beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro)

In vitro [beta]-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Gossypium hirsutum) fiber cells. The reaction mixture favoring [beta]-1,4-glucan synthesis included the following effectors: Mg2+, Ca2+, cellobiose, cyclic-3[prime]:5[prime]-GMP, and digitonin. The ethanol insoluble fraction from this reaction contained [beta]-1,4-glucan and [beta]-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the [beta]-1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated product favoring [beta]-1,4-glucan synthesis strongly resembled that of cellulose II. On the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan product was found to be exclusively [beta]-1,4-linked. Enzymic hydrolysis confirmed that the product was hydrolyzed only by cellobiohydrolase I. Autoradiography proved that the product was synthesized in vitro. The degree of polymerization (DP) of the in vitro product was estimated by nitration and size exclusion chromatography; there were two average DPs of 59 (70%) and 396 (30%) for the [beta]-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble product. On the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.     

A Comparative Analysis of in vitro cellulose synthesis from cell-free extracts of mung bean (Vigna radiata,Fabaceae) And Cotton (Gossypium hirsutum,Malvaceae)

A comparison of cellulose synthesized in vitro from primary walls of etiolated mung bean (Vigna radiata) seedlings and secondary walls of cotton fibers (Gossypium hirsutum) was made by applying conditions found to be essential for in vitro cellulose I assembly from cotton (Kudlicka et al., 1995, Plant Physiology, vol. 107, pp. 111–123). Mung bean fractions including the plasma membrane (PM), the first solubilized fraction (SE₁), and the second solubilized fraction (SE₂), incorporated more radioactive UDP-Glc into the total product than the same fractions from secondary walls. A significant difference was found with the mild digitonin solubilized fraction (SE₁), which produced eight times more total product than the SE₁ fraction of cotton. However, the SE₁ fraction from cotton produced a larger quantity of cellulose (32.1%) than from mung bean (6.9%). Treatment of the in vitro product by acetic/nitric acid reagent (AN) for varying periods of time demonstrated that cellulose synthesized in vitro from mung bean was more easily degraded than cellulose from cotton fibers. This would suggest that cellulose I produced in vitro from the cotton SE₁ fraction may have a higher crystallinity and DP than cellulose I produced in vitro from mung bean. The fibrils of cellulose produced by the SE, fraction of mung bean were loosely associated and not arranged into a compact bundle as in case of cellulose I synthesized by the cotton SE₁ fraction. The electron diffraction patterns (ED) of both products show reflections characteristic for cellulose I. Products from the SE₂ fraction of mung bean and cotton reveal similarities with the cellulose II allomorph synthesized, as well as abundant β-1,3-glucan.     

beta]-Glucan Synthesis in the Cotton Fiber (II. Regulation and Kinetic Properties of [beta]-Glucan Synthases

The regulation and kinetic properties of cellulose synthase as well as [beta]-1,3-glucan synthase have been studied. The cellulose was detected using acetic/nitric acid insolubility as an indicator of cellulose (this product contained only [beta]-1,4-linked glucans; K. Okuda, L. Li, K. Kudlicka, S. Kuga, R.M. Brown, Jr. [1993] Plant Physiol 101: 1131-1142). These studies reveal that (a) [beta]-1,3-glucan synthesis is enhanced up to 31-fold by cellobiose with a Ka of 1.16 mM; (b) cellulose synthesis is increased 12-fold by a combination of cellobiose (Ka = 3.26 mM) and cyclic-3[prime]:5[prime]-GMP (Ka = 100 [mu]M); (c) the common components in the reaction mixture required by both enzymes are cellobiose, calcium, and digitonin; (d) cellulose synthase has an essential requirement for magnesium (Ka = 0.89 mM); (e) cellulose synthase also requires a low concentration of calcium (Ka = 90 [mu]M); (f) the optimal pH for cellulose synthase (7.6-8.0) is slightly higher than that for [beta]-1,3-glucan synthase (7.2-7.6); (g) the Km for UGP-Glc for cotton (Gossypium hirsutum) cellulose synthase is 0.40 mM; (h) the Km for UDP-Glc for for [beta]-1,3-glucan synthase is 0.43 mM.     

beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed.     

Cellulose and Callose Biosynthesis in Higher Plants (I. Solubilization and Separation of (1->3)- and (1->4)-[beta]-Glucan Synthase Activities from Mung Bean)

(1->3)- and (1->4)-[beta]-glucan synthase activities from higher plants have been physically separated by gel electrophoresis in nondenaturing conditions. The two glucan synthases show different mobilities in native polyacrylamide gels. Further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a different polypeptide composition in these synthases. Three polypeptides (64, 54, and 32 kD) seem to be common to both synthase activities, whereas two polypeptides (78 and 38 kD) are associated only with callose synthase activity. Twelve polypeptides (170, 136, 108, 96, 83, 72, 66, 60, 52, 48, 42, and 34 kD) appear to be specifically associated with cellulose synthase activity. The successful separation of (1->3)- and (1->-4)-[beta]-glucan synthase activities was based on the manipulation of digitonin concentrations used in the solubilization of membrane proteins. At low dipitomin concentrations (0.05 and 0.1%), the ratio of the cellulose to callose synthase activity was higher. At higher digitonin (0.5-1%) concentrations, the ratio of the callose to cellulose synthase activity was higher. Rosette-like particles with attached product were observed in samples taken from the top of the stacking gel, where only cellulose was synthesized. Smaller (nonrosette) particles were found in the running gel, where only callose was synthesized. These findings suggest that a higher level of subunit organization is required for in vitro cellulose synthesis in comparison with callose assembly.     

