大鼠红细胞葡萄糖代谢的异头物选择性

为研究大鼠红细胞对葡萄糖利用的异头物选择性及其作用机制,应用大鼠红细胞,对葡萄糖的两种异头物作了异构化速率、乳酸生成量、内流速度和大鼠红细胞已糖激酶作用下的磷酸化速度等进行了测定．结果指出,３７℃时大鼠红细胞的Ｄ－葡萄糖β－异头物和α－异头物代谢成乳酸的速度分别是０．２７μｍｏｌ／ｇＨｂ（３ｍｉｎ）和０．２１μｍｏｌ／ｇＨｂ（３ｍｉｎ）,即前者快于后者３０％．同时β－Ｄ－葡萄糖向红细胞内转运速度也快于后者：分别是５．０和３．５μｍｏｌ／ｇＨｂ（３ｍｉｎ）．大鼠红细胞已糖激酶的葡萄糖磷酸化速率实验结果指出：β－异头物比α－异头物快３０％;对于该两种异头物已糖激酶的Ｋｍ值均为５３μｍｏｌ／Ｌ．红细胞与α－和β－Ｄ－葡萄糖保温１ｍｉｎ后,其葡萄糖浓度均达到１ｍｍｏｌ／Ｌ左右,说明至少在１ｍｉｎ内对于已糖激酶的磷酸化此两种异头物的葡萄糖浓度均已饱和．这些结果提示,大鼠红细胞葡萄糖利用的β－异头物优选性主要与其磷酸化速度有关,而与其转运速度关系不大．     

Reciprocal influence of glucose anomers upon their respective phosphorylation by hexokinase

The phosphorylation of D-glucose (1.0mM) was measured in homogenates of tumoral islet cells incubated at 7 degrees C in the presence of labelled alpha- and/or beta-D-glucose, with or without exogenous glucose 6-phosphate. The close-to-maximal reaction velocity of hexokinase was higher with beta- than alpha-D-glucose. The latter anomer inhibited beta-D-glucose phosphorylation more than the beta-anomer decreased the phosphorylation of alpha-D-glucose. This behaviour was accounted for by the higher affinity of hexokinase for alpha- than for beta-D-glucose. These direct measurements of the relative contribution of each anomer to the overall rate of glucose phosphorylation in the presence of mixed populations of alpha- and beta-D-glucose validate the concept that the phosphorylation of D-glucose displays anomeric specificity even when the hexose is used at anomeric equilibrium. Glucose 6-phosphate inhibited the phosphorylation of the two anomers more severely when alpha-D-glucose rather than beta-D-glucose was the most abundant anomer.     

Obtention de I-D-ribofuranosylade´nines

The I-D-ribosyladenines have been obtained by treatment of 5-amino-4-cyanoimidazole (N-substituted or not) with ethyl N-(2,3-O-isopropylidene-D-ribofuranosyl)formimidate. The anomeric mixtures of the corresponding O-isoprophylidene nucleoside have been separated and the anomer fully characterized. In neutral aqueous medium, these compounds are transformed into an anomeric mixture of the corresponding 6-D-ribofuranosyladenine. In basic medium, however, anomerisation of the starting compounds to give an α,β equilibrium, in which the α anomer preponderates, takes place.     

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.     

Zn2+, Mg2+, and H+ binding to d-fructose 1,6-bisphosphate studied by 31P and 1H NMR

The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by ³¹P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s^?1 for the β → α anomerization and (14.9 ± 0.5) s^?1 for the reverse reaction. A D₂O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg²⁺ and Zn²⁺ bind preferentially to the 1-phosphate in the α-anomeric position. Zn²⁺ has a higher affinity for this phosphate group than Mg²⁺ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg²⁺/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg²⁺ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C₁O₁ bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn²⁺, Mg²⁺, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.     

Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Phosphorylation by liver glucokinase of D-glucose anomers at anomeric equilibrium

The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate.     

