMR studies of silenan SV indicated that its molecule contains a linear alpha-1,4-D-galacturonan backbone and ramified regions. The core of the ramified regions is composed of residues of alpha-1,4-D-galacturonic acid along with 2-substituted alpha-rhamnopyranose residues. The NMR data showed that the silenan SV side chains are composed of the blocks built from the terminal alpha-1,5-linked arabinofuranose and beta-1,4-linked galactopyranose residues; these most likely are the side chains of rhamnogalacturonan, characteristic of other pectic polysaccharides. The nonreducing ends of these side chains contain alpha-arabinofuranose residues.     

The structure of carbohydrate unit B of porcine thyroglobulin.

The oligosaccharide fraction was obtained from porcine thyroglobulin by hydrazinolysis. Four fractions of unit B-type oligosaccharides were purified by successive chromatographies on columns of DEAE-cellulose and concanavalin A-Sepharose, and their structures were investigated by the combination of endo- and exo-glycosidase digestions, methylation analysis and Smith degradation. From the results of these studies, the structures of the unit B oligosaccharides were proposed to be as follows: see formula in text. Thus the glycoprotein was found to have triantennary and biantennary complex-type oligosaccharides as acidic sugar chains. Concerning the triantennary oligosaccharides, the following structural features were shown: (1) the sialic acid residues were not localized on certain specific branches but distributed on all three branches; (2) however, alpha (2 leads to 3)-linked sialic acid residues were exclusively located on the terminal of the branch arising from C-4 of the branching alpha-mannose residue, whereas alpha (2 leads to 6)-linked sialic acid residues occupied terminals of the other branches; (3) the outer branching alpha-mannose residue was attached to C-3 or C-6 of an inner branching beta-linked mannose residue, and both types were observed to exist.     

Structural Studies on Pectin from Marsh Cinquefoil <Emphasis Type="Italic">Comarum palustre</Emphasis> L.

Pectin with [alpha]D(20) +192 degrees (c 0.1; water), named comaruman, was isolated from marsh cinquefoil Comarum palustre L., which is widespread in the European North. The sugar chain of comaruman contains residues of D-galacturonic acid (64%), D-galactose (13%), L-rhamnose (12%), L-arabinose (6%), and trace amounts of xylose and glucose. Partial acid hydrolysis and digestion with pectinase demonstrated that comaruman composed of the backbone comprised regions of linear alpha-1,4-D-galactopyranosyl uronan interconnected by numerous residues of alpha-1,2-L-rhamnopyranose. In addition to the backbone (core of the macromolecule), ramified regions are involved in comaruman and comprise alpha-2,4-L-rhamno-alpha-4-D-galacturonan with side chains consisting mainly of beta-1,4-linked residues of D-galactopyranose. The ramified region contains additionally residues of 5-O-substituted arabinofuranose and 3- and 6-O-substituted galactopyranose. The present 3,4- and 4,6-di-O-substituted residues of galactopyranose appear to be branching points of the side chains. Some galactopyranose residues were found to occupy the terminal positions of the side chains or appeared to be single sugar residues attached to the side chains. Methylation analysis data indicated that comaruman contains residues of terminal, 3- and 3,4-di-O-substituted galactopyranosyl uronic acid, which appeared to be constituents of the side chains, and the latter represented additionally branching points of the backbone.     

Structural features of an arabinogalactan from the seeds of Becium filamentosum

The Cetavlon non-precipitable fraction of Becium filamentosum seed mucilage on DEAE-cellulose column chromatography yielded three fractions. The major polysaccharide was composed of L-rhamnose, D-galactose and L-arabinose (1:2:2). Structural analysis revealed a (1 → 4)-linked D-galactopyranose backbone with occasional side chains at O-6 of (1 → 5)-linked L-arabinofuranose terminating in rhamnopyranosyl residues.     

Structural and immunochemical studies on D-arabino-D-mannans and D-mannans of Mycobacterium tuberculosis and other Mycobacterium species.

Serologically active D-arabino-D-mannas ([alpha]D, +82 degrees approximately 89 degrees; ratio of D-arabinose to D-mannose, 1-2:1) were isolated from the soluble fraction of disintegrated cells of M. tuberculosis, M. smegmatis, and several other Mycobacterium species. These arabinomannans had similar structures, consisting of alpha-(1 leads to 5)-linked D-arabinose residues and alpha-(1 leads to 6)-, and (1 leads to 2)-linked D-mannose residues. Methylation and enzymic degradation studies using Arthrobacter sp. alpha-D-mannosidase and M-2 enzyme (D-arabinan hydrolase) indicated that the arabinomannan of M. tuberculosis Aoyama B possesses short side chains built up from alpha-(1 leads to 2)-D-mannosidic linkages which are attached to an alpha-(1 leads to 6)-linked mannan back-bone chain. The alpha-(1 leads to 5)-linked D-arabinose residues located in the side chains were shown, by comparison of the immunochemical activities of the native and enzyme-degraded polysaccharides, to be the main immunodeterminants, as in the cell-wall arabinogalactan. There appeared to be variations in the ratio of arabinose and mannose residues, and also in the proportion of (1 leads to 2)-linked D-mannose units, depending on the individual strain; no (1 leads to 2)-mannosidic linkage was found in M. smegmatis arabinomannan. In addition to arabinomannan, a serologically inactive alpha-D-mannan ([alpha)D, +65 degrees approximately 68 degrees), whose structure may resemble that of the core mannan of the arabinomannan, was isolated as a copper hydroxide complex from the soluble fraction of disintegrated mycobacterial cells.     

