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.     

The Formation of beta, 1 --> 4 Glucan from UDP-alpha-d-Glucose Catalyzed by a Phaseolus aureus Enzyme

Particulate enzyme preparations from Phaseolus aureus hypocotyls catalyze the formation of an alkali insoluble β, 1 → 4 linked [¹⁴C]-glucan using UDP-α-d [¹⁴C]-glucose as substrate. Particulate enzymes prepared from root tissue also catalyzed the production of β, 1 → 4 glucan. UDP-β-d-[¹⁴C]-glucose would not serve as a substrate for these enzymes. The presence or absence of β, 1 → 4 glucan synthetase activity was independent of tissue source, substrate concentration, or homogenization method.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Substrate Activation of beta-(1 --> 3) Glucan Synthetase and Its Effect on the Structure of beta-Glucan Obtained from UDP-d-glucose and Particulate Enzyme of Oat Coleoptiles

UDP-d-glucose, at a micromolar level in the presence of MgCl₂ and oat (Avena sativa) coleoptile particulate enzyme which contains both β-(1 → 3) and β-(1 → 4) glucan synthetases, produces glucan with mainly β-(1 → 4) glucosyl linkages. An activation of β-(1 → 3) glucan synthetase by UDP-d-glucose and a decrease in the formation of β-(1 → 3) glucan in the presence of MgCl₂ have been observed. However, at high substrate concentration (≥ 10⁻⁴m), the activation of β-(1 → 3) glucan synthetase is so pronounced that the formation of β-(1 → 3) glucosyl linkage predominates in synthesized glucan regardless of the presence of MgCl₂. These observations may explain the striking shift in the composition of glucan of particulate enzyme from a β-(1 → 4) to β-(1 → 3) glucosyl linkage when UDP-d-glucose concentration is raised from a low concentration (≤ 10⁻⁵m) to a higher concentration (≥ 10⁻⁴m).     

Fission Yeast Rgf2p Is a Rho1p Guanine Nucleotide Exchange Factor Required for Spore Wall Maturation and for the Maintenance of Cell Integrity in the Absence of Rgf1p

Schizosaccharomyces pombe Rho1p is essential, directly activates β-1,3-glucan synthase, and participates in the regulation of morphogenesis. In S. pombe, Rho1p is activated by at least three guanine nucleotide exchange factors (GEFs): Rgf1p, Rgf2p, and Rgf3p. In this study we show that Rgf2p is a Rho1p GEF required for sporulation. The rgf2⁺ deletion did not affect forespore membrane formation and the nuclei were encapsulated properly. However, the mutant ascospores appeared dark and immature. The rgf2Δ zygotes were not able to release the ascospores spontaneously, and the germination efficiency was greatly reduced compared to wild-type (wt) spores. This phenotype resembles that of the mutants in bgs2⁺, which encodes a sporulation-specific glucan synthase subunit. In fact, glucan synthase activity was diminished in sporulating rgf2Δ diploids. Rgf2p also plays a role in β-glucan biosynthesis during vegetative growth. Overexpression of rgf2⁺ specifically increased GTP-bound Rho1p, caused changes in cell morphology, and elicited an increase in β-1,3-glucan synthase activity. Moreover, the simultaneous disruption of rgf1⁺ and rgf2⁺ was lethal and both Rgf1p and Rgf2p were able to partially substitute for each other. Our results suggest that Rgf1p and Rgf2p are alternative GEFs with an essential overlapping function in Rho1p activation during vegetative growth.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : II. Product Inhibition by UDP

The mode of inhibition of UDP, one of the products of the reaction catalyzed by (1→3)-β-d-glucan synthase in sugar beet (Beta vulgaris L.) was investigated. In the absence of added UDP, the enzyme, in the presence of Ca²⁺, Mg²⁺, and cellobiose, exhibited Michaelis-Menten kinetics and had an apparent K_m of 260 micromolar for UDP-glucose. Complex effects on the kinetics of the (1→3)-β-d-glucan synthase were observed in the presence of UDP. At high UDP-glucose concentrations, i.e. greater than the apparent K_m, UDP behaved as a competitive inhibitor with an apparent K_i of 80 micromolar. However, at low UDP-glucose concentrations, reciprocal plots of enzyme activity versus substrate concentration deviated sharply from linearity. This unusual effect of UDP is similar to that reported for fungal (1→3)-β-d-glucan synthase. However, papulacandin B, a potent inhibitor of this fungal enzyme, had no effect on the plant (1→3)-β-d-glucan synthase isolated from sugar beet petioles. The inhibitory effect of UDP was also compared with other known inhibitors of glucan synthases.     

