Glucan Synthetase Research Articles

Plasma Membranes from Oats Prepared by Partition in an Aqueous Polymer Two-Phase System

Presumptive plasma membrane fractions have been prepared from oat (Avena sativa L. cv. Brighton) roots and shoots, respectively, by partition of microsomal fractions in a dextran-polyethylene glycol two-phase system. The plasma membranes had a high affinity for the polyethylene glycol-rich upper phase, whereas membranes from mitochondria and other organelles partitioned in the dextran-rich lower phase or at the interface. Thus, relatively pure plasma membranes were obtained by only two partition steps, and within 3 hours from homogenization of the material.The plasma membranes from both organs were enriched in K(+)-stimulated Mg(2+)-dependent ATPase and glucan synthetase II, two tentative markers for the plant plasma membrane. Silicotungstic acid, an indicative stain for the plasma membrane, stained the vesicles recovered from the upper phase, but failed to stain the membranes partitioning in the lower phase or at the interface.The plasma membranes were also enriched in a light-reducible b-cytochrome. This b-cytochrome can be measured by its light-induced absorbance change and may serve as a marker for the plant plasma membrane.

Levels of membrane components regulated by cytokinins and by temperature and aging pretreatments of cotyledons

Membrane preparations from Amaranthus cotyledons show increased carotenoid content and increased specific activities of NADPH-cytochrome c reductase, K +-ATPase and glucan synthetase after treatment of the explants with cytokinin. Some selectivity is apparent since all membrane components assayed are not increased. The greatest effects of benzyladenine are on glucan synthetase I and carotenoids, markers for Golgi and etioplasts respectively. The effect of pretreating the half-seedlings at 40°C followed by a period of aging at 25°C is to increase the carotenoid content and the glucan synthetase activity (other markers not investigated). This supports the idea that the potentiation of cytokinin-dependent betacyanin synthesis by such pretreatment is due to synthesis of functional membrane. Both the potential for and b inhibited by cerulenin, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO 1618) and filipin.

Synthesis of noncellulosic cell-wall β-glucan by cell-free extracts from zoospores and cysts of Phytophthora palmivora

Zoospores of Phytophthora palmivora, induced to encyst by vigorous agitation, synthesize alkali-insoluble β-glucans at a linear rate for ca. 10 min. Cell-free extracts of zoospores and cysts of P. palmivora contained particulate enzymes capable of synthesizing alkali-insoluble, noncellulosic β-1,3-, β-1,6-glucans from uridine diphosphate glucose. Similar levels of activity were detected in zoospores and cysts. Enzymatic activity was stimulated by Mg 2+; Ca 3+ and Mn 2+ were less effective. Cellobiose and other β-linked glucose disaccharides were strong activators. Evidently, the swimming zoospore is equipped with the enzymatic ability to make insoluble, noncellulosic β-glucan—a major component of the cyst wall; hence, a mechanism must exist which prevents the operation of glucan synthetase(s) until the time of encystment. The cell-free extracts also had the ability to synthesize soluble β-1,3-glucan. Most of this material was characterized as neutral mycolaminaran and mycolaminaran phosphate.

Interaction between yeast beta-(1 goes to 3)glucan synthetase and activating phosphorylated compounds. A kinetic study.

Yeast beta-(1 goes to 3)glucan synthetase is stimulated by ATP or GTP. The structural requirements for the activation were investigated by testing several phosphorylated compounds. The simplest substance with stimulatory ability was inorganic pyrophosphate. Addition of a nucleoside, as in GDP, decreased the concentration required for half-maximal stimulation; a third phosphate group, as in GTP, further enhanced the stimulatory capacity. On the other hand, esterification of the terminal phosphate of GTP with a nucleoside or a methyl group led to a total loss of activating ability: dinucleoside triphosphates and the gamma-phosphate methyl ester of GTP acted as competitive antagonists of the activators. alpha,beta- and beta,gamma-imino and -methylene derivatives of both ATP and GTP stimulated the enzymatic activity, suggesting that activation can occur without covalent transfer either of the terminal phosphate or pyrophosphate, or of the nucleotidyl residue. The stimulatory effect of the beta,gamma-imino derivatives of ATP and GTP was not additive. The inhibition constants obtained with gamma-phosphate esters of GTP were the same for either one of the two imino analogs. It is concluded that adenosine and guanosine derivatives bind to the same domain of the enzyme. It is also postulated that activators may interact with the enzyme or with a regulatory protein at two locations, a binding site for the nucleoside moiety and a "functional" site for the pyrophosphate residue.

