Rapid and sensitive,colorimetric determination of the anomers of d-glucose with d-glucose oxidase,peroxidase, and mutarotase

A modification, utilising mutarotase, of an enzymic, colorimetric system for determining d-glucose with d-glucose oxidase, peroxidase, and ABTS was satisfactory for the assay of the anomers of d-glucose in aqueous solution. The time required for a single assay is ≈ 10 min, and the lower limit is 0.4 μg of d-glucose. The method is applicable to the anomer analysis of d-glucose released by enzymic hydrolysis of d-glucosides.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

The hydrothermolysis of cellobiose and its reaction-product d-glucose

The hydrothermolysis of cellobiose in the range 180–249° has been studied. Kinetic analysis of the reaction showed that 60% of the cellobiose is converted into d-glucose, and 40% into other products. The rate (k₁) of cellobiose disintegration is approximately eight times that (k₂) of d-glucose. Thus, hydrothermolysis differs from acidic hydrolysis. Hydrothermolysis is not dependent on pH, at least in the range 3–7.     

X-ray diffraction study of the molecular association in aqueous solutions of d-fructose, d-glucose,and sucrose

X-ray diffractograms of aqueous solutions of d-fructose, d-glucose, and sucrose, and of two amorphous “solid” forms of these carbohydrates are recorded in the angular range of O 2-16°. The formation, in these diffractograms, of one or two intensity maxima as the concentration is varied is interpreted in terms of modification of the molecular association. A model is proposed that takes account of the state of organization of the solutions of the three sugars as a function of the concentration. Sucrose shows an “anomaly”, as it is the only sugar to establish intramolecular bonds. The number of these intramolecular bonds also depends on the concentration.     

Synthesis of 4-deoxy-d-xylo-hexose and 4-azido-4-deoxy-d-glucose and their effects on lactose synthase

Syntheses are reported of 4-deoxy-d-xylo-hexose and 4-azido-4-deoxy-d-glucose as potential inhibitors for lactose synthase [uridine 5′-(α-d-galactopyranosyl pyrophosphate):d-glucose 4-β-d-galactopyranosyltransferase, EC 2.4.1.22]. These syntheses involved SN2 displacement of the 4-methylsulfonyloxy group of methyl 2,3,6-tri-O-benzoyl-4-O-methylsulfonyl-α-d-galactopyranoside by iodide and azide ions. In both cases, inversion in configuration was observed. The resulting intermediates, methyl 2,3,6-tri-O-benzoyl-deoxy-4-iodo-α-d-glucopyranoside and methyl 4-azido-2,3,6-tri-O-benzoyl-deoxy-α-d-glucopyranoside, were obtained in crystalline form. Both 4-deoxy-d-xylo-hexose and 4-azido-4-deoxy-d-glucose were found to be inhibitors for lactose synthase in the presence of α-lactalbumin, but had no effect in the absence of α-lactalbumin. Both d-glucose analogues bind to the enzyme system far more weakly than d-glucose, suggesting that the recognition of the 4-OH group of the acceptor substrate is an important factor in binding.     

Specific d-glucose transport in sarcolemma vesicles

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate d-glucose in preference to l-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific d-glucose uptake by these vesicles is a saturable process, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both d- and l-glucose into the vesicles.Stereospecific d-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca²⁺ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific d-glucose transport. Apparent uptake of both d- and l-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.     

Synthesis and growth regulator activity of fatty acyl derivatives of d-glucose and d-galactose

In an attempt to gain information about one or more components of the brassin complex, fatty acid esters of d-glucose and d-galactose were prepared and tested for growth regulator activity in a bean hypocotyl bioassay. 4-O-Acyl-d-glucoses and, perhaps, 1-O-acyl- d-galactoses had a similar qualitative activity to that of the brassin complex. 3-O-Acyl- d-galactoses inhibited elongation of bean hypocotyls and stimulated rooting. 3- And 6-O- acyl-d-glucoses both stimulated and inhibited elongation, depending on the source of fatty acids; in both cases, stimulation was observed when safflower oil was used as the source of fatty acids and inhibition was observed when peanut oil was used as the source of fatty acids. Fatty alkyl β-d-galactopyranosides were inactive.     

