Glucan Synthetase Activity Research Articles

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.

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.

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.

Studies on the Mechanism of Action of Dinitramine

The effect of dinitramine, a selective herbicide, on the plasma membrane of the soybean (Glycine max L.) root was studied. Used as marker systems to observe the herbicide effect were two plasma-membrane-specific enzymes, pH 6.5 ATPase and glucan synthetase.The activity of pH 6.5 ATPase decreased significantly in membrane vesicles prepared from roots harvested 15 minutes after treatment with dinitramine. Maximum inhibition occurred in roots harvested 2 hours after treatment. Glucan synthetase activity decreased similarly within 2 hours of treatment.Membrane permeability to (86)Rb was rapidly increased by dinitramine.The activity of pH 6.5 ATPase returned to the control level within 8 hours of treatment with dinitramine.These results show dinitramine's initial site of action to be the plasma membrane, producing an over-all reduction in membrane function through inactivation of membrane-associated proteins.

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers

A uridine diphosphate(UDP)-glucose:glucan synthetase can be demonstrated in detached cotton fibers (Gossypium hirsutum L.) and in an isolated particulate fraction from such fibers. When assayed with detached fibers, the kinetics of the glucan synthetase activity with respect to variation in substrate concentration is complex and indicates activation of the enzyme by the substrate. Activity is stimulated by Ca(2+) or Mg(2+) and beta-linked glucosides; the effect of the beta-linked glucosides is to shift the range in which substrate activation occurs to lower concentrations of UDP-glucose. At concentrations of UDP-glucose below 50 mum, addition of uridine triphosphate, in addition to beta-linked glucoside, results in significant stimulation of activity. This effect can be explained by the conversion of uridine triphosphate to UDP-glucose by UDP-glucose pyrophosphorylase, thereby raising substrate concentration to the activating range. In detached fibers, glucan synthetase activity is high at all stages of fiber development. The properties of the glucan synthetase of the isolated particulate fraction closely resemble those of the enzyme assayed in detached fibers; however, in contrast to detached fibers, the ability to detect enzyme activity is more dependent on fiber age, showing maximal activity between 16 and 18 days postanthesis, coincident with the time of rapid onset of secondary wall cellulose deposition.

Adenosine Triphosphatase from Soybean Callus and Root Cells

The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+) > Sr(2+)) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K(+) > Rb(+) > Choline(+) > Na(+) > Li(+) > NH(4) (+) > Cs(+) > tris(+). Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na(+) over K(+). No synergism between K(+) and Na(+) was found with preparations from either cell source.The pH optimum for ATP hydrolysis in the presence of 50 mm KCl and 3 mm MgSO(4) was 6.5 for both preparations and slightly higher in the presence of 3 mm MgSO(4) alone. The order of nucleotide preference was found to be: ATP >> ADP > GTP > CTP > UTP. Maximal glucan synthetase activity at high (1 mm), but not at low (1 mum), substrate was found to be coincident with the position of this fraction on the sucrose gradient.

The influence of gibberellic acid and abscisic acid on cell and tissue differentiation of bean callus.

Bean callus was induced to form roots (tissue differentiation) and vascular nodules (cell differentiation) by lowering the ratio of auxin to cytokinin in the growth medium. Both types of differentiation were inhibited by the addition of abscisic acid (at concentrations greater than I muM) to induction medium. Initiation of differentiation was inhibited, but its subsequent development was not, and the inhibition was not affected by the addition of gibberellic acid. Addition of gibberellic acid (GA) alone to induction medium stimulated tissue differentiation, although cell differentiation was unaffected (30 muM GA) or inhibited (45 muM GA) and its onset was delayed at both concentrations. Root initiation was also stimulated by gibberellic acid (0.I-45 muM) at an auxin-to-kinin ratio 10 times that normally optimal for cell differentiation. The phenylalanine ammonia lyase (PAL) activity of the calluses was closely correlated with the amount of cell differentiation which had occurred, and measurement of this confirmed that gibberellic acid delayed the initiation of cell differentiation. The increase and subsequent decline of PAL and betaI leads to 3 glucan synthetase activities, normally induced by transfer to induction medium, was abolished by abscisic acid. Addition of gibberellic acid did not affect the betaI leads to 3 glucan synthetase activity.

Synthesis of Yeast Wall Glucan

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.

Quantitative measurement of the course of bean callus differentiation.

Two strains of callus have been isolated from bean hypocotyl and grown on a defined maintenance medium supplemented with 2 mg/l. 2:4-dichlorophenoxyacetic acid (2:4D) and 2% sucrose. Root initiation was observed in one strain and formation of nodules containing xylem and phloem in both strains after transfer to an induction medium supplemented with 1 mg/l. naphthyleneacetic acid, 0-2 mg/l. kinetin and 3% sucrose, after 3 transfers to maintenance medium. The number of nodules per gramme increased 10-fold between 6 and 12 days after transfer, and thereafter remained constant. Phenylalanine ammonia lyase (PAL) activity rose to a maximum value when the rate of nodule formation was greatest, and decreased after the maximum nodule concentration was reached. The final constant value for PAL activity was above that of callus grown on maintenance medium. Beta I leads to 3 glucan synthetase activity rose to a maximum 15 days after transfer, and then fell gradually to a level above that measured in callus on maintenance medium. Callus was transferred from maintenance medium after 3, 4, 5 and 6 transfers. The concentration of nodules after 21 days on induction medium decreased as the callus was kept in culture. No further differentiation could be induced after 6 transfers. The fall in nodule formation was paralleled by a decrease in PAL and betaI leads to 3 glucan synthetase activities measured 21 days after transfer.

Indoleacetic acid stimulates cellulose deposition and selectively enhances certain β-glucan synthetase activities

Particulate preparations from growing regions of 8-day old Pisum sativum epicotyls catalysed glucosyl transfer to β-glucan from UDPglucose and GDP-glucose. The activities assayed with GDPglucose (6 or 600 μM) or low (6μM) concentrations of UDPglucose disappeared from decapitated epicotyls within 3 days, but were maintained when the cut apex was treated with the hormone indoleacetic acid. These activities re-appeared when indoleacetic acid was added 3 days after decapotation; cycloheximide prevented this response. The activity assayed with high (600 μM) concentrations of UDPglucose, in contrast, remained in the decapitated epicotyl unaffected by indoleacetic acid or cycloheximide during incubation periods of upt to 5 days. In competition experiments with the two substrates, the individual synthetase activities were not additive, and part of the activity with one substrate was still detectable in the presence of a large excess of the other. These observations indicate the existence in pea particles of at least 4 glucan synthetase activities which differ in substrate affinities, stability and developmental responses to treatments that affect growth and protein synthesis. Such treatments alo markedly influence the deposition of cellulose, e.g. indoleacetic acid caused an 8-fold increase in cellulose laid down in a 3-day period. It is suggested that indoleacetic acid-regulated synthetase activities account for the extra cellulose evoked by indoleacetic acid during sustained growth, and a different non-regulated synthetase activity is responsible for a basal rate of cellulose deposition which proceeds in the presence or absence of indoleacetic acid.

A New Working Hypothesis for the Structure and Function of the Golgi Apparatus in Plant Cell Wall Biogenesis

It is the general belief of many investigators that the pathway of cellulose biogenesis occurs via soluble pools of hexose phosphate monomers and the plasma membrane which is thought to be the site of polymerization and/or crystalization. Brown and coworkers and Ray (see 1) have proposed to the contrary that the Golgi apparatus is the site of cellulose biogenesis in certain scale-producing algae and higher plants respectively. While it has been fairly well established that cellulose can be biosynthesized by the Golgi apparatus, considerable doubt has been expressed that this type of system could be applicable to cellulose biogenesis in higher plant systems. Conversely, the problems associated with in vitro cellulose biosynthesis in Golgi-enriched homogenates raises questions about the in vivo localization of B-(1,4)-glucan synthetase activity.

Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose-dependent beta-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 mum IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate-xylose as substrates, and to a lesser extent with guanosine diphosphoglucose-dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control.

Particulate glucan synthetase activity: Generation and inactivation after treatments with indoleacetic acid and cycloheximide

Growing regions from epicotyls of Pisum sativum L. var Alaska contain a particulate enzyme which transfers glucose from guanosine diphosphate glucose to alkali-soluble and -insoluble products (glucan synthetase activity). When the epicotyl is decapitated to remove the source of natural hormone, the tissue below ceases growth and loses synthetase activity as well as the capacity to continue forming cellulose in vivo. If indoleacetic acid (IAA) is added to the cut apex, massive amounts of cellulose are deposited in the next few days. Particulate glucan synthetase activity is either maintained or greatly increased depending on whether endogenous activity levels are relatively high or low at the time of hormone addition. These effects appear to be due in part to IAA-dependent generation of a protein essential for synthetase activity since they are severely inhibited by concentrations of cycloheximide which are effective at preventing protein synthesis. Nevertheless, the addition of cycloheximide alone to the epicotyl reduces the rate of disappearance of synthetase activity, i.e., a protective effect. Also, a soluble thermolabile component is present in the aging epicotyl which promotes loss of synthetase activity when added to the particulate enzyme in vitro. Accordingly, turnover of pea glucan synthetase activity may be controlled in part by an inactivating protein which is itself subject to turnover.

The Formation of β, 1 → 4 Glucan from UDP-α-d-Glucose Catalyzed by a Phaseolus aureus Enzyme

Particulate enzyme preparations from Phaseolus aureus hypocotyls catalyze the formation of an alkali insoluble beta, 1 --> 4 linked [(14)C]-glucan using UDP-alpha-d [(14)C]-glucose as substrate. Particulate enzymes prepared from root tissue also catalyzed the production of beta, 1 --> 4 glucan. UDP-beta-d-[(14)C]-glucose would not serve as a substrate for these enzymes. The presence or absence of beta, 1 --> 4 glucan synthetase activity was independent of tissue source, substrate concentration, or homogenization method.The particulate enzyme also catalyzes the formation of a beta, 1 --> 3 linked glucan from UDP-d glucose which is usually soluble in hot alkali. The solubility of the beta, 1 --> 3[(14)C]-glucan decreased when the enzyme was obtained from hypocotyls germinated at a higher temperature. The water-soluble material resulting from the catalyzed reaction includes a large proportion of what appears to be [(14)C]-laminaribiose, and smaller proportions of [(14)C]-laminaritriose and [(14)C]-glucose. There is no detectable quantity of [(14)C]-cellobiose or [(14)C]-cellotriose in the water-soluble material.

Effect of L-Sorbose on Polysaccharide Synthetases of Neurospora crassa

Neurospora glycogen synthetase (EC 2.4.1.11) occurs in 100,000 x g particles. The two forms (glucose-6-phosphate dependent-independent) of glycogen synthetase were solubilized and separated by digitonin treatment of the 100,000 x g particles. Glucan synthetase activity of Neurospora was found only in a cell-wall preparation. These two enzymes have been characterized in relation to the paramorphogenic action of sorbose. Sorbose-grown cultures showed a marked decrease in the specific activity of both enzymes, as compared to sucrose-grown wild-type cultures. Sorbose inhibited the activity of the wild-type enzymes both in vivo and in vitro. In the presence of 5 mM sorbose incorporation of [(14)C]glucose from [(14)C]uridinediphosphate glucose into glycogen by the dependent form of glycogen synthetase was completely inhibited. Thus, the paramorphogenic action of sorbose seems to result from its inhibition of these enzymes of cell-wall biosynthesis. Activities of the enzymes from the sorbose-resistant mutant, patch, were not affected by sorbose either in vivo or in vitro.