An L-arabino-D-galactan and an L-arabino-D-galactan-containing proteoglycan from radish (Raphanus sativus) seeds

An l-arabino-d-galactan and an l-arabino-d-galactan-containing proteoglycan were isolated from hot phosphate-buffered saline extracts of radish seeds by ethanol fractionation, ion-exchange chromatography, and gel filtration, and found homogeneous by ultracentrifuge analysis and high-voltage electrophoresis. The proteoglycan consisted of 86% of a polysacchraide component containing β-l-arabinose and d-galactose as major sugar constituents, together with small proportions of d-xylose, d-glucose, and uronic acids, and 9% of a hydroxyproline-containing protein. Methylation analysis, periodate oxidation, and enzymic degradations indicated a backbone chain of (1å3)-linked β-dgalactosyl residues with side chains at O-6 of (1å6)-linked β-d-galactosyl residues and uronosyl groups. The α-l-arabinofuranosyl residues were located mainly in the outer regions as nonreducing groups, as well as O-2- or -5-linked inner chain residues, and O-2,5- or -3,5-linked branching residues. Reductive, alkaline degradation of the proteoglycan indicated that the polysaccharide chains were partly linked through O-glycosyl linkages to the threonine residues of the polypeptide chains. The proteoglycans from radish leaves and seeds appeared to share common antigenic determinant(s). The radish-seed arabinogalactan had a high content (81%) of l-arabinose and its basic structure seemed to be similar to that of the polysaccharide component of the proteoglycan.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

Structural studies on hairy region of pectic polysaccharide from campion Silene vulgaris (Oberna behen)

Using enzymic digestion with pectinase, controlled Smith degradation and NMR-spectroscopy, some structural features of the hairy region of pectic polysaccharide termed silenan SV from the aerial part of campion Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn) were elucidated.

Silenan was subjected to enzymic digestion with pectinase to furnish the polysaccharide fraction (SVP). The contained residues of -galacturonic acid (43%), arabinose, galactose and rhamnose as main constituents. The backbone of the hairy region of silenan was found to consist of -1,4-galactopyranosyl uronic acid and 2-O-glycosylated rhamnopyranose residues. The side chains contained linear regions of residues of -1,5-linked arabinofuranose and β-1,3-, β-1,4-linked galactopyranose. Silenan SV and its fragment SVP were subjected to Smith degradation to give fractions SVS and SVPS. These contain the residues of terminal and 2-substituted -arabinofuranose as well as residues of terminal, 3-, and 2,3-substituted β-galactopyranose. In addition, NMR-spectral data confirmed that the residues of -rhamnopyranose 2-O-glycosylated with the residues of -1,4-galactopyranosyl uronic acid of the backbone occurred in the core of SVPS and, therefore, in the backbone of silenan SV.

On the basis of data obtained, the hairy regions of silenan were suggested to contain mainly the linear chains of β-1,3-, β-1,4-galactopyranan and -1,5-arabinofuranan. The chains of -1,5-linked arabinofuranose, β-1,3- and β-1,4-linked galactopyranose were shown to be involved in the side chains of the hairy region having branching points at 2,3-substituted β-galactopyranose residues.     

Structural and immunological studies of a major polysaccharide from spores of Ganoderma lucidum (Fr.) Karst

A polysaccharide isolated from spores of the fungus, Ganoderma lucidum, was found to be a complex glucan. On the basis of compositional and methylation analyses, periodate oxidation, Smith degradation, 1D and 2D NMR, and ESIMS experiments of the native polysaccharide and its degraded products, the polysaccharide was shown to have a backbone of beta-(1-->3)-linked D-glucopyranosyl residues, with branches of mono-, di- and oligosaccharide side chains substituting at the C-6 of the glucosyl residues in the main chain. Conformational analysis in aqueous solution and immunological activities of the native and degraded glucans were also investigated. The results suggested that the degree of substitution on the main chain and the length of side chains may be very important factors in determining the conformation and the biological activities of beta-(1-->3)-linked glucans.     

