Β-glucan Synthetase Research Articles

A role for F-BAR protein Rga7p during cytokinesis in S. pombe.

F-BAR proteins are known to participate in cytokinesis, but their mechanisms are not well understood. Here we investigated Rga7p, an Schizosaccharomyces pombe F-BAR protein with a RhoGAP domain. Localization of Rga7p to the cytokinetic cleavage furrow depends on its F-BAR domain, actin filaments, the formins Cdc12p and For3p, and the presence of a contractile ring. Rga7p is not required for the constriction of the contractile ring but does participate in the transport of a β-glucan synthetase (Bgs4p) from the late Golgi compartments to plasma membrane that is adjacent to the contractile ring. Cells without Rga7p moved Bgs4p normally from the poles to the Golgi complex near to the cell center, but Bgs4p then moved slowly from the late Golgi compartments to the cleavage site. The late arrival and lower than normal numbers of Bgs4p result in septal defects late in cytokinesis, and in the lysis of separating cells, similar to that in cells with mutations in the cwg1(+) gene (which encodes Bgs4p).

The Pneumocystis Ace2 Transcription Factor Regulates Cell Wall-remodeling Genes and Organism Virulence

Pneumocystis carinii (Pc) β-glucans are major components of the organism cell wall; yet, the regulation of Pc cell wall genesis and remodeling is not well understood. Ace2 transcription factors, which are present in many fungi, regulate glucanases and other enzymes needed for cell wall remodeling. The cloning and heterologous expression of PcAce2 in ace2Δ Saccharomyces cerevisiae demonstrated that PcAce2 can restore the defective glucanase and endochitinase gene expression of the mutant as well as regulate cell wall β-glucan biosynthetic genes. Furthermore, when a reconstructed yeast system was used, PcAce2 activated the transcription of the Pneumocystis gsc1 β-glucan synthetase, confirming the activity of a Pc transcription factor on a native Pneumocystis promoter and gene for the first time. We further observed that Pneumocystis binding to host extracellular matrix proteins and lung epithelial cells induced the phosphorylation (activation) of the PcAce2 transcription factor. Finally, we present a novel method that confirms the role of PcAce2 in modulating organism virulence using ace2Δ Candida glabrata infection in neutropenic mice. Together, these results indicate that the adherence of Pc to lung matrix proteins and epithelial cells leads to the activation of the Ace2 transcription factor, which regulates cell wall degradation and biosynthesis genes that are required for cell wall remodeling.

β-Glucan synthetase II activity upon callose formation in the flower ofArabidopsis thaliana

Callose formation was observed in the pollens during flower development and pollen tube grown in the pistil ofA. thaliana. The accumulation of callose occurred in the tetrad in the flower bud and pollen tube. Therefore, the activity of β-glucan synthetase II (GS II), which is responsible for synthesizing the callose, was measured in the flowers on the same developmental stages. The enzyme activity was increased by about 10% while the level of callose contents was increased by about 70% in tetrads. Then, callose accumulation was increased during pollen tube growth by about 30% higher than the other stages and enzyme activity was detected, 30% more too. These results suggest that callose plays an important role in the growth of pollen and pollen tube by increasing GS II activity.

Pneumocystis cariniipneumonia: Current therapy and future prospects: Pneumocystis carinii pneumonia: Current therapy and future prospects

Pneumocystis carinii is virtually ubiquitous in its exposure to man throughout the world, and thus represents a most common opportunistic infection in patients with immunocompromised immune systems, such as in cancer, transplant, or AIDS patients. Therapy for P. carinii pneumonia, a life-threatening infection, is limited, with pentamidine and cotrimoxazole (trimethoprim-sulfamethoxazole) as primary therapeutic choices. New therapies such as 566C80 (a hydroxynaphthaquinine), pneumocandins and echinocandins (lipopeptide inhibitors of β-glucan synthetase), new combination therapies and new analogues of pentamidine (that may inhibit topoisomerase II) may offer new therapy options.

Paracoccidioides brasiliensis: Current Research on Morphogenesis

The involvement of cell wall glucans in the process of dimorphism in Paracoccidioides brasiliensis has been proposed for many years. The control of glucan synthetase activity through cytoplasmic factors is also considered. Current research on biochemical aspects of dimorphism in P. brasiliensis suggests that proteinases are involved in the control of β-glucan synthetase activity in the generation of the fungal cell wall. The probable involvement of protein disulfide reductase is also suggested. A complementary model of yeast mycelium transformation is proposed.