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.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Mode of action of a xylanase and its significance for the structural investigation of the branched l-arabino-d-glucurono-d-xylan from redwood (Sequoia sempervirens)

The substitution pattern of the water-soluble l-arabino-(4-O-methyl-d-glucurono)-d-xylan from redwood (Sequoia sempervirens) has been studied by enzymic degradation. Exhaustive hydrolysis by an endo-xylanase (EC 3.2.1.8) from a Basidiomycete Sporotrichum dimorphosporum left a residue accounting for 20% of the original d-xylan. In the dialyzable material, oligosaccharides having arabinose or 4-O-methylglucuronic acid residues attached to the non-reducing d-xylosyl end-group of xylobiose or xylotriose, respectively, were the smallest branched oligomers released. Action of the xylanase appears to involve a region of the polysaccharide backbone having three xylosyl residues. A mode of action is proposed that requires unsubstituted hydroxyl groups at C-2, C-3, and C-2′ of a xylobiosyl residue. The binding site seems to correspond to a shallow cavity. The composition and structure of the final residue of attack shows that the enzyme has no action when the xylosyl residues branched through O-2 are separated by only one, unsubstituted xylose residue. This pattern of action, the nature of the dialyzable products, and the production of a final residue in which the substituents are accumulated, suggest that the arabinosyl and glucosyl-uronic groups are irregularly distributed on the main chain of the xylan from redwood and that in some regions they are in close vicinity when not actually on adjacent xylosyl residues.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Laser-raman study of solute-solvent interactions in aqueous solutions of d-fructose, d-glucose,and sucrose

The solute-solvent interactions of d-fructose, d-glucose, and sucrose in aqueous solution were studied by comparison of characteristic, Raman of the water and the sugar components. Shifts in frequency and intensity were observed in both the bending and the stretching regions of CH₂ and H₂O. The ratios of integrated, Raman intensities I(CH₂)/I(H₂O) of the CH₂ peak and the H₂O bending band, and I(CH)/I(OH) of the C-H stretching line to O-H stretching band were determined. Their evolutions in terms of mass-concentration display discontinuities at specific concentrations for each of the three sugars. These breaks were interpreted as changes in the hydrogen bonding of the various species.     

Syntheses of methyl 2,6-diacetamido-2,3,6-trideoxy-α-d-ribo-hexopyranoside (methyl di-N-acetyl-α-d-tobrosaminide) and methyl 2-acetamido-2,3,6-trideoxy-β-l-lyxo-hexopyranoside

The title glycosides were synthesised from d-glucose, via the common intermediate methyl 2-acetamido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-α-d-ribo-hexopyranoside.     

Isolation and some properties of extracellular d-glucosyltransferases and d-fructosyltransferases from Streptococcus mutans serotypes c, e, and f