Glucans in the Cell Walls Regenerated from Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere cultured for 5 days. The cell walls regenerated from theprotoplasts were mainly composed of glucans having 1,3- and1,4-linkages. To investigate the molecular species, these glucanswere separated into four fractions: EDTA (50 mM, pH 4.5)-soluble(fraction E), KOH (24%)- soluble but not precipitatable by neutralizationwith acetic acid (fraction K-S), KOH (24%)-soluble and precipitatableby neutralization with acetic acid (fraction K-P), and KOH (24%)-insoluble(fraction C). By means of sugar composition analysis, methylationanalysis, periodate oxidation and enzymatic digestion, the molecularspecies of the glucans contained in the regenerated cell wallswere deduced to be ß-1,4-glucan (cellulose) and ß-1,3-glucan.Fraction C was mainly composed of ß-1,4-glucan; ß-1,3-glucanwas mainly recovered in fraction K-P. The ß-l,3-glucanwas soluble in dilute alkali solution, but was only slightlysoluble in water. The ß-1,3-glucan had an essentiallyunbranched structure, and its weight average molecular weightestimated by gel permeation chromatography was 4.5–5.0x 10⁴. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan (Received May 21, 1981; Accepted October 13, 1981)     

Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content.

The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins. The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety. In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.     

beta-1,3-Glucan in Developing Cotton Fibers: Structure, Localization, and Relationship of Synthesis to That of Secondary Wall Cellulose

Evidence is presented for the existence of a noncellulosic β-1,3-glucan in cotton fibers. The glucan can be isolated as distinct fractions of varying solubility. When fibers are homogenized rigorously in aqueous buffer, part of the total β-1,3-glucan is found as a soluble polymer in homogenates freed of cell walls. The proportion of total β-1,3-glucan which is found as the soluble polymer varies somewhat as a function of fiber age. The insoluble fraction of the β-1,3-glucan remains associated with the cell wall fraction. Of this cell wall β-1,3-glucan, a variable portion can be solubilized by treatment of walls with hot water, a further portion can be solubilized by alkaline extraction of the walls, and 17 to 29% of the glucan remains associated with cellulose even after alkaline extraction. A portion of this glucan can also be removed from the cell walls of intact cotton fibers by digestion with an endo-β-1,3-glucanase. The glucan fraction which can be isolated as a soluble polymer in homogenates freed of cell walls is not associated with membranous material, and we propose that it represents glucan which is also extracellular but not tightly associated with the cell wall. Enzyme digestion studies indicate that all of the cotton fiber glucan is β-linked, and methylation analyses and enzyme studies both show that the predominant linkage in the glucan is 1 → 3. The possibility of some minor branching at C-6 can also be deduced from the methylation analyses. The timing of deposition of the β-1,3-glucan during fiber development coincides closely with the onset of secondary wall cellulose synthesis. Kinetic studies performed with ovules and fibers cultured in vitro show that incorporation of radioactivity from [¹⁴C]glucose into β-1,3-glucan is linear with respect to time almost from the start of the labeling period; however, a lag is observed before incorporation into cellulose becomes linear with time, suggesting that these two different glucans are not polymerized directly from the same substrate pool. Pulse-chase experiments indicate that neither the β-1,3-glucan nor cellulose exhibits significant turnover after synthesis.     

Specific and direct measurement of theβ-1,3-glucan in developing cotton fiber

The reaction of N,N-diethylaziridinium chloride with raw cotton (Gossypium hirsutum L.) seed fibers to introduce N,N-diethylaminoethyl (DEAE) substituents at a low degree of substitution was used for demonstrating the presence of O(4)H, characteristic of a -1,3-glucan. The derivatized 1,3-glucan/cellulose was hydrolyzed to DEAE-glucoses that were analyzed by gas-liquid chromatography. Capillary columns proved effective for measuring the small amounts of 4-O-DEAE-glucose in the presence of major amounts of 2-O- and 6-O-DEAE-glucoses. Analyses of raw cotton fibers were carried out through fiber development (20, 27, 34, 41 and 48 d post anthesis, DPA) and field exposure (62, 83 and 104 DPA) periods. The yields of 4-O- and other individual DEAE-glucoses and the yield of 4-O-DEAE-glucose in relation to 2-O-DEAE-glucose were particularly informative concenring the role of the -1,3-glucan in cellulose. The results confirmed the early production and almost immediate decrease of the -1,3-glucan and demonstrated continued production of accessible cellulose followed by a sharp decrease in accessibility after boll opening. The -1,3-glucan content of the raw cotton fiber, estimated from the yield of 4-O-DEAE-glucose (representing 1,3-glucan) and the yield of 2-O-DEAE-glucose (approximating 1,3-glucan plus cellulose) was 10%, 4%, 1% and 0.6% at, in the order given, 20, 27, 48, and 104 DPA. These results are in general agreement with other conventional analyses.Abbreviations DPA days post-anthesis - DEAE diethylaminoethyl     

A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.     