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model

The substrate specificity of immobilized D-glucose isomerase (EC 5.3. 1.5) is investigated with an immobilized enzyme-packed reactor. A series of isomerization experiments with alpha-, beta-, and equilibrated D-glucose solutions indicates that beta anomer as well as alpha anomer is a substrate of the glucose isomerase at pH 7.5 and 60 degrees C. For substrate concentration of 0.028 mol l(-1) (1% w/v), the initial conversion rate of alpha-D-glucose was 43% higher than that with equilibrated glucose at the same concentration and 113% higher than beta-D-glucose conversion rate. This anomeric reactivity of glucose isomerase is mathematically described with a set of kinetic equations based on the reaction steps complying with Briggs-Haldane mechanism and the experimentally determined kinetic constants. The proposed reaction mechanism includes the mutarotation and the isomerization reactions of alpha- and beta-D-glucose with different rate constants.     

Changes of Hexosamine and Acetyl Contents during Cultivation of Extracellular Galactosamine-containing Polymer from Aspergillus parasiticus

The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method.

1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing β anomer formation.

2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.     

Anomeric specificity in the response of gut glucagon-like immunoreactive materials to glucose.

An anomeric specificity of the glucose sensors of A cells and B cells of the pancreas has been reported. In this context the present authors investigated, using the canine intestinal loop prepared from the terminal portion of the ileum, how glucagon-like immunoreactive materials (GLI) of the gut would respond to glucose anomers in an attempt to explore a possible anomeric specificity of glucose-stimulated gut GLI secretion. As a result GLI was found to be more readily released into the blood stream after an intestinal alpha-glucose load than following beta-gluocse during a 15-minute observation period. It is thus suggested that gut GLI-secreting cells have glucose sensors similar to those of pancreatic A or B cells which are specific for the alpha-glucose anomer.     

Anomeric specificity of D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.     

Effect of conformation and configuration of arabinose residues on the circular dichroism of C-glycosylflavones

Examination of a variety of arabinose containing C-glycosylflavones has shown that the sign and intensity of the CD band at 250–275 nm (charge-transfer band) reflect not only the point of attachment of the sugar to the flavone but also depend upon the absolute and anomeric configuration, ring-size and ring-conformation in addition to the preferred rotameric conformation of the sugar about the C-aryl, C-l″ bond. A change in stereochemistry of arabinose from the α to β anomer resulted in sign inversion of the 250–275 nm CD band for 6-C-l-arabinosylflavones. Furthermore, a 6-C-arabinosylflavone containing α-l-arabinose exhibited an oppositely signed charge-transfer CD band in comparison to one which contained α-d-arabinose. 6,8-Di-C-glycosylflavones containing arabinose and glucose exhibited CD bands resulting from contributions due to both sugars, if the arabinose was not present as the β-pyranose form (¹C₄, conformation).     

B3LYP/6-311++G** geometry-optimization study of pentahydrates of alpha- and beta-D-glucopyranose

Five water molecules were placed in 37 different configurations around alpha- and beta-D-glucopyranose in the gt, gg, and tg conformational states, and the glucose-water complexes were geometry optimized using density functionals at the B3LYP/6-311++G** level of theory. The five water molecules were organized in space and energy minimized using an empirical potential, AMB02C, and then further geometry optimized using DFT algorithms to minimum energy positions. Electronic energy, zero point vibrational energy, enthalpy, entropy, stress energy on glucose and the water cluster, hydrogen-bond energy, and relative free energy were obtained for each configuration using thermodynamic procedures and an analytical Hessian program. The lowest energy complex was that of a clustering of water molecules around the 1- and 6-hydroxyl positions of the beta-gt anomer. Configurations in which the water molecules created a favorable network completely around and under glucose were found to have low energy for both alpha and beta anomers. Calculation of the alpha/beta anomeric ratio using the zero point corrected energy gave, approximately 32/68%, highly favoring the beta anomer in agreement with the experimental approximately 36/64% value. This ratio is better than the approximately 50/50% ratio found in our previous monohydrate study. An approximate hydroxymethyl population was obtained by noting average relative energies among the three conformational states, gt, gg, and tg. In the beta anomer complexes the gt conformation was favored over the gg state, while in the alpha anomer complexes the gg state was favored over the gt conformation, with the tg conformations all being of higher energy making little or no contribution to the rotamer population. Some geometry variances, found between glucose in vacuo and glucose after interaction with water molecules, are described and account for some observed C-5-C-6 bond length anomalies reported by us previously for the vacuum glucose structures.     