Extracellular d-glucosyltransferases (GTase) and d-fructosyltransferases (FTase) were isolated from Streptococcus mutans IB (serotype c), B14 (e), and OMZ175 (f) by chromatofocusing, followed by hydroxyapatite column chromatography. The GTases isolated from serotypes c, e, and f are basic proteins (pI 7.4). The serotype c and e enzymes have two protein components having M_r 173 000 and 158 000 and the enzyme of the serotype f one component having M_r 156 000. The GTases of all the serotypes showed a K_m value for sucrose of 10–14mm and an optimum pH 5.5–6.0 for enzyme activity, and their activities were enhanced by the presence of primer Dextran T10. The α-d-glucans synthesized by the purified GTases are water soluble and primarily consist of (1→6)-α-d-glucosidic linkage (41–66 mol/100 mol) and α-d-(1→3,6)-branch linkage (6–20 mol/100 mol), but significant proportions of α-d-(1→3), α-d-(1→4), and α-d-(1→3,4) linkages (11, 6, and 14 mol/100 mol, respectively) were detected in the serotype c α-d-glucan. The isolated FTases of the serotypes c, e, and f are acidic enzymes (pI 4.6) and consist of two components having M_r 84 000 and 76 000 for the serotype c enzyme, and 106 000 and 84 000 for the serotypes e and f enzymes, respectively. The K_m value for sucrose was 6, 10, and 17mm for the serotypes c, e, and f enzymes, respectively, and the optimum pH of enzymic activity 5.5–6.0. Reactivity with Concanavalin A, susceptibility to acid hydrolysis, and paper chromatography of the hydrolyzates suggested that the water-soluble β-d-fructans synthesized by the purified FTases were of the inulin-type and had chemical structures somewhat different among the serotypes.     

Laser-raman spectra of d-glucose and sucrose in aqueous solution

Laser-Raman spectra of d-glucose in water at various concentrations were recorded below 1700 cm^?1. Assignments of the frequencies of d-glucose were proposed, based on earlier work on the vibrational spectra of this sugar, and determination by other techniques of the anomeric composition of aqueous solutions of d-glucose. The proportions of the anomers found from the ratio of the Raman intensities for the same models of vibrations were similar to those found by other techniques. Assignments of the frequencies for sucrose were proposed, in the light of previous results on d-glucose and d-fructose, and the effect, on their Raman spectra, of the condensation of these two monosaccharides was pointed out.     

Temperature dependence of d-glucose transport in reconstituted liposomes

Sodium-dependent d-glucose uptake into proteoliposomes reconstituted from dimyristoylphosphatidylcholine (DMPC) and hog kidney brush border membrane extract is strongly affected by temperature and the physical state of the membranes. This dependence is defined by a nonlinear Arrhenius plot with a break point at 23°C, a temperature not significantly different from the phase transition temperature of the pure lipid (24°C). The transport process is characterized by different activation energies: 35.1 kcal/mol below and 5.5 kcal/mol above the transition temperature. The shift in the break point for the d-glucose transport activity from 15°C, in the brush border membranes, to 23°C in the reconstituted system leads us to conclude that the lipids surrounding the sodium/d-glucose cotransport system can exchange readily with the bulk lipid used for reconstitution. The results thus provide no evidence for the presence of an annulus of specific lipids surrounding the transport system.     

The crystal structure of d-glucose 6-(sodium hydrogen-phosphate)

The title compound, when recrystallised from water, is monoclinic, space group P2₁, with a = 5.774(4), b = 7.189(5), c = 12.69(1) Å, β = 106.66(5)°, and Z = 2. The crystal structure was determined from three-dimensional X-ray diffraction data taken on an automatic diffractometer with CuKα, and refined by least-squares techniques to R = 0.034 for 977 reflexions. The pyranose ring adopts the ⁴C₁ conformation. The conformation about the exocyclic C-5-C-6 bond is gauche-trans [the torsion angles O-6-C-6-C-5-O-5 and O-6-C-6-C-5-C-4 are 64.2(8) and ?175.6(7)°, respectively], which is significantly different from the gauche-gauche geometry in d-glucose 6-(barium phosphate). The phosphate ester bond, P-O-6, is 1.584(3) Å. All of the oxygen-bonded hydrogen atoms are involved in intermolecular hydrogen-bonds.     

Enzymic determination of d-galactose, d-arabinose,and their homologs

The enzymes d-galactose dehydrogenase and d-arabinose dehydrogenase were demonstrated to be applicable to the quantitative determination of d-galactose (and homologs) and d-arabinose (and homologs), respectively. The enzymic reactions were quite specific. When coupled with β-galactosidase, d-galactose dehydrogenase could be used in the quantitative determination of β-galactosides.