Branched glucan from the fruiting bodies of Piptoporus betulinus (Bull.:Fr.) Karst

A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination ofphysicochemical methods, it was established that piptoporane I was a branched glucan with a backbone consisting of alpha-( 1->3)-glucopyranose residues substituted at the C-6 position by single residues of beta-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from the fungus genus Piptoporus.     

Production and physicochemical characterization of β-glucan produced by<Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JB115

This study was conducted to develop a bacterial glucan as an animal feed additive. A novel glucan-producing bacterium.Paenibacillus polymyxa JB115, was isolated from Korean soil. The glucan, JB115-BG, produced byP. polymyxa JB115, was confirmed by TLC to be composed of glucose only. By examining FT-IR,¹H NMR, and¹³C NMR spectra, it was proven that JB115-BG has a β-(1→3)- and β-(1→6)-linked glucan structure. The particle size of JB115-BG was distributed in the range of 4–800 μm, with a mean value of 149.1 μm, and its molecular distribution ranged from 6.9∼3,103.7 kDa. It was also observed that 80% of the purified JB115-BG had a molecular distribution above 100 kDa. The obtained results suggest that the glucan JB115-BG can be used as an animal feed additive for the purpose of enhancing immunity.     

Cell-wall Polysaccharides of Immature Barley Plants. I. Isolation and Characterization of a β-d-Glucan

A β-d-glucan was isolated on fractionation of a 4% potassium hydroxide extract (hemi-celluloses) of immature barley plants (Hordeum distichum L.). Most of the glucose residues in the extract were found to be derived from the glucan. Methylation analysis and enzyme degradation studies showed that the glucan had (l-→3)-and (1-→4)-linked d-glucopyranosyl residues in an approximate molar ratio of 1.0:2.3. The molecular weight of the glucan was estimated to be 1.8 x 10⁵ by gel filtration on Sepharose CL-6B.     

High level accumulation of α-glucan in maize kernels by expressing the <Emphasis Type="Italic">gtfD</Emphasis> gene from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Glucosyltransferases (GTFs, EC.2.4.1.5) are bacterial enzymes that catalyze the polymerization of glucose residues from sucrose, leading to the production of high molecular weight glucan with α-1,3 /α-1,6 linkages. Such glucans, with many potential food and industrial applications, do not normally exist in higher plants. We fused a mutant form of the gtfD gene from Sreptococcus mutans with the maize (Zea mays L.) chloroplastic Brittle 1 transit peptide for amyloplast targeting. This construct, driven by the ubiquitin promoter, was introduced into maize by Agrobacterium-mediated transformation. We developed a novel HPLC-based method that enabled us differentially to distinguish transgene glucan from other endogenous polysaccharides in maize kernels. Using this method, we screened over 100 transgenic plants for the presence of GTF-produced glucan whose content varied between 0.8 and 14% of dry weight in the mature transgenic seeds. The mature transgenic plants were indistinguishable from wildtype plants in growth rate and morphology. Furthermore, starch granule size in the transgenic maize kernel was unaffected by the accumulation of the foreign polysaccharide. Mutation in Sh2, which encodes a subunit of ADP-glucose pyrophosphorylase, had no effect on glucan accumulation caused by gtfD expression. Our results indicated that high levels of novel carbohydrate polymer can be accumulated in crop plants through transgene technology.     

Host-Pathogen Interactions: XI. Composition and Structure of Wall-released Elicitor Fractions

The structures of the four wall-released elicitor fractions isolated from the Phytophthora megasperma var. sojae mycelial walls have been examined. The results demonstrate that fraction I is primarily composed of a branched β-1,3-glucan, similar in structure to the extracellular elicitors described previously (Ayers, A., J. Ebel, F. Finelli, N. Burger, and P. Albersheim. 1976. Plant Physiol. 57: 751-759). Fractions II and IV are primarily composed of a highly branched mannan-containing glycoprotein, with fraction IV richer in protein than fraction II. Fraction III contains, attached to protein, a mixture of the two polysaccharide types found in fraction I and in fractions II and IV. The structural data presented here, in concert with the biological data presented in the previous two papers (Ayers et al. 1976. Plant Physiol. 57: 751-759; 760-765), demonstrate that the only compound produced by P. megasperma var. sojae which contains elicitor activity is the glucan. Evidence is presented that the terminal glycosyl residues of the glucan are required for elicitor activity. In addition, it is demonstrated that 90% of the glucan can be removed enzymically without any loss of biological activity. The active residue of the enzymic digestion is a highly branched 3- and 3,6-linked glucan containing about 4% mannosyl residues. The results presented suggest that the mannosyl residues of the glucan, which represent only about 1% of the undegraded glucan, are likely to participate in the active site of this molecule. The role of elicitors and phytoalexins in host-pathogen interactions is discussed. Evidence for the existence of and possible identity of another factor, which determines race specificity of host-pathogen interactions, is summarized.     