Purification par séparation de phase et caractérisation du plasmalemme de l'épicotyle de pois

The validity of phase partition to obtain a substantial proportion of vesicles of plasmalemma origin from the microsomal fraction of pea epicotyl has been demonstrated. In the fractions enriched with plasma membranes, N-naphthyl phtalamic acid binding and β-glucan synthetase II activity, showed a yield of about 60% and an enrichment of 2.3 and 2.2, respectively, in comparison with the microsomal fraction. When such plasmalemmic vesicles are permabilized by Triton X-100, an intense Mg2+-ATPase activity is obtained in presence of K+ at acid as well as alkaline pH. Inhibition of Mg2+-ATPase by vanadate in presence of K+ and its variations in relation to pH were shown. Dicyclohexylcarbodiimide and diethylstilbestrol inhibit 40–55% of this enzymatic activity, both at acid and neutral pH. The data show a slight contamination of the plasmalemmic fraction by endomembranes and suggest an asymmetry of the two sides of the plasmalemma.

β-Glucan synthetase activities in regenerating protoplasts from carrot suspension cultures

The activities of β-glucan synthetases I and II from particulate fractions of protoplast homogenates were compared to the phases of cell wall regeneration in cultivated carrot protoplasts. β-1,4-Glucan synthetase (I), assayed at low UDP-glucose (UDPG) and high Mg 2+ levels showed a lag phase of 15–20 h. The specific activity increased 2-fold during the first 3 days of cultivation. Cultivated carrot cells from the exponential growth period did not exhibit any increase in the activity of the particulate fraction. Inhibitors of RNA and protein synthesis prevented an increase suggesting that enzyme protein synthesis is a prerequisite for this process. β-Glucan synthetase II, presumably producing β-1,3-glucans at high substrate concentrations in the absence of Mg 2+, showed an immediate rise for 20 h, but then declined. The phase of cell wall regeneration was determined by the incorporation of [ 14C]glucose into cellulose.

?-1-3- and ?-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The β-glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various β-glucanases and by chromatography show that both β-1-3- and β-1-4-linkages are formed at high substrate concentrations. In the absence of MgCl2, β-1-3-linked glucans are mainly produced. By increasing MgCl2 concentrations the total synthesis activity and β-1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl2, β-1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl2.

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 β-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 β-glucan synthesis. Those products whose synthesis is stimulated are mainly β-1,3-glucan, but there is also a considerable increase in β-1,4-glucan. No α-1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on β-glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.

Influence of sucrose on the characterization of higher plant membranes by β-glucan synthetase activity and its relation to osmotic pressure

Increasing concentrations of sucrose stimulate β-GS activity in heavy and light membranes of roots of Lupinus. Measured β-GS activity in fractions obtained by sucrose gradient centrifugation should hence be corrected when used as a test for plasmalemma identification. After applying corrected β-GS values, fractions enriched in plasmalemma can be detected in both heavy and light membrane preparations at lower densities of sucrose than those usually admitted. The sucrose effect seems largely due to an osmotic shock and so it would be preferable to use non-osmotic media for membrane purification on gradients.

Cooperative action of β-glucan synthetase and UDP-xylose xylosyl transferase of Golgi membranes in the synthesis of xyloglucan-like polysaccharide

Golgi membranes of pea seedling tissue contain a UDP xylose:polysaccharide xylosyl transferase, the action of which is stimulated by UDP glucose. In the presence of both nucleotide-sugars a heteropolysaccharide containing both xylose and glucose (xyloglucan) is produced. Transfer of xylose and glucose units is presumed to be due to separate enzymes, because their properties differ in a number of respects. Xylosyl units appear to be transferred to a glucan core polysaccharide that is produced from UDP glucose by β-1,4-glucan synthetase. This, rather than cellulose biosynthesis, is inferred to be the in vivo role of Golgi membrane β-1,4-glucan synthetase.

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.

Tissue Slice and Particulate β-Glucan Synthetase Activities from Pisum Epicotyls

beta-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg(2+) optimum, apparent K(m)) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various beta-glucanases all show that both beta-1,3 and beta-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. beta-1,4 Linkages predominate at low substrate (5 mum) concentration. Kinetic data indicate that the capacity to synthesize beta-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.

-Glucan Synthetases from Saprolegnia monoica

Cell extracts of Saprolegnia monoica Pringsheim contained enzymes which synthesized glucans from uridine diphosphoglucose. Both MgCl2 and cellobiose stimulated enzyme activity. The pH optimum was about 5.8. High substrate concentrations increased the proportion of alkali-insoluble glucans. Paper chromatography of cellulase-hydrolysed alkali-insoluble glucans revealed cellobiose indicating the presence of a (1#4)-β-glucan (cellulose) synthetase. Glucan synthetases were mainly located in the wall and microsomal fractions. They were more active in branched hyphae than in unbranched mycelium. Subcellular fractions were characterized by density gradient ultracentrifugation, enzymic tests and electron microscopy. Synthetase activities were associated with dictyosomes which, by histochemical staining, were shown to contain (1#4)-linked polysaccharides. The results were consistent with the current interpretation of the role of the Golgi apparatus in hyphal morphogenesis.

Biosynthesis of β-glucans by cell-free extracts from saccharomyces cerevisiae

Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U- 14C]glucose into β-1, 3-glucans which contained a significant proportion of β-1, 6-glycosidic linkages. When GDP-[U- 14C]-glucose was used as substrate only trace amounts of glucose were incorporated. Activity of β-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. β-Glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of β-1, 6-glycosidic linkages respectively. A marked decrease in the activity of β-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.

?-Glucan synthetase activity in Golgi vesicles ofPetunia hybrida

β-Glucan synthetase activity has been demonstrated in a Golgi vesicle fraction isolated from pollen tubes ofPetunia hybrida. Thisβ-glucan synthetase activity differs from that of most other higher plants in its inability to incorporate [(14)C]glucose from GDP-[(14)C]glucose. UDP-[(14)C]glucose, however, is an appropriate glucose donor for this enzyme. The optimum conditions for thisβ-glucan synthetase activity are: 1 mg Golgi vesicle protein/ml reaction mixture; pH=±8 and a temperature of 25°C. The newly synthesized alkali-insoluble glucan containsβ-1,3- as well as β-1,4-glucosidic linkages.

Sterol composition of plasma membrane enriched fractions from maize coleoptiles

Membrane fractions have been isolated from a 100 000 g pellet of corn ( Zea mays L.) coleoptiles by density gradient centrifugation. Using the following markers: succinate and NADPH cytochrome c oxidoreductase, β-glucan synthetase and N-1 napthylphthalamic acid (NPA) binding assay, two fractions enriched in endoplasmic reticulum and in plasma membrane, respectively were identified. The plasma membrane contained more sterols than other membrane fractions. The relative proportions of the different sterols (stigmasterol, sitosterol, campesterol and cholesterol) were similar in all fractions.

Effects of Heat Shock on Growth and on Lipid and β-Glucan Synthetases in Leaves of Phaseolus vulgaris and Vigna sinensis

Primary leaves of intact plants of Phaseolus vulgaris and Vigna sinensis 7 to 9 days old were dipped in water for 2 minutes at 47.4 +/- 0.2 C and 48.7 +/- 0.5 C, respectively. Leaf growth was inhibited within the first 20 hours, and growth partially or completely recovered in a subsequent 20-hour period. Analysis of the glucolipid and beta-1, 3- and beta-1, 4-glucan synthetases showed that the most evident effects of heat shock were the severe inhibition of the synthesis of beta-1, 4-glucosyl glycosidic linkages and stimulation of beta-1, 3-linkage synthesis. The stimulation appeared either immediately after treatment or within the first 20 hours and lasted at least 60 hours after treatment. Phaseolus plants reacted more intensely to the heat shock, reflecting a possible genetic difference in the response mechanisms.

β-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem

Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. beta-1,3- and beta-1, 4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 mum) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mm) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of beta-1, 4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, beta-1, 4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of beta-1, 3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations.

Association of β-Glucan Synthetases with Plant Plasma Membranes: Quantitative Electron Microscopy as an Aid to Enzyme Localization in Cell Fractionation

Phosphotungstic acid - chromic acid (PTA-CrO3), an electron-dense stain specific for plant plasma membranes, has made possible the identification and purification of this cell component in isolated membrane fractions. In studies of plant cell wall biogenesis, localization of β-glucan synthetases by in vitro assay of membrane fractions has shown their association with dictyosome-rich fractions when assayed at low (1 to 5 μM) UDP-glucose concentrations. However, when assayed at 1 mM UDP-glucose, plasma membrane-rich fractions from onion stem synthesized β-glucans most actively. Glucans synthesized were primarily β,1-4 at low substrate concentrations and primarily β,1-3 at 1 mM UDP-glucose. Analysis of glucolipid synthesis by both membrane fractions indicated that at low substrate concentrations competition for substrate for the synthesis of sterol glucosides was responsible for relatively low β-glucan synthesis by plasma membranes.