Fucokinase, its anomeric specificity and mechanism of phosphate group transfer

Fucokinase phosphorylates L-fucose at the anomeric position and, as such, might use either the alpha or beta anomer as its substrate. Examination of the utilization of radiolabelled alpha and alpha,beta mixtures established beta-L-fucose as the required substrate. Phosphorylation at the anomeric center might involve either the loss or retention of the anomeric oxygen. The mechanism has been shown to involve anomeric oxygen retention through mass spectrometric analysis of the product phosphate derived from 18O-labelled L-fucose.     

Effective Anomerisation of 2′‐Deoxyadenosine Derivatives During Disaccharide Nucleoside Synthesis

The formation of a disaccharide nucleoside (11) by O3′‐glycosylation of 5′‐O‐protected 2′‐deoxyadenosine or its N ⁶‐benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding α‐anomeric product (12). The latter reaction can be explained by instability of the N‐glycosidic bond of purine 2′‐deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2′‐deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3′‐O‐β‐d‐ribofuranosyl‐2′‐deoxyadenosines and its α‐anomer.     

13C- and 1H-nuclear magnetic resonance spectroscopy of permethylated disaccharides

The equilibrium composition of D-psicose in water, as determined from its ¹³C n.m.r. spectrum, is 7:2:5:5 α-furanose:β-furanose:α-pyranose:β-pyranose. These data, which are discussed in relation to the anomeric and ring-form equilibria of the other 2-hexuloses, are in general agreement with expectations based on conformational analysis. However, although the ¹³C chemical-shift pattern of the β-pyranose is closely consistent with the 1C(D) conformation predicted for this anomer, that of the α-pyranose is less readily reconciled with its predicted C1(D) conformation. Usually, carbon-13 nuclei of the furanose rings are substantially less shielded than those of their pyranose counterparts; for 2-hexulopyranoses in general, overall ¹³C shielding is close to that of those aldopyranoses expected to have similar conformational free-energies. Spectral data are also reported for several derivatives (glycosides, ethers, and selectively deuterated compounds) that were utilized in analysis of the D-psicose spectrum.     

Substrate form of D-frutose 1,6-bisphosphate utilized by fructose 1,6-bisphosphatase.

Rapid quench kinetic experiments on fructose 1,6-bisphosphatase demonstrate a stereospecificity for the alpha anomer of fructose 1,6-bisphosphate relative to the beta configuration. The beta anomer is only utilized after mutarotation to the alpha form in a process that is not enzyme catalyzed. Studies employing analogues of the acyclic keto configuration indicate that the keto form is utilized at a rate less than 5% that of the alpha anomer, a finding also confirmed by computer simulation of the rapid quench data. Chemical trapping experiments of the keto analogue, xylulose 1,5-bisphosphate, and the normal substrate suggest that interconversion of the acyclic and anomeric configurations is retarded by their binding to the enzyme. A hypothesis is advanced attributing substrate inhibition of fructose 1,6-bisphosphatase to possible binding of the keto species.     

Kinetically controlled Ferrier rearrangement of 3-O-mesyl-d-glycal derivatives

The Ferrier rearrangement, which is widely used in carbohydrate chemistry, is generally performed under acidic conditions to give an α anomer with high stereoselectivity. We have found that 3-O-mesyl-d-glycals 2-4 were smoothly reacted with alcohols in the presence of triethylamine. The present reaction was shown to proceed under kinetic control to give ∼1.3:1.0 mixture of α and β anomers, indicating that a kinetic anomeric effect does not operate.     

Anomeric specificity of L-fucose dehydrogenase: a stereochemical imperative in aldopyranose dehydrogenases?

A set of hypotheses is proposed that explains the anomeric specificity of aldopyranose dehydrogenases in terms of an evolutionarily selected function. The first hypothesis, based on stereoelectronic theory, argues that, in the "allowed" transition state for oxidation at the anomeric carbon, the two oxygens attached to the anomeric carbon each bear a lone pair of electrons antiperiplanar to the departing "hydride". The second hypothesis is that the dehydrogenase is functionally constrained to bind the anomer that has this arrangement of lone pairs in its lowest energy chair conformer. The anomeric specificity of L-fucose dehydrogenase is experimentally examined. The enzyme oxidizes preferentially the beta-anomer, consistent with the prediction made by these hypotheses. Available experimental data for other enzymes (D-glucose-6-phosphate dehydrogenase, D-glucose dehydrogenase, D-galactose dehydrogenase, D-abequose dehydrogenase, and D-arabinose dehydrogenase) are found to be also consistent with the proposed hypotheses.