Structures and Antitumor Activities of the Polysaccharides Isolated from Fruiting Body and the Growing Culture of Mycelium of Ganoderma lucidum

Several β-D-glucans, appertaining to the same molecular species but having different degrees of branching, were isolated from water and alkali extracts of the fruiting body of Ganoderma lucidum (Reishi). The purified glucans that were mostly water-insoluble had a backbone of (1 →3)-linked D-glucose residues, attached mainly with single D-glucosyl units at 0-6 and also with a few short (l→4)-linked glucosyl units at 0-2 positions. However, their degrees of branching appeared to differ in the range of d.b. 1/3 ~ 1/23, depending on the extracted glucan fractions. In addition to the ^-glucans, the fruiting body contained water-soluble heteropolysaccharides, comprising D-glucose, D-galactose, D-mannose, L-(or D)-arabinose, D-xylose, and L-fucose.

A branched (1 →3)-β-D-glucan was also isolated from the culture filtrate of G. lucidum grown in a glucose-yeast extract medium. The extracellular β-D-glucan was less soluble in water after purification, but soluble in dilute alkali. This glucan has essentially the same structure as that of hot-water extracted polysaccharide from the fruiting body. The repeating unit of the glucan contains a backbone chain of (1 →3)-linked D-glucose residues, five out of sixteen D-glucose residues being substituted at 0-6 positions with single D-glucosyl units and one D-glucose residue at 0-2 positions probably with a cellobiose unit.

The hot-water extractable fruiting body glucan and the extracellular glucan of the culture of growing mycelium showed relatively high growth-inhibition activities against Sarcoma 180 solid tumor in mice, when administered by. successive intraperitoneal injections. When the moderately branched glucans were modified to D-glucan-polyols by periodate oxidation and borohydride reduction, they exhibited higher antitumor activities, confirming the previous conclusion that the attachment of polyol groups to the (1 →3)-lmked backbone significantly enhances its host-mediated antitumor effect.     

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae

 A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of l-glutamine but also of d-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137–143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5′-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae. Received: 23 August 1999 / Received last revision: 7 January 2000 / Accepted: 14 January 2000     

Composition of the cell walls of several yeast species

Cell walls, representing 26%–32% of the cell dry weight, were prepared from several strains of the yeasts Kloeckera apiculata, Debaryomyces hansenii, Zygosaccharomyces bailii,Kluyveromyces marxianus and Saccharomyces cerevisiae. Extraction of the walls with potassium hydroxide at 4 °C, followed by saturation of the alkali-soluble extract with ammonium sulphate gave fractions of mannoprotein, alkali-soluble glucan and alkali-insoluble glucan. Chitin was associated with the alkali-insoluble glucan. The proportions of the different fractions within the walls varied with the species and strain. Mannoprotein comprised between 25% and 34% of the walls, the content of alkali-insoluble glucan ranged from 15% to 48%, and the content of alkali-soluble glucan ranged from 10% to 48%. There was significant variation in the physical appearance of the alkali-soluble glucans and the relative viscosity of suspensions of these glucans. The yeasts could represent novel sources of polysaccharides with industrial and medical applications. Received: 30 December 1997 / Received revision: 24 March 1998 / Accepted: 27 March 1998     

Catalytic properties and amino acid sequence of endo-1→3-β-D-glucanase from the marine mollusk <Emphasis Type="Italic">Tapes literata</Emphasis>

A specific 1→3-β-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45dgC. The 1→3-β-D-glucanase catalyzes hydrolysis of β-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K _m for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-β-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-β-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-β-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.     

Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.     

Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide:adenosine glycosidase activity, from leaves of Phytolacca dioica L.

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29 407 ± 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the β-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M_r values of 32 715 ± 1 (PD-L1), 31 542 ± 1 (PD-L2), 30 356 ± 1 (PD-L3) and 29 185 ± 1 Da (PD-L4). The 1171 kDa difference in M_r, within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin I and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%. Received: 5 August 1998 / Accepted: 28 October 1998     

Degradation of beta-hexachlorocyclohexane by haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205

The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal γ-isomer and noninsecticidal α-, β-, and δ-isomers, among which β-HCH is the most recalcitrant and has caused serious environmental problems. A γ-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded β-HCH more rapidly than the well-characterized γ-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, β-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB_MI) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB_UT) was identified as an enzyme responsible for the two-step conversion of β-HCH to TCDL. This property of LinB_MI contrasted with that of LinB_UT, which catalyzed only the first step conversion of β-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB_MI. However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB_MI. Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB _MI gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.     

A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography

A new screening method for β-(1,3–1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml⁻¹ in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45°C over 1 h and was stable between pH 5.8 and 12 at 4°C for 24 h and up to 40°C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l⁻¹) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.     