Changes in glucan synthetase activity and plasma membrane proteins during encystment of the cellular slime mold Polysphondylium pallidum

The activity of glucan synthetase increased dramatically during encystment of Polysphondylium pallidum cells. The majority of activity was present in purified plasma membranes. Activity, measured as glucose incorporation from UDPG into NaOH-insoluble glucan, increased 30-40 fold in the membranes. Increases in activity within the cells preceded plasma membrane increases and the enzyme appeared to be rapidly transported to the plasma membrane. Intracellular activity was relatively low. When cells were incubated with UDPG and when phloretin was included to inhibit glucose uptake, no NaOH-insoluble glucan was synthesized. Hence, the UDPG-binding site was not exposed at the cell-surface. When the NaOH-insoluble glucan was digested with endo-β-1,4-glucanase the products were cellobiose and glucose. The glucan could also be precipitated from Schweizer's reagent with acetic acid. These results suggest that the glucan contained predominantly β-1,4-linkages and may be cellulose. Experiments with cycloheximide confirmed that protein synthesis was required for encystment. Labeling of cells with [1-(14)C]-acetate showed that the synthesis of certain plasma membrane proteins was developmentally regulated. A number of proteins (e.g., myosin heavy chains and actin) were synthesized during the lag phase and their synthesis was subsequently reduced or ceased altogether. Immediately prior to the commencement of cyst wall formation seven new plasma membrane proteins were synthesized. These proteins were not detected intracellularly, indicating rapid transfer to the plasma membrane. The possible relationship between the seven developmentally regulated proteins and a postulated "multi-enzyme-complex" involved in cellulose synthesis is discussed. Their synthesis may be related to the increase in particles in the outer leaflet of the plasma membrane observed during encystment with freeze-etching (G.W. Erdos and H.R. Hohl, 1980, Cytobios, 29, 7-16).

Partial Purification and Characterization of a Blue Light-Sensitive Cytochrome-Flavin Complex from Corn Membranes

A membrane fraction which contains a blue light-sensitive flavin-cytochrome complex (Brain et al. 1977 Plant Physiol 59:948) has been partially purified by sucrose and Renografin gradient centrifugations. Assays for marker enzymes show that this membrane fraction is distinct from endoplasmic reticulum, golgi, and mitochondria. This membrane fraction co-sedimented with glucan synthetase II activity, a proposed marker for plasma membrane in higher plants. The purified membrane fraction shows virtually identical light minus dark and dithionite reduced minus oxidized difference spectra with difference bands near 427 and 557 nanometers, suggesting that contamination by other cytochrome-containing membrane fractions is not significant. The photoactivity can be completely solubilized by 0.1% Triton X-100, leaving the bulk of the membrane undissolved. The kinetics for cytochrome photoreduction are not significantly affected by solubilization, indicating that both flavin and cytochrome could be associated with the same protein moiety.

Identification of glucan synthetase glycosidically bound to amylopectin from cotton leaf starch

Starch-granule bound glucan synthetase was isolated from cotton leaves by a sucrose density technique. Starch granules were successively washed with 0.02 M Na-phosphate buffer containing 0.9% NaCl. The alkaline hydrolysate of washed materials and acid hydrolysate of protein-bound amylopectin were separated by use of Con A-Sepharose. Amylolytic hydrolysate of starch granules also was carried out with α-amylase and fractionated by chromatography on Sephadex G-25. Amylopectin was the only glucan glycosidically linked with proteins, in which glucan synthetase activity was identified. The amylose component was free from covalently bound proteins.

