Water-soluble (1→3), (1→4)-β-d-glucans from barley (Hordeum vulgare) endosperm. III. Distribution of cellotriosyl and cellotetraosyl residues

Cellotriosyl and cellotetraosyl residues, linked by single (1→3)-β-linkages, account for more than 90% of the 40°C water-soluble (1→3), (1→4)-β-d-glucan from barley flour. We have analysed their sequence dependence by treating the polymer as a two-state Markov chain with stationary distribution. Quantitation of the penultimate oligosaccharides released during hydrolysis of the (1→3), (1→4)-β-d-glucan with (1→3), (1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73) by analytical gel filtration chromatography enabled the relative abundance of two adjacent cellotriosyl, two adjacent cellotetraosyl and adjacent cellotetraosyl/cellotriosyl residues to be estimated and the sequence dependence to be evaluated.Within the theoretical and practical constraints of the method it is concluded that the cellotriosyl and cellotetraosyl residues are arranged in an essentially independent (random) fashion. Thus, any mechanism proposed for the biosynthesis of the molecule should explain this apparently random distribution of cellotriosyl and cellotetraosyl residues as well as the presence, in relatively low frequency, of blocks of up to 10 or more adjacent (1→4)-linkages.     

Evolution and differential expression of the (1→3)-β-glucan endohydrolaseencoding gene family in barley, Hordeum vulgare

The (1→3)-β-d-glucan glucanohydrolases [(1→ 3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1→3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1→3,1→4)-β-glucan 4-glucanohydrolases [(1→3,1→4)-GGH; EC 3.2.1.73] further indicate that the (l→3)-GGHs and (l→3,1→4)-GGHs are derived from a single ‘super’ gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1→3,1→4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1→3,1→4)-β-glucans that are the major constituents in barley walls, while the (1→3)-GGHs, which are unable to degrade the plant (1→3,1→4)-β-glucans, can hydrolyse the (1→3)- and (1→3,1→6)-β-glucans of fungal cell walls.     

Optimized methodology for extraction of (1 → 3)(1 → 6)-β-d-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative

The cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with immunostimulant properties. The standard methodologies described for its extraction involve acid and alkaline washings, which degrade part of its glucose chains and reduce the final yield. In the present study, an optimized methodology for extraction of β-d-glucan from S. cerevisiae cells, involving sonication and enzyme treatment, with a yield of 11.08 ± 0.19%, was developed. The high-purity (1 → 3)(1 → 6)-β-d-glucan was derivatized to carboxymethyl-glucan (CM-G). In vitro tests with CM-G in Chinese hamster epithelial cells (CHO-k1) did not reveal any cytotoxic or genotoxic effects or influences of this molecule on cell viability. The method described here is a convenient alternative for the extraction of (1 → 3)(1 → 6)-β-d-glucan under mild conditions without the generation of wastes that could be potentially harmful to the environment.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Enzymatic synthesis of α- -fucosyl-N-acetyllactosamines and 3′-O-α- -fucosyllactose utilizing α- -fucosidases

An α- -fucosidase from porcine liver produced α- -Fuc-(1→2)-β- -Gal-(1→4)- -GlcNAc (2′-O-α- -fucosyl-N-acetyllactosamine, 1) together with its isomers α- -Fuc-(1→3)-β- -Gal-(1→4)- -GlcNAc (2) and α- -Fuc-(1→6)-β- -Gal-(1→4)- -GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl α- -fucopyranoside and β- -Gal-(1→4)- -GlcNAc. The enzyme formed the trisaccharides 1–3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. α- -fucosidase led to the regioselective synthesis of trisaccharides containing a (1→3)-linked α- -fucosyl residue. When β- -Gal-(1→4)- -GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and α- -Fuc-(1→3)-β- -Gal-(1→4)- -Glc (3′-O-α- -fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

Synthesis, and carbon-13 n.m.r. study, of O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose and O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl (1→3)- -rhamnopyranose, constituents of bacterial, cell-wall polysaccharides

O-α- -Rhamnopyranosyl-(1→3)- -rhamnopyranose (19) and O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α- -rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α- -rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β- -galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyl]-β- -galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-β- -rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β- -galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α- -rhamnopyranosylation at HO-2 or -3 of a free α- -rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α- -Rhamnopyranosylation of HO-6 of gb- -galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O₂,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Sequential synthesis and C-N.m.r. spectra of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides

The reaction of 2,3-di-O-acetyl-4-O-benzyl-α,β-d-xylopyranosyl bromide (2) with methyl 2,3-di-O-acetyl-β-d-xylopyranoside gave methyl O-(2,3-di-O-acetyl-4-O-benzyl-β-d-xylopyranosyl)-(1→4)-2,3-di-O-acetyl-β-d-xylopyranoside (22). Catalytic hydrogenolysis of 22 exposed HO-4′ which was then condensed with 2. This sequence of reactions was repeated three more times to afford, after complete removal of protecting groups, a homologous series of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides. ¹³C-N.m.r. spectra of the synthetic methyl β-glycosides (di- to hexa-saccharide) are presented together with data for six other, variously substituted, homologous series of (1→4)-d-xylo-oligosaccharides.     

Medicinal importance of fungal β-(1→3), (1→6)-glucans

Non-cellulosic β-glucans are now recognized as potent immunological activators, and some are used clinically in China and Japan. These β-glucans consist of a backbone of glucose residues linked by β-(1→3)-glycosidic bonds, often with attached side-chain glucose residues joined by β-(1→6) linkages. The frequency of branching varies. The literature suggests β-glucans are effective in treating diseases like cancer, a range of microbial infections, hypercholesterolaemia, and diabetes. Their mechanisms of action involve them being recognized as non-self molecules, so the immune system is stimulated by their presence. Several receptors have been identified, which include: dectin-1, located on macrophages, which mediates β-glucan activation of phagocytosis and production of cytokines, a response co-ordinated by the toll-like receptor-2. Activated complement receptors on natural killer cells, neutrophils, and lymphocytes, may also be associated with tumour cytotoxicity. Two other receptors, scavenger and lactosylceramide, bind β-glucans and mediate a series of signal pathways leading to immunological activation. Structurally different β-glucans appear to have different affinities toward these receptors and thus generate markedly different host responses. However, the published data are not always easy to interpret as many of the earlier studies used crude β-glucan preparations with, for the most part, unknown chemical structures. Careful choice of β-glucan products is essential if their benefits are to be optimized, and a better understanding of how β-glucans bind to receptors should enable more efficient use of their biological activities.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

β-N-Acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides

A large panel of fungal β-N-acetylhexosaminidases was tested for the regioselectivity of the β-GlcNAc transfer onto galacto-type acceptors ( -galactose, lactose, 2-acetamido-2-deoxy- -galactopyranose). A unique, non-reducing disaccharide β- -GlcpNAc-(1→1)-β- -Galp and trisaccharides β- -GlcpNAc-(1→4)-β- -GlcpNAc-(1→1)-β- -Galp, β- -Galp-(1→4)-β- -Glcp-(1→1)-β- -GlcpNAc and β- -Galp-(1→4)-α- -Glcp-(1→1)-β- -GlcpNAc were synthesised under the catalysis of the β-N-acetylhexosaminidase from the Aspergillus flavofurcatis CCF 3061 with -galactose and lactose as acceptors. The use of 2-acetamido-2-deoxy- -galactopyranose as an acceptor with the β-N-acetylhexosaminidases from A. flavofurcatis CCF 3061, A. oryzae CCF 1066 and A. tamarii CCF 1665 afforded only β- -GlcpNAc-(1→6)- -GalpNAc.     

Purification and some properties of a (1→4)-β- -glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β- -glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β- -glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 m , respectively. -Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β- -glucopyranoside (K_i 2.1 μ ), cellobiose (K_i 1.95 μ ), and cellotriose (K_i 7.9 μ ) [cf. -glucose (K_i 1756 μ )]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β- -glucopyranoside appeared to be competitively inhibited by -glucono-1,5-lactone. However, inhibition of hydrolysis by -glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β- -glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β- -glucanase found in the same cellulase, namely, the (1→4)-β- -glucan cellobiohydrolase.     

Evaluation of oat bran as a soluble fibre source. Characterization of oat β-glucan and its effects on glycaemic response

Oat β-glucan, present in oat bran in greater concentrations than in the whole oat groat, is mainly composed of β-(1 → 3)-linked cellotriosyl and cellotetraosyl units, present at 52 and 34% by weight of the molecule, respectively. The remaining structure consits of β-(1 → 3)-linked blocks composed of four or more consecutive β-(1 → 4)-linked -glucopyranosyl units. Size-exclusion chromatography indicated a molecular weight for oat β-glucan of 2–3 × 10⁶. This was significantly reduced during digestion in the small intestine of rats and chicks. In healthy human volunteers, oat β-glucan reduced the postprandial glucose response to an oral glucose load similarly to guar gum. The effectiveness of oat β-glucan was proportional to the logarithm of the viscosity of the solution fed.     

Conversion of amylose into a (1→4)-2-amino-2-deoxy-α-D-glucopyranuronan and its 2-N-sulfo derivative, a heparin analog

By a modification of a previously established reaction-sequence involving successive oxidation with methyl sulfoxide-acetic anhydride, oximation, and reduction with lithium aluminum hydride, 6-O-tritylamylose (1) was converted into a 6-O-tritylated (1→4)-α-D-linked glucan (3) containing 2-amino-2-deoxy-D-glucose residues and some O-(methylthio)methyl groups. Removal of the ether groups from this product gave a 2-aminated amylose (4) of degree of substitution (d.s.) by amine of 0.54 that underwent cleavage by fungal alpha-amylase to give oligosaccharides containing amino sugar residues. N-Trifluoroacetylation of 3 followed by removal of the ether groups, oxidation at C-6 with oxygen-platinum, and removal of the N-substituent, gave a (1 →4)-2-amino-2-deoxy-α-D-glucopyranuronan 7 having d.s. by amine of up to 0.65, and by carboxyl, of 0.46. Sulfation of this product with sulfur trioxide-pyridine and then with chlorosulfonic acid-pyridine gave a (1→4)-2-deoxy-2-sulfoamino-α-D-glucopyranuronan, isolated as its sodium salt 8, which showed appreciable blood-anticoagulant activity.     

Triple helix conformation of botryosphaeran, a (1→3;1→6)-β-d-glucan produced by Botryosphaeria rhodina MAMB-05

Botryosphaeran, a (1→3;1→6)-β-d-glucan produced by Botryosphaeria rhodina MAMB-05, was found to be present in a triple helix conformation from helix–coil transition studies using Congo Red. The triple helix conformation was disrupted at increasing alkali concentrations. Conformational changes were also observed using phenanthrene as a fluorescent probe, and the fluorescence intensity decreased 80% in the presence of dimethyl sulfoxide. The results confirmed the triple helix conformation of botryosphaeran, an important property manifesting biological response modifying activity.     

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Water-soluble (1→3),(1→4)-β-d-glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted.The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β-d-glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β-d-glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.     

Synthesis of Neu5Ac- and Neu5Gc-α-(2→6′)-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-α-(2→6′)-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-α-(2→6′)-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4′,6′-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Topology of the maize mixed linkage (1->3),(1->4)-beta-d-glucan synthase at the Golgi membrane

Mixed-linkage (1-->3),(1-->4)-beta-d-glucan is a plant cell wall polysaccharide composed of cellotriosyl and cellotetraosyl units, with decreasingly smaller amounts of cellopentosyl, cellohexosyl, and higher cellodextrin units, each connected by single (1-->3)-beta-linkages. (1-->3),(1-->4)-beta-Glucan is synthesized in vitro with isolated maize (Zea mays) Golgi membranes and UDP-[(14)C]d-glucose. The (1-->3),(1-->4)-beta-glucan synthase is sensitive to proteinase K digestion, indicating that part of the catalytic domain is exposed to the cytoplasmic face of the Golgi membrane. The detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid] (CHAPS) also lowers (1-->3),(1-->4)-beta-glucan synthase activity. In each instance, the treatments selectively inhibit formation of the cellotriosyl units, whereas synthesis of the cellotetraosyl units is essentially unaffected. Synthesis of the cellotriosyl units is recovered when a CHAPS-soluble factor is permitted to associate with Golgi membranes at synthesis-enhancing CHAPS concentrations but lost if the CHAPS-soluble fraction is replaced by fresh CHAPS buffer. In contrast to other known Golgi-associated synthases, (1-->3),(1-->4)-beta-glucan synthase behaves as a topologic equivalent of cellulose synthase, where the substrate UDP-glucose is consumed at the cytosolic side of the Golgi membrane, and the glucan product is extruded through the membrane into the lumen. We propose that a cellulose synthase-like core catalytic domain of the (1-->3),(1-->4)-beta-glucan synthase synthesizes cellotetraosyl units and higher even-numbered oligomeric units and that a separate glycosyl transferase, sensitive to proteinase digestion and detergent extraction, associates with it to add the glucosyl residues that complete the cellotriosyl and higher odd-numbered units, and this association is necessary to drive polymer elongation.     

The endo-(1→4)-β- -glucanase system of Penicillium pinophilum cellulase: Isolation, purification, and characterization of five major endoglucanase components

Five major endo-(1→4)-β- -glucanases (I–V) have been isolated from a cellulase preparation of P. pinophilum. The pI values for I–V were 7.4, 4.8, 4.1, 3.7, and 4.0, respectively, and the respective molecular weights were 25,000, 39,000, 62,500, 54,000, and 44,500, when determined by SDS-gel electrophoresis. Endoglucanase V was optimally active at 65–70° and I–IV were most active at 50–60°. The pH optima of I and III–V were in the range 4.0–5.0. Antiserum prepared to I reacted only with I; II antiserum reacted only with II. Endoglucanases I and V were more random in their attack on CM-cellulose and H₃PO₄-swollen cotton cellulose, and showed no activity against cello-oligosaccharides containing less than five -glucose residues, whereas III and IV were active against all the cello-oligosaccharides tested and acted in a less random manner, and II was intermediate in its catalytic action. III was adsorbed completely on both Avicel PH101 and H₃PO₄-swollen cellulose, whereas IV was not adsorbed. The endoglucanases I–V have distinct roles in the digestion of cellulose.