Structure of Plant Cell Walls : XII. Identification of Seven Differently Linked Glycosyl Residues Attached to O-4 of the 2,4-Linked l-Rhamnosyl Residues of Rhamnogalacturonan I

Seven differently linked glycosyl residues have been found to be glycosidically linked to O-4 of the branched 2,4-linked l-rhamnosyl residues contained in the rhamnosyl and galacturonosyl backbone of the cell wall pectic polysaccharide rhamnogalacturonan I. These seven glycosyl residues are, therefore, the first residues of at least seven different side chains attached to the rhamnogalacturonan backbone. These first side chain glycosyl residues are 5-linked l-arabinofuranosyl and terminal 3-, 4-, 6-, 2,6-, and 3,6-linked d-galactopyranosyl residues. The existence of at least seven different side chains in rhamnogalacturonan I indicates that rhamnogalacturonan I is either an exceedingly complex polysaccharide or that rhamnogalacturonan I is a family of polysaccharides with similar or identical rhamnogalacturonan backbones substituted with different side chains.     

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.     

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

Treatment of rhamnogalacturonan I with lithium in ethylenediamine

Rhamnogalacturonan I is a pectic polysaccharide that is solubilized from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus) by the action of a highly purified endo-1,4-α-polygalacturonanase. Rhamnogalacturonan I has a linear backbone consisting of the diglycosyl repeating unit, →4)-α-d-GalpA-(1→2)-α-l-Rhap-(1→. Approximately half of the α-l-rhamnosyl residues of the backbone are branched at O-4. Selective cleavage at the galactosyluronic acid residues of the backbone by treatment of rhamnogalacturonan I wit lithium in ethylenediamine resulted in the release of the neutral glycosyl-residue sidechains that had been attached to the backbone. Various analytical techniques, including combined liquid chromatography-mass spectrometry, combined gas-liquid chromatography-mass spectrometry, and ¹H-nuclear magnetic resonance spectroscopy, were used to determine the structure of the side chains. The majority of the sidechains were isolated as oligoglycosylalditols, with rhamnitol at the “reducing” end. Terminal 2-, 4-, or 6-linked galactosyl residues were found attached to O-4 of the rhamnitol residues The 2-, 4-, and 6-linked galactosyl residues had terminal or 2-linked arabinosyl, or additional galactosyl, residues attached to them. Based on the results of fast-atom-bombardment mass spectrometry, the side chains were found to range in size from one to fourteen glycosyl residues. The side-chain structures suggest that there are four or more distinct families of side chains attached to the backbone of rhamnogalacturonan I.     

Structural study of bergenan, a pectin from Bergenia crassifolia

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of aqueous solutions they form. The results of enzymatic hydrolysis by alpha-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear alpha--1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-1). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constituent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the main carbohydrate chain by 1,4-link and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked (-arabinofuranose, and 1,4-and 1,6-linked beta-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC-MS, ESMS and ESMS/MS.

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.     

Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801

A 21-kDa heteropolysaccharide, coded as GFPS1b, was obtained from the cultured mycelia of Grifola frondosa GF9801 by hot-water extraction, ethanol precipitation, and fractioned by DEAE Sepharose Fast-flow, followed by the purification with Sephadex G-100 column chromatography using an AKTA purifier. It exhibited more potent anti-proliferative activity on MCF-7 cells than other polysaccharide fractions. GFPS1b was an acidic polysaccharide with approximately 16.60% protein and 4.3% uronic acid. Gas chromatography of absolute acid hydrolysate of GFPS1b suggested that it was composed of D-glucose, D-galactose, and L-arabinose with a molar ratio of 4:2:1. Periodate oxidation, Smith degradation, partial acid hydrolyzation, methylation analysis, FT-IR, and (1)H, (13)C NMR spectroscopy analysis revealed that GFPS1b had a backbone consisting of alpha-(1-->4)-linked D-galacopyranosyl and alpha-(1-->3)-linked D-glucopyranosyl residues substituted at O-6 with glycosyl residues composed of alpha-L-arabinose-(1-->4)-alpha-D-glucose (1--> linked residues.     

An Antioxidant Acidic Polysaccharide from Cuscuta chinensis

An acidic polysaccharide, H2, was isolated from the alkali-extract CHC of seeds of Cuscuta chinensis Lam. with the molecular weight more than 1.0×106. Chemical and spectroscopic studies led to the structure determination as follows: the backbone chain consists of 1,6-linked-β- D Galp, 1,4-linked-β- D Galp, 1,4-linked-β- D GalA and 1,2- or 1,4-linked-β- L Rhap having branching points at position O-3 of some 1,6-linked-β- D Galp residues (one among eight) and O-4 or O-2 of 1,2- or 1,4-linked-β- L Rhap residues to terminal β-D-galactopyranose. The side chains composed of terminal Galp, 1,6-linked-β- D Galp, 1,4-linked β- D Galp and 1,3,6-linked-β- D Galp also linked at position O-3 of 1,6-linked-β- D Galp residues in the backbone chain. β- L -arabinofuranosyl and terminal β- L -rhamnopyranosyl residues existed in the periphery of this polysaccharide linked to O-3 of 1,6-linked-β- D Galp residues in the backbone chain and the side chains. The polysaccharide H2 increased significantly the survival rate of PC12 cells indicating that it had protective effects against H2O2 insult.     

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.