The effect of malathion on amino acid incorporation into plasma membrane proteins of Vigna sinensis (L): Effect of plant growth hormone supplementation.

Amino acid incorporation into plasma membrane proteins of Vigna sinensis (L) was inhibited by malathion, an organophosphorous insecticide, whereas some stimulatory effect was elicited by plant growth hormones, viz., IAA, GA3 and kinetin. The inhibitory effect of malathion towards amino acid incorporation into plasma membraneproteins was marked at concentrations above 50 ppm, and at 400 ppmthe amino acid incorporation was muchless. Up to 100 ppm of malathion treatment, the plant growth hormones could counteract the malathion-induced inhibition on amino acid incorporation into plasma membrane protein but above this concentration, the plant hormones gradually lost their effectiveness. Plasma membrane fraction was characterized by studying the activities of enzymes like IDPase, Oligomycin treated ATPase, Cytochrome ooxidase and glucan synthetase, and also by a sterol/phospho lipid ratio study. From the above studies it appears that glucan synthetase may be the marker enzyme of the plasma membrane of V. sinensis (L), and some change at the membrane level on malathion exposure is suggested.

Isolation of starch granules and starch-granule-bound glucan synthetase from cotton leaves

A simple sucrose density technique has been developed for starch granule isolation from leaves. With this procedure starch-granule-bound glucan synthetase (EC 2.4.1.21) was successfully isolated from phenolic-rich plant tissue. This enzyme is firmly bound to starch granules and free from soluble glucan synthetase. The enzyme specific activity is more than 100-fold higher than that prepared by the previously reported procedure.

The role of NDP-glucose pyrophosphorylases in growing mung bean seedlings in relation to cell wall biosynthesis

NDP-glucose pyrophosphorylase activities were measured in different parts of growing mung bean seedlings. Amongst the different enzymic activities only UDP-glucose pyrophosphorylase proved to be active, showing highest activity in the lower, no-longer elongating, sections of the hypocotyl. In contrast UDP-glucose : glucan synthetase activity was highest in the upper, still-elongating sections of the hypocotyl. Using hypocotyls of 2–6-day-old seedlings it could be demonstrated that UDP-glucose pyrophosphorylase activity was increasing as elongation ceased. At the same time UDP-glucose : glucan synthetase activity was decreasing.

Biosynthesis of the yeast cell wall. I. Preparation and properties of beta-(1 leads to 3)glucan synthetase.

The synthesis of the major linkage found in yeast cell wall structural polysaccharides, glucosyl-beta-(1 leads to 3)-glucosyl, was studied with a membrane preparation from Saccharomyces cerevisiae. The sugar donor was UDP-glucose, and the reaction required addition of glycerol bovine serum albumin, and ATP or GTP for maximal activity. Under optimal conditions, extremely efficient glucose transfer was obtained, with 20 to 50% of the substrate utilized in 20 min at 30 degrees C. The polysaccharide formed in the reaction was insoluble in water and soluble in alkali; it was characterized enzymatically and chemically as a beta-(1 leads to 3)-linked linear glucan of chain length 60 to 80. The terminal reducing group was found to be labeled with 14C, as was the substrate used; therefore, the polysaccharide is synthesized de novo. For each glucosyl group transferred, one equivalent of UDP was formed. No evidence was found for a lipid-linked intermediate. When yeast protoplast lysates were subjected to fractionation by centrifugation in Renografin gradients, glucan synthetase was found in the plasma membrane fraction, with the same distribution and sidedness as chitin synthetase. Because of the spatially restricted growth of the cell wall during cell division in budding yeasts, this result suggests localized and reversible activation of the enzyme during the cell cycle.

Biosynthesis of the yeast cell wall. II. Regulation of beta-(1 leads to 3)glucan synthetase by ATP and GTP.

Biosynthesis of the yeast cell wall. II. Regulation of beta-(1 leads to 3)glucan synthetase by ATP and GTP.