Changes in Molecular Weight of Cellulose in the Pea Epicotyl during Growth

A procedure is described for preparing cellulose nitrate from pea tissues (Pisum sativum L. var. Alaska) in quantitative yield, undegraded and uncontaminated by other polysaccharides. The average degree of polymerization of this product, estimated from viscosity measurements, increased during cell growth and development from a value of about 5000 glucose units in the apical meristem (plumule plus hook) to values near 8000 in fully grown maturing tissues (>20 mm from apex). The cellulose content per cell also increased (approximately 10-fold) during growth in these tissues, as did particulate glucan synthetase activity (3-fold rise). Since the yield of soluble cellulase activity is known to decrease from high values in the meristem to barely detectable amounts in mature tissues, it is suggested that the relative levels and properties of these hydrolytic and synthetic enzyme activities control the amount and degree of polymerization of cellulose formed during cell expansion in the pea epicotyl.Degree of polymerization distribution patterns showed that a low molecular weight component of cellulose (degree of polymerization < 500) was prominent in young tissues whereas high molecular weight components (degree of polymerization > 7000) predominated in mature tissues. Also, cellulose which was formed from radioactive sucrose during 30 minutes of incubation showed a remarkably similar degree of polymerization distribution to cellulose which was present in the tissue at the time of synthesis. It is concluded that new and old parts of the epicotyl cellulose framework are subject to constant modification and equilibration by cellulose-metabolizing enzymes.

Auxin (2,4-D) stimulation ( [formula omitted] and [formula omitted]) of polysaccharide synthesis in plasma membrane fragments isolated from onion stems

Glucan synthetase activity associated with plasma membrane of onion stem was increased by adding the auxin herbicide 2,4-D. The effect was expressed either by pre-incubating the tissue with 2,4-D ( in vivo ) or by adding 2,4-D to the synthetase assay ( in vitro ). The 2,4-D effect was most pronounced on the synthesis of polysaccharides soluble in hot water. This is the first biochemical deomonstration of an in vitro response to auxin of magnitude comparable to that observed in vivo . The results show that initial responses to the hormone occur at the plasma membrane.

Particulate glucan synthetase activity: dependence on acceptor, activator, and plant growth hormone.

Particulate fractions isolated from the growing region of the epicotyl of Pisum sativum L. var Alaska are capable of transferring glucose from uridine or guanosine diphosphate glucose-14C to buffer-insoluble products which are partly alkali-soluble and partly alkali-insoluble. Cellobiose activates the reaction; carboxymethylcellulose and cellodextrins act as competitive acceptor molecules.When the epicotyl is decapitated, glucan synthetase activity disappears from particulate fractions of the growing region within 3 days unless the hormone indoleacetic acid (IAA) is added to the tissue, in which event activity is retained. Other growth regulators (gibberellic acid, benzyladenine) have no such effect. Loss of activity is not due to a change in solubility of enzyme or product or to the absence of substrate, activator, or acceptor molecules, nor is it accompanied by comparable losses in total protein or cellulase activity from the particulate fraction. It is concluded that IAA is needed for the formation and/or protection of an essential part of the insoluble synthetase complex.

Solubilization and Separation of Uridine Diphospho-d-glucose: β-(1 → 4) Glucan and Uridine Diphospho-d-glucose:β-(1 → 3) Glucan Glucosyltransferases from Coleoptiles of Avena sativa

The particulate glucan synthetase preparation isolated from a homogenate of oat coleoptiles at 4 C lost 65% of its original activity after 1 day when the UDP-d-glucose substrate concentration was 5 x 10(-7)m to 1.0 x 10(-6)m. Storage of the particulate enzyme at -20 C or in liquid nitrogen did not prevent the enzyme from losing its activity. Incorporation of 0.5% hovine serum albumin into the medium stabilized the particulate enzyme at 0 C for 6 days and for at least 2 weeks in liquid nitrogen.When the particulate enzyme was treated with 8% digitonin, 40 to 50% of its activity appeared in the 100,000g supernatant fraction. The particulate and digitonin-solubilized enzyme preparations synthesized both beta-(1 --> 4) and beta-(1 --> 3) glucosyl linkages from UDP-d-glucose, but beta-(1 --> 3) glucan was the main product at 1 x 10(-3)m UDP-d-glucose substrate. The activity of beta-(1 --> 4) glucan synthetase was stimulated at least 10-fold in the presence of MgCl(2). A separation of beta-(1 --> 4) and beta-(1 --> 3) glucan synthetase activities could be achieved at 1 x 10(-3)m UDP-d-glucose when the digitonin-solubilized enzyme was adsorbed on a hydroxylapatite gel and then eluted with concentrated potassium phosphate buffer. The results indicate that the particulate enzyme contains two enzymes, one responsible for the synthesis of beta-(1 --> 4) and another beta-(1 --> 3) linkages in the glucan or glucans synthesized from UDP-d-glucose.