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 μm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.     

Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum.

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of partial amino acid sequences of the purified enzyme, two degenerate oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb fragment of corresponding genomic DNA. The PCR product was used to isolate the genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA confirmed that the PCR product corresponded to EXG1. A strain of the fungus specifically mutated in the EXG1 gene was constructed by homologous integration of an internal fragment of EXG1. In the mutant, enzymatic activity and the corresponding peak of UV absorption during high-pressure liquid chromatography purification were reduced by at least 98%. However, crude culture filtrates of the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This residual activity was due to two additional activities which were chromatographically separable from the product of EXG1 and which were coeluted with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 mutant was still pathogenic to maize.     

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers: II. Structure of the Reaction Product

The solubility properties, composition, and structure of the radioactive product synthesized from UDP-[¹⁴C]glucose by a highly active cotton fiber glucan synthetase have been determined. Product obtained under the following three different conditions was analyzed: at high and low substrate concentrations by detached fibers, and at high substrate concentrations with an isolated particulate preparation. The results of acetic and nitric acid digestion, enzyme digestion, total acid hydrolyses, periodate oxidation, partial acid hydrolyses, and methylation analyses all support the conclusion that the product of the glucan synthetase produced under all three assay conditions is a linear β-(1→3)-glucan.     

The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)-derived structure to beta 1,6-glucan, which is linked in turn to beta 1, 3-glucan. When isolated cells walls were digested with beta 1,6-glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve beta 1,6-glucan. The beta 1,6-glucanase-resistant protein fraction contained the recently identified GPI-lacking, O-glycosylated Pir-CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to beta 1,3-glucan through an alkali-sensitive linkage, without beta 1,6-glucan as an interconnecting moiety. In beta 1,6-glucan-deficient mutants, the beta 1,6-glucanase-resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of beta 1,6-glucan.     

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.     

Isolation and properties of β-glucan synthetase from the aquatic fungus, Aphanomyces astaci

A β-glucan synthetase was isolated from a membrane fraction of the crayfish parasitic fungus Aphanomyces astaci Schikora, strain Si. [¹⁴C]-UDP-glucose was incorporated linearly for about 1 h at 30°C into an acid insoluble product. The apparent K_m for UDP-glucose was found to be approximately 4.5 m M and the apparent K_i for UDP, a competitive inhibitor of the reaction, was 1 m M . The acid insoluble product obtained after incubating this glucan synthetase with[₁₄C]-UDP-glucose was partially characterized by glucanase treatment. This product mainly consisted of β-1,3-linked glucosyl units. Synthetase activity was not stimulated by MgCl₂, but cellobiose as well as GTP and EDTA in combination or ATP alone enhanced enzyme activity. A high proportion of the A. astaci synthetase was probably already activated during preparation and not accessible to further stimulation by nucleotide additions as found for glucan synthetase of Saccharomyces cerevisiae and Candida albicans. No synthetase activity or any factors affecting this enzyme was present in the cytosol. An exudate prepared from the cuticle of the crayfish host, did not inhibit glucan synthetase activity in vitro.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4–12-fold) stimulation of the activity of membrane-associated -glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of -glucan synthesis. Those products whose synthesis is stimulated are mainly -1,3-glucan, but there is also a considerable increase in -1,4-glucan. No -1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on -glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.Abbreviations UDP-glucose uridine-5-diphosphoglucose - PEG polyethylene glycol - BTP bistrispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane) - MES 2(N-morpholino)ethanesulfonic acid - VAL valinomycin     

Isolation and polypeptide composition of l,3-ß-glucan synthase from plasma membranes of Brassica oleracea

The l,3-ß-glucan synthase (callose synthase, EC 2.4.1.34) was solubilized from cauliflower ( Brassica oleracea L.) plasma membranes with digitonin, and partially purified by ion exchange chromatography and gel filtration [fast protein liquid chromatography (FPLC)] using 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) in the elution buffers. These initial steps were necessary to obtain specific precipitation of the enzyme during product entrapment, the final purification step. Five polypeptides of 32, 35, 57, 65 and 66 kDa were highly enriched in the final preparation and are thus likely components of the callose synthase complex. The purified enzyme was activated by Ca²⁺, spermine and cellobiose in the same way as the enzyme in situ, indicating that no essential subunits were missing. The polyglucan produced by the purified enzyme contained mainly 1,3-linked glucose.