Structural studies of arabinogalactan and pectin from Silene vulgaris (M.) G. callus

Arabinogalactan and pectin (named silenan) were isolated from Silene vulgaris (M.) G. callus. Fractionation by ion-exchange chromatography on DEAE-cellulose and digestion with pectinase demonstrated that silenan from S. vulgaris callus (80% of D-galacturonic acid) and silenan from the aerial part of the campion S. vulgaris are similar: both pectins contain a high quantity of homogalacturonan segments. The NMR spectral data and mass spectrometry of the purified polysaccharide and its fragment obtained by Smith degradation confirmed that the core of the arabinogalactan consisted of the different segments of β-1,3-D-galactopyranan. Some of the β-galactopyranose residues of the backbone are branched at O-6. The side chains of the arabinogalactan were shown to contain residues of terminal and 3-O-substituted β-galactopyranose, terminal α-arabinofuranose and α-rhamnopyranose, and 2-O-substituted α-rhamnopyranose. The α-rhamnopyranose residues in the sugar chain appeared to be 2-O-glycosylated by the β-1,4-D-galactopyranosyl uronic acid residues. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 798–807.     

UDPGLUCOSE PYROPHOSPHORYLASE ACTIVITY IN THE DIATOM CYCLOTELLA CRYPTICA. PATHWAY OF CHRYSOLAMINARIN BIOSYNTHESIS 1

UDPglucose pyrophosphorylase activity was detected in cell-free extracts of the diatom Cyclotella cryptica TI3L Reimann, Lewin and Guillard. When assayed in the direction of UDPglucose formation, the enzyme had maximal activity at pH 7.8 and was stimulated by Mg²⁺and Mn²⁺ions. 3-Phosphoglycerate and inorganic phosphate had little effect on enzymatic activity, and the enzyme was relatively insensitive to feedback inhibition from UDPglucose (K, > I millimolar). A glucan was formed from UDP-[¹⁴C]glucose in cell-free extracts of C. cryptica. This glucan had a median molecular weight of 4600 (as determined by gel filtration chromatograbhy) and could be hydrolyzed by laminarinase. Partial acid hydrolysis of the glucan resulted in the formation of glucose and laminaribiose. but not cellobiose. These results suggest that the synthesis of chrysolaminarin (the major storage carbohydrate of diatoms) occurs via the activity of UDPglucose pyrophosphorylase. followed by glucosyl transfer from UDPglucose to the growing β-(1–3)-linked glucan.     

Host-Pathogen Interactions : XVII. HYDROLYSIS OF BIOLOGICALLY ACTIVE FUNGAL GLUCANS BY ENZYMES ISOLATED FROM SOYBEAN CELLS

The ability of β-glucosylase I, a soybean cell wall β-glucosyl hydrolase, to degrade elicitors of phytoalexin accumulation was studied. Extensive β-glucosylase I treatment of the glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae results in hydrolysis of 77% of the glucosidic bonds of the elicitor and destruction of 94% of its activity. Soybean cell walls contain some additional factor, probably one or more additional enzymes, which can assist β-glucosylase I in hydrolyzing the glucan elicitor. This was demonstrated by the more rapid hydrolysis of the glucan elicitor by a mixture of soybean cell wall enzymes (containing β-glucosylase I). In a single treatment, the mixture of cell wall enzymes hydrolyzed 91% of the glucosidic bonds and destroyed 85% of the activity of the elicitor. The enzymes from soybean cell walls will also hydrolyze elicitor-active oligoglucosides prepared from the mycelial walls of Phytophthora megasperma var. sojae. The active oligoglucosides are more susceptible than the glucan elicitor to hydrolysis by these enzymes. The mixture of cell wall enzymes or β-glucosylase I, by itself, hydrolyzes more than 96% of the glucosidic bonds and destroys more than 99% of the activity of the oligoglucoside elicitor. Two possible advantages for the existence of these enzymes in the walls of soybean cells are discussed.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Characterization of Copper-Resistant Rhizosphere Bacteria from <Emphasis Type="Italic">Avena sativa</Emphasis> and <Emphasis Type="Italic">Plantago lanceolata</Emphasis> for Copper Bioreduction and Biosorption

Copper is a toxic heavy metal widely used to microbial control especially in agriculture. Consequently, high concentrations of copper residues remain in soils selecting copper-resistant organisms. In vineyards, copper is routinely used for fungi control. This work was undertaken to study copper resistance by rhizosphere microorganisms from two plants (Avena sativa L. and Plantago lanceolata L.) common in vineyard soils. Eleven rhizosphere microorganisms were isolated, and four displayed high resistance to copper. The isolates were identified by 16S rRNA gene sequence analysis as Pseudomonas putida (A1), Stenotrophomonas maltophilia (A2) and Acinetobacter sp. (A6), isolated from Avena sativa rhizosphere, and Acinetobacter sp. (T5), isolated from Plantago lanceolata rhizosphere. The isolates displayed high copper resistance in the temperature range from 25°C to 35°C and pH in the range from 5.0 to 9.0. Pseudomonas putida A1 resisted as much as 1,000 mg L⁻¹ of copper. The isolates showed similar behavior on copper removal from liquid medium, with a bioremoval rate of 30% at 500 mg L⁻¹ after 24 h of growth. Speciation of copper revealed high copper biotransformation, reducing Cu(II) to Cu(I), capacity. Results indicate that our isolates are potential agents for copper bioremoval and bacterial stimulation of copper biosorption by Avena sativa and Plantago lanceolata.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA