Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

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.     

Action of Endo-(1 → 6)-α-d-glucanases on the soluble dextrans produced by three extracellular α-d-glucosyltransferases of Streptococcus sobrinus

Four different α-d-glucosyltransferases (GTF) have been obtained from culture filtrates of Streptococcus sobrinus strains grown in the chemostat at pH 6·5 in complex medium supplemented with Tween 80. Three of the enzymes, GTF-S1, GTF-S3 and GTF-S4, converted sucrose into soluble glucans. Their limit of hydrolysis with endodextranase, the proportion of linear to branched oligosaccharides among the end products of enzymic degradation, and methylation analysis, all supported the view that the glucans were dextrans. The S1-dextrans were highly branched (32% of α-(1 → 3)-branch points), S3-dextrans were linear, and the branching of S4-dextrans was intermediate in value (9%). The enzymes that catalyze the synthesis of three such diverse dextrans were thus proved to be three different GTF, each with a characteristic specificity. Conditions of growth in the chemostat could be varied to provide maximum yields of either GTF-S1, -S3 or -S4.     

β-d-glucosidase-catalysed transfer of the glycosyl group from aryl β-d-gluco- and β-d-xylo-pyranosides to phenols

The effect of phenols on the hydrolysis of substituted phenyl β-d-gluco- and β-d-xylo-pyranosides by β-d-glucosidase from Stachybotrys atra has been investigated. Depending on the glycon part of the substrate and on the phenol substituent, the hydrolysis is either inhibited or activated. With aryl β-d-xylopyranosides, transfer of the xylosyl residue to the phenol, with the formation of new phenyl β-d-xylopyranosides, is observed. With aryl β-d-glucopyranosides, such transfer does not occur when phenols are used as acceptors, but it does occur with anilines. A two-step mechanism, in which the first step is partially reversible, is proposed to explain these observations. A qualitative analysis of the various factors determining the overall effect of the phenol is given.     

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.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-d-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Relative stability of poly(β-hydroxy-l-proline)

The semiempirical CNDO/2 SCF MO method using the tight-binding approximation for polymers has been applied to poly(β-hydroxy-l-proline), β-PHP, to compare the electronic structure of β-PHP with that of poly(γ-hydroxy-l-proline), γ-PHP, which we have described in a previous publication. The results obtained show the preferred orientation of the OH group at the β-position of the pyrrolidine ring. The different situation between β-PHP and γ-PHP is briefly discussed. Analysis of the calculated results shows that the energy difference between the two species is not sufficient to deny the existence of either form. This agrees well with the experimental results. The conformational stability between the trans and cis forms of the H---:C---:O---:H group is explained by using the calculated results in connection with the previous experimental and theoretical treatments. From the analysis of the total energy, the dominant stabilizing factors are discussed.     

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(d-Val-l-Pro-l-Val-d-Pro)3

The cyclic dodecapeptide PV, cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃, a structural analogue of the ion-carier valinomycin, increase the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na⁺ < NH₄⁺ < K⁺ < Cs⁺ < Rb⁺. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanims). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.     

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.     

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.     

Efficient synthesis of d-xylo and d-ribo-phytosphingosines from methyl 2-amino-2-deoxy-β-d-hexopyranosides

A general and flexible synthetic approach to biologically important 5,6-unsaturated C₁₈-phytosphingosines was developed via olefin cross-metathesis employing truncated C₆-phytosphingosines as the key intermediates. These were efficiently prepared in high yields by zinc-mediated reductive opening of methyl 2-amino-2-deoxy-β-hexopyranosides.

Full-size image (6K)

     

Synthesis and glycogen phosphorylase inhibitory activity of N-(β-d-glucopyranosyl)amides possessing 1,4-benzodioxane moiety

A series of N-(β-d-glucopyranosyl)amides 5d–i were synthesized by PMe₃ mediated Staudinger reaction of O-peracetylated β-d-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d–i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure–activity relationships of these compounds are also discussed in this paper.     

ω-Aminoalkyl β-glycosides of N-acetylmuramyl-l-alanyl-d-isoglutamine,and their conjugates with meningococcal group C polysaccharide

The 6-aminohexyl β-glycoside of N-acetylmuramyl-l-alanyl-d-isoglutamine and its spacer-arm-linked analog (3.8 nm) were synthesized from 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)-[2,1-d]-2-oxazoline, and coupled with meningococcal group C polysaccharide in attempts to enhance the immunogenicity of the polysaccharide antigen.     

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.     

Crystal and molecular structure of potassium β-d-glucopyranose 6-sulphate

The crystal and molecular structure of potassium β-d-glucopyranose 6-sulphate has been determined by direct methods. The sugar ring has the expected ⁴C₁ conformation although the sulphate group causes flattening of the ring. The potassium ion has octahedral co-ordination involving oxygen atoms of five independent β-d-glucopyranose 6-sulphate molecules. The n.m.r. spectrum of the 6-sulphate in the solid state is consistent with the occurrence of two molecules in the unit cell related by a 2₁ symmetry axis.