Glucan Synthetase Activity Research Articles

Effect of α-naphthylacetic and gibberellic acids on inhibitor protein content and fiber-forming enzyme activity of gymnosperm and fuzzy cotton

The effect of the phytohormones α-naphthylacetic acid and gibberellic acid on the protein content; glucansynthetase, peroxidase, and cellulase enzyme activity; and development of gymnosperm and fuzzy cotton ovules was studied. The effect on cellulose synthesis of an inhibitor protein isolated from integument of gymnosperm cotton treated with gibberellic acid was investigated.

Cellulose biosynthesis of cotton treated with retardants

The effect of retardants on cellulose biosynthesis and protein composition in seedlings and fiber of two cotton species is investigated. The activity of glucansynthetase and the synthesis of “de novo” polypeptides increase under the influence of morphonol and Pix.

Effect of bentonite clay on biosynthesis of cotton cellulose

Bentonite clay affects biochemical processes occuring during synthesis of cotton-fiber cellulose by increasing the activity of glucansynthetase, peroxidase, and cellulase at 20% clay concentration. The content of soluble proteins increases and their composition changes.

Action de la désertomycine sur la synthèse des polymères pariétaux du Saccharomyces uvarum

The desertomycin action upon Saccharomyces uvarum wall synthesis has been studied. Spheroblast regeneration was carried out in a liquid medium containing labeled glucose to monitor the synthesis of different wall components. In the presence of desertomycin, wall synthesis was affected; this was expressed as a net reduction of insoluble alkali constituents content, more precisely the insoluble acido-alkali fraction that, in yeasts, is constituted by chains of beta(1,3)-glucans linked among themselves by beta(1,6) bonds. Mannan formation was not inhibited such polymers that cannot be fixed to the glucan matrix of the wall were liberated in the regeneration medium. Because of desertomycin action, the decrease in insoluble alkali content revealed an interference with the enzymatic systems catalyzing glucan synthesis. In vitro, however, this antifungal had little effect upon glucan synthetase activity: doses 5 times superior to the subinhibiting level used in vivo caused only 30% inhibition. This result can be explained by an indirect action of desertomycin. Parietal disorders were the result of membrane structure disturbance, notably the phospholipids and localized enzymatic systems. This antifungal presents an analogical structure with macrolides with recognized membrane action.

Kinetic characterization of the synthesis of cotton cellulose

The influence of the temperature, pH, and inhibitors on the synthesis of cellulose from cottonplant shoots has been investigated. The maximum activity of glucan synthetase was exhibited at 27°C, pH 8. O. The greatest inhibition of the formation of cellulose was shown by EDTA.

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.

Direct isolation of vacuoles from leaf tissue of lettuce (Lactuca sativa) retaining protoplasts within the leaves

A procedure is described in which vacuoles are isolated from leaf tissue of lettuce (Lactuca sativa L.). After incubation in an enzyme solution, the vacuoles are directly extracted from the leaf tissue by osmotic shock using a phosphate buffer. In this method no protoplasts are released from the leaf tissue. This procedure avoids the problems of separating vacuoles from protoplasts with similar density. To evaluate the purity of the vacuoles, the activity of glucan synthetase 11 (EC 2.4.1.34), NAD(P) H-cytochrome c reductase (EC 1.6.99.3) and malate dehydrogenase (EC 1.1.1.37) was measured. To measure vanadate- and nitrate-sensitive ATPase activity (EC 3.6.1.8) vesicles were prepared from the vacuoles and ATP-dependent vesicle acidification was measured as acridine orange quenching. Nitrate inhibited the quenching, while addition of vanadate had no effect. It was concluded that the vacuoles were not contaminated with plasma membranes. To evaluate the viability of the vacuoles [(14) C]-malate uptake was measured. The vacuoles showed a constant rate of [(14) C]-malate uptake during 45 min. This rate was maximal at pH 6.8.

The effect of papulacandin B on (1→3)-β- d-glucan synthetases. A possible relationship between inhibition and enzyme conformation

The antibiotic, papulacandin B, inhibited growth or (1→3)-β- d-glucan synthetase (or both) in the fungi Saccharomyces cerevisiae, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune and Wangiella dermatitidis. No efect was observed on Achlya ambisexualis. There was no apparent correlation between the inhibition of growth and that of the synthetase. With most of the fungal extracts, the inhibition of glucan synthetase by papulacandin B became less pronounced as the substrate (UDP-glucose) concentration was decreased. At very low levels of UDP-glucose, with the enzymes from S. cerevisiae and W. dermatitidis, the antibiotic stimulated the activity of glucan synthetase. As further studied with the W. dermatitidis enzyme, those low concentrations of UDP-glucose corresponded to a sigmoidal portion of the rate vs. substrate curve. The sigmoid segment of the curve extended to higher concentrations of UDP-glucose as the temperature was increased. Concomitantly, the range of substrate concentrations at which papulacandin B stimulated the reaction or was noninhibitory was broadened. It is tentatively concluded that glucan synthetase may exist in more than one interconvertible form. The stimulatory effect of papulacandin B is possibly due to preferential binding to the active form of the enzyme. The equilibrium between these forms could be shifted by structural changes in the membrane in which the enzyme is embedded. The lack of correlation between the effects of papulacandin B in whole cells and in extracts is discussed in terms of the variations in membrane structure in the two situations.

Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast

Hansenula mrakii secretes extracellularly a killer toxin which kills sensitive Saccharomyces cerevisiae. In protoplasts of this yeast, the killer toxin selectively inhibited the synthesis of alkali-insoluble acid-insoluble polysaccharides consisting mainly of β-glucan, but did not inhibit either the synthesis of other cell wall polysaccharides, such as mannan, chitin and alkali-insoluble acid-soluble polysaccharides, or the synthesis of protein. Consistent with these results, the toxin was inhibitory to the β-(1,3)-glucan synthetase activity of a cell-free extract from sensitive S. cerevisiae.

Effect of nucleotides on glucan synthesis inParacoccidioides brasiliensis

The activity of Paracoccidioides brasiliensis glucan synthetase was partially inhibited by guanosine 5'-triphosphate, adenosine 3'5'-phosphate and adenosine 5'-triphosphate. This inhibition was more pronounced in the mycelial system than in the yeast one, being higher at 23 degrees C than at 37 degrees C. Addition of ethene diamine tetracetic acid to the incubation mixture inhibited partially the enzymatic activity in mycelial preparations but stimulated it in the yeast system.

Effects of Water Stress on Starch and Its Metabolizing Enzyme Activities in Cotton Leaves

AbstractCotton plants were subjected to water stress ranging from –1.0 to –2.2 MPa. Starch content decreased during the initial decline in water potential from –1.1 to –1.4 MPa. This change was related to an increase in soluble α‐amylase activity and a decrease in bound glucan synthetase activity. Starch content then increased sharply to nearly 200% of control levels with a decrease in water potential to about –1.6 MPa. With a further reduction in leaf water potential to –2.2 MPa, both bound glucan synthetase activity and starch content decreased rapidly, while soluble α‐amylase activity continued to increase. The enzymic data demonstrate that effects of water stress on cotton are adverse, because starch degradation products are not translocated, but accumulate as reassembled starch in the source leaves.

Alterations in the biosynthesis of chitin and glucan in the slime mutant of Neurospora crassa

The slime mutant of Neurospora crassa lacks both chitin and insolubleβ(1 → 3)glucans either cellbound or secreted to the medium. Failure to synthesize both polymers is not due to deficiencies in the precursor sugar nucleotides, UDP-GlcNAc and UDP-Glc, since these are present in higher amounts in the mutant than in the wild type. The slime mutant also contains higher levels of chitin synthetase than the wild type, but lacks detectable glucan synthetase activity. an os-1 mutant grown in hypertonic medium contained reduced amounts of chitin and glucan, but its glucan and chitin synthetase activities were nearly normal. It is concluded that the absence of a cell wall in the slime mutant is due to a mutational event(s) which affects the mechanism of activation or delivery of chitin synthetase to the cell surface and the formation of a functionalβ(1 → 3)glucan synthetase. This mutational event must be different in the os-1 strain.

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 Km for UDP‐glucose was found to be approximately 4.5 mM and the apparent Ki for UDP, a competitive inhibitor of the reaction, was 1 mM. 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.

Inhibition by light of growth and Golgi-localized glucan synthetase in the maize mesocotyl

When dark-grown maize (Zea mays L.) seedlings were exposed to red light (R), Golgi-localized glucan synthetase activity in the mesocotyl began to decrease within 1 h, and fell by approx. 70% in 12 h. The response required at least 10(-2) μmol m(-2) R and saturated at 100 μmol m(-2). Far-red light (FR) alone inhibited glucan synthetase, and FR reversed the inhibition by R back to the level caused by FR alone. Density gradient fractionation indicated that of the major membrane markers only the Golgi-localized glucan-synthetase activity was affected by R. Golgi-localized latent inosine-diphosphatase activity was unaffected. The kinetics of the response, the photon fluence dependence, and the reversibility by FR all correlated with the inhibition by light of elongation of the mesocotyl, indicating that light inhibits growth and glucan synthetase activity by a similar mechanism.

Auxin controls Golgi-localized glucan synthetase activity in the maize mesocotyl

Decapitation or red light irradiation (R) inhibited growth and Golgi-localized glucan synthetase (GS I) activity in the mesocotyl of intact maize (Zea mays L.) seedlings. Applied auxin (indole-3-acetic acid) prevented the effects of R and of decapitation on both growth and GS I. Auxin applied several hours after irradiation prevented any further decline in GS I but did not restore it. Mesocotyl segments incubated in solution elongated in response to auxin but lost GS I with time regardless of the presence of exogenous auxin. An attached seed was necessary for maintenance of GS I in the dark-grown mesocotyl.

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.

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).

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 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.

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.