Isolation and Characterization of Concanavalin A-labeled Plasma Membranes of Carrot Protoplasts

The plasma membranes of protoplasts released from carrot suspension culture cells were labeled with [(14)C]acetyl-concanavalin A. After homogenization a single labeled membrane fraction was isolated in a continuous isopycnic Renografin gradient. The labeled membranes peaked at an apparent density of 1.14 grams per cubic centimeter between the Golgi fraction at a density of 1.11 grams per cubic centimeter as determined by latent IDPase activity and the mitochondria at a density of 1.16 grams per cubic centimeter as determined by the cytochrome c oxidase activity. This method provided a very discrete peak of putative plasma membrane. On discontinuous Renografin gradients a relatively pure fraction of labeled plasma membranes could be readily isolated at the 1.122 to 1.146 grams per cubic centimeter interface. The labeled fraction was enriched in both an ATPase (pH 6.5) and a glucan synthetase with a pH optimum of 6.5 whose activity was promoted by magnesium and cellobiose. Enzyme activities were not altered by the membrane label.

Digitonin Solubilization and Protease Stimulation of β Glucan Synthetases of Saprolegnia

Summary β-glucan synthetases from particulate preparations of Saprolegnia monoica were partially solubilized by digitonin treatment. The enzymes which were still associated with the membrane fraction were increased in total and specific activities. Glucan synthetases assayed at low substrate concentration in the presence of Mg Cl 2 , were inhibited by trypsin. On the contrary, enzymes assayed at high substrate concentration in the absence of Mg Cl 2 , were stimulated by trypsin but inhibited by other proteases and by a trypsin inhibitor. Activation followed by inactivation of the synthetases was observed in the presence of high trypsin concentration. Regulation of glucan synthetase activities is discussed in relation to hyphal morphogenesis.

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K(+)-stimulated Mg(2+)-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557-571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm(3). This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10-1.11 g/cm(3)), Golgi (1.12 g/cm(3)) and mitochondria (1.18 g/cm(3)). Furthermore, other recent literature indicates that the 1.13 g/cm(3) buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K(+)-stimulated Mg(2+)-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm(3) density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm(3). Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm(3) particulate components with minimal contamination by particles of higher densities.

Isolation of plasma membrane from a maize coleoptile homogenate: Possible use of a mitochondrial fraction

Summary A «mitochondrial pellet» has been prepared from a maize coleoptile homogenate by centrifugation at 10,000 × g. Subsequent application of sucrose and Ficoll-sucrose density gradients yields a fraction enriched in both K-ATPase and glucan synthetase activities and in indoleacetic acid binding capacity.

Regulation of Glucose Metabolism and Cell Wall Synthesis in Avena Stem Segments by Gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent beta-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only beta-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only beta-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K(+)-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.

The distribution of glucan synthetase in maize coleoptiles: A comparison with K-ATPase

The distribution of glucan synthetase, assayed at high substrate concentration, has been investigated in maize coleoptiles. Enzyme activities have been assayed in the coleoptile homogenate and in various sub-cellular fractions obtained by differential centrifugation. The distribution of enzyme activities is affected by the method of homogenization. The major glucan synthetase-containing fractions, a 10 000 × g and a 153 000 × g sediment, have been further examined on linear and non-linear sucrose gradients. 63% of the glucan synthetase activity is located on particulate material sedimenting at 10 000 × g and having an equilibrium density in sucrose of 1.169 g cm −3. A further 28% of the enzyme sediments at 153 000 × g, the equilibrium density of this material being approx. 1.146 g cm −3. Sedimentation analyses on sucrose density gradients show that the glucan synthetase activity is, at least partially, non-mitochondrial and is not specifically associated with NADH-cytochrome c reductase, IDPase or carotenoid containing membrane fractions. Distribution of glucan synthetase over the differential centrifugation fractions and its profiles on sucrose gradients have been compared to those of K-ATPase, as both enzymes are reported to be located on plant plasma membranes. However, significant differences in distribution occur, indicating the possibility that these enzymes are not uniquely associated with the same subcellular fraction. The value of the use of both enzymes as universal markers for plasma membranes in plants is discussed.