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.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic β-(1,2)-Glucan Biosynthesis

The cyclic β-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic β-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic β-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic β-(1,2)-glucan biosynthesis.     

UDP-Glucose: (1,3)-beta-Glucan Synthase from Daucus carota L. : Characterization, Photoaffinity Labeling, and Solubilization

The membrane-bound UDP-glucose-β-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca²⁺, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5′-β-[³²P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.     

Nanoscopic cell-wall architecture of an immunogenic ligand in Candida albicans during antifungal drug treatment

The cell wall of Candida albicans is composed largely of polysaccharides. Here we focus on β-glucan, an immunogenic cell-wall polysaccharide whose surface exposure is often restricted, or “masked,” from immune recognition by Dectin-1 on dendritic cells (DCs) and other innate immune cells. Previous research suggested that the physical presentation geometry of β-glucan might determine whether it can be recognized by Dectin-1. We used direct stochastic optical reconstruction microscopy to explore the fine structure of β-glucan exposed on C. albicans cell walls before and after treatment with the antimycotic drug caspofungin, which alters glucan exposure. Most surface-accessible glucan on C. albicans yeast and hyphae is limited to isolated Dectin-1–binding sites. Caspofungin-induced unmasking caused approximately fourfold to sevenfold increase in total glucan exposure, accompanied by increased phagocytosis efficiency of DCs for unmasked yeasts. Nanoscopic imaging of caspofungin-unmasked C. albicans cell walls revealed that the increase in glucan exposure is due to increased density of glucan exposures and increased multiglucan exposure sizes. These findings reveal that glucan exhibits significant nanostructure, which is a previously unknown physical component of the host–Candida interaction that might change during antifungal chemotherapy and affect innate immune activation.     

Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP-Pyridoxal: Evidence for an Active-Site Amino Group

UDP-pyridoxal competitively inhibits the Ca²⁺-, cellobiose-activated (1→3)-β-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K_i of 3.8 ± 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V_max of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 ± 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (1→3)-β-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca²⁺ and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1→3)-β-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[³H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca²⁺ and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP-[³H]pyridoxal in the presence of Mg²⁺ and copurifies with (1→3)-β-glucan synthase activity.     

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Bgs3p,a putative 1,3-beta-glucan synthase subunit,is required for cell wall assembly in Schizosaccharomyces pombe

β-Glucans are the main components of the fungal cell wall. Fission yeast possesses a family of β-glucan synthase-related genes. We describe here the cloning and characterization of bgs3⁺, a new member of this family. bgs3⁺ was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis. Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the β-glucan, and raises the amount of α-glucan polymer. These cells finally died. bgs3⁺ is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations. Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas. We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination. We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.     

Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide.     

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

β-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 Mg²⁺ 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 β-glucanases all show that both β-1,3 and β-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. β-1,4 Linkages predominate at low substrate (5 μm) concentration. Kinetic data indicate that the capacity to synthesize β-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.     

Hydrolytic Activity and Substrate Specificity of an Endoglucanase from Zea mays Seedling Cell Walls

An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1→3),(1→4)-β-d-glucosyl sequences. Substrates with (1→4)-β-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1→3)-β-glucans such as laminarin were not hydrolyzed. When (1→3),(1→4)-β-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 × 10⁴). Activity against a water soluble (1→3),(1→4)-β-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1→3),(1→4)-β-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1→3),(1→4)-β-d-glucan that contained blocks of (1→4) linkages and/or more than a single contiguous (1→3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1→3),(1→4)-β-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase.