Anomeric Specificity Research Articles

Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy.

[99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1-13C]- and [2-13C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1-13C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO.

Anomeric specificity of glucose metabolism in the pentose cycle.

The production of 3H2O from alpha- and beta-D-[5-3H]glucose and that of 14CO2 from either alpha- and beta-D-[1-14C] or alpha- and beta-D-[6-14C]glucose were measured in rat pancreatic islets and tumoral insulin-producing cells incubated at 7 degrees C. The ratio in 14CO2 output from D-[1-14C]glucose/D-[6-14C]glucose, the fraction of glucose metabolism occurring through the pentose cycle, and the flow rate through such a cycle were always higher in the presence of beta- than alpha-D-glucose. This indicates that the anomeric specificity of glucose-6-phosphate dehydrogenase is operative in intact islet cells.

Is glucokinase responsible for the anomeric specificity of glycolysis in pancreatic islets?

At a low concentration of D-glucose (3.3 mM), the phosphorylation rate of this hexose in rat pancreatic islet homogenates incubated at 8 degrees C is higher with the beta- than with the alpha-anomer, as expected from the anomeric specificity of hexokinase. In the presence of a high concentration of glucose 6-phosphate (3.0 mM), which inhibits hexokinase but not glucokinase, the phosphorylation rates of the two anomers are not significantly different from one another. Nevertheless, in intact islets exposed at 8 degrees C to the same low concentration of D-glucose, the alpha-anomer augments, more than the beta-anomer, the production of lactic acid and net uptake of 45Ca. At the same concentration (3.3 mM), the alpha-anomer is also more potent than the beta-anomer in enhancing insulin release from perfused pancreases stimulated at 37 degrees C by L-leucine or by the combination of Ba2+ and theophylline. It is concluded that the participation of glucokinase is not essential for the anomeric specificity of glycolysis and insulin release in rat pancreatic islets.

Environmental modulation of the anomeric specificity of glucose metabolism in normal and tumoral cells

In rat pancreatic islets and erythrocytes, α- d-glucose (2.8–5.6 mM) is better metabolized than β- d-glucose, as judged from the conversion of d-[5- 3H]glucose to 3H 2O, augmentation in lactic acid production (or output) or oxidation of d-[U- 14C]glucose. In tumoral cells, however, whether of the insulin-producing or lymphocytic leukemia type, the anomeric preference for α- d-glucose utilization is no longer present when the cells are incubated at comparable glucoṡe concentrations (2.8–4.0 mM). Nevertheless, the tumoral insulin-producing cells are able to display preference for either α- d-glucose (at very low glucose concentrations in the 0.14–0.82 mM range) or β- d-glucose (in the presence of 16.7 mM glucose). These findings indicate that the anomeric specificity of glucose metabolism may differ in distinct cell types, and can be modulated by the ambient glucose concentration.

Anomeric specificity of hexokinase and glucokinase activities in liver and insulin-producing cells.

Conflicting data have been reported concerning the anomeric specificity of glucokinase. In the present study, liver hexokinase (Km for D-glucose 0.4 mM) displayed a higher affinity for but lower Vmax. with alpha- than with beta-D-glucose. The velocity of the reaction catalysed by liver glucokinase was higher with with beta- than with alpha-D-glucose, whatever the glucose concentration. The apparent Km of glucokinase was somewhat lower, however, with alpha- than with beta-D-glucose. Comparable results were obtained for the high-Km glucokinase-like enzymic activity present in normal pancreatic islets or insulin-producing tumoral cells. These results suggest that the anomeric specificity of glucokinase cannot account for the higher rate of glycolysis found in islets exposed to alpha- as distinct from beta-D-glucose.

Anomeric specificity of mannose phosphorylation by hexokinase

In rat parotid or pancreatic islet homogenates incubated at 7°C, hexokinase displayed a greater affinity for but a lower maximal velocity with the α-anomer, as distinct from β-anomer, of d-mannose. The anomeric specificity of mammalian hexokinase was similar in the case of d-mannose and d-glucose, but represented a mirror image of that of yeast hexokinase.

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 α or β anomer as its substrate. Examination of the utilization of radiolabelled α and α,β mixtures established β- 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.

Anomeric specificity of hexose metabolism in pancreatic islets.

Anomeric specificity of hexose metabolism in pancreatic islets.

The specificity of viral and bacterial sialidases for α(2–3)- and α(2–6)-linked sialic acids in glycoproteins

The anomeric specificity of six sialidases ( Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, Newcastle disease virus, fowl plaque virus and influenza A 2 virus sialidases) was assessed with sialylated antifreeze glycoprotein, ovine submandibular gland glycoprotein and α 1-acid glycoprotein, resialylated specifically in α(2–3) or α(2–6) linkage with N-acetylneuraminic acid or N-glycolylneuraminic acid using highly purified sialyltransferases. The rate of release of sialic acid from these substrates was found to correlate well with the specificity observed earlier with the same sialidases using small oligosaccharide substrates, i.e., α(2–3) glycosidic linkages are hydrolyzed faster than α(2–6) linkages,w ith the exception of the enzyme from A. ureafaciens. Sialidase activity was higher with N-acetylneuraminic acid when compared with N-glycolylneuraminic acid. The studies also showed that the core oligosaccharide and protein structure in glycoproteins may influence the rate of release for different glycosidic linkages.

13C nuclear magnetic resonance studies of anaerobic glycolysis in Trypanosoma brucei spp.

Anaerobic glycolysis in Trypanosoma brucei spp. has been studied by 13C NMR at 50 and 75.5 MHz. The uptake of [U-13C]glucose by cell suspensions of T. b. brucei was monitored by time-course spectroscopy, and while no anomeric specificity was found, the end-products of glycolysis were confirmed as glycerol and pyruvate together with alanine and dihydroxypropionate. The intermediacy of L-glycerol-3-phosphate was also ascertained. The incorporation of C-1 of [1-13C]glucose and of C-6 of [6-13C]glucose into glycerol and pyruvate in T. b. gambiense was quantified by measurement of the longitudinal relaxation times of the species involved. An incorporation to the extent of 66% of each substrate into equimolar amounts of glycerol and pyruvate indicate that Keq for the triosephosphate-isomerase-mediated reaction approaches unity.

Metabolism of glucose and mannose anomers in pancreatic islets.

The alpha-anomers of D-glucose and D-mannose stimulate insulin release more efficiently than the corresponding beta-anomers. This coincides with higher glycolytic and oxidative fluxes in pancreatic islets exposed to alpha- than to beta-anomers. This situation may be attributable, in the case of alpha-D-glucose, not solely to the alpha-stereospecificity of phosphoglucose isomerase but also to that of phosphoglucomutase resulting in a higher islet content of glucose-1,6-bisphosphate, an activator of phosphofructokinase. Likewise, more aldohexose-bisphosphate accumulates in the islets exposed to alpha-D-mannose than in those incubated with beta-D-mannose. The anomeric specificity of hexose metabolism in pancreatic islets supports the fuel hypothesis for insulin release.

Carbohydrate binding specificity and purification by biospecific affinity chromatography of [formula omitted][formula omitted] traust. Hemagglutinins

The carbohydrate specificities of Ascidia malaca serum hemagglutinins were determined by hemagglutination inhibition tests. Analysis of agglutinins against rabbit and human A, B, O erythrocytes suggests that the size of the combining site corresponds to a disaccharide with a specificity for saccharides containing a D-galacto configuration (D-melibiose, D-raffinose, D-galactose, α-lactose, lactulose, L-arabinose). No anomeric specificity was observed with oligosaccharides. Hydroxyl groups probably involved in hydrogen-bond formation with agglutinin binding site, were identified as carbons C2, C4, C5 and C6 of D-galactose. Absorption experiments showed that two distinct agglutinins with similar sugar specificity ranges were responsible for rabbit and human erythrocyte agglutination. The D-galactose specificity of the agglutinins allowed the isolation, by biospecific affinity chromatography of the serum, of a protein fraction demonstrated by polyacrylamide gel electrophoresis.

Anomeric specificity of D-mannose metabolism in pancreatic islets

Summary The α-anomer of D-mannose is better able than the β-anomer to stimulate insulin release, protect against alloxan, induce reduction of pyridine nucleotides, augment lactate output, undergo oxidation and provoke the accumulation of aldohexose-bisphosphates in rat pancreatic islets.

Purification of 2,5-anhydro- d-hexitol bis(phosphates) and identification of a major 1,4,6-tris(phosphate) contaminant by 31P-, 13C-, and 1H-N.M.R. spectroscopy

The reaction of two equivalents of diphenylchlorophosphate in cold pyridine with 2,5-anhydrohexitols has been assumed to result in only 1,6-bis(diphenylphosphate) products. However, by thin-layer, silica gel dry-column, and DEAE-Sephadex A-25 column chromatography, the products of this reaction have been shown to contain three major components; monophosphates (32 or 30%, by weight), 1,6-bis(phosphates) (40 or 56%), and 1,4,6-tris(phosphates) (28 or 14%): the former percentages for the product from 2,5-anhydro- d-mannitol ( 1) and the latter for the product from 2,5-anhydro- d-glucitol ( 10). The identity of each bis- and tris-(phosphate) of 1 or 10 was established by 31P- and 13C-n.m.r. spectroscopy. Acetylated bis- and tris-(diphenylphosphates) of 1 were also examined by 1H-n.m.r. The significance of these findings on the interpretation of studies of the anomeric specificity of enzymes and on the specificity of the reagent diphenylchlorophosphate are discussed. The formation of only a 1,4,6-tris(phosphate) of 10 suggests that the 1,6-bis(diphenylphosphate) of 10 may undergo formation of a 1,3-cyclic phosphate triester by transesterification with elimination of phenol. A method for the determination of the number of cyclohexylammonium groups crystallizing with a sugar phosphate is proposed that simplifies the elemental analysis of this type of salt.

Anomeric specificity of the monosaccharide carrier in yeasts and yeast-like organisms.

The anomeric specificity of monosaccharide uptake was investigated in 42 species of yeasts and related mycelium-forming fungi. Differences in the uptake of anomers were determined by the following methods. (1) Shift of anomeric equilibrium in the outer medium caused by preferential uptake of one of the anomeric forms was monitored polarimetrically as induced mutarotation. (2) The uptake of 14C-D-glucose by cells was examined after addition of freshly prepared solutions of alpha- or beta-D-glucose. Most of the organisms examined display the Saccharomyces-type preference for the alpha-anomers of glucose and xylose which is caused by the higher affinity of the monosaccharide carrier for the alpha-pyranose configuration. The following genera show this type of preference (the number of species is given in parenthesis): Saccharomyces (5), Schizosaccharomyces (1), Endomycopsis (2), Eremascus (1), Endomyces (1), Pichia (1), Hansenula (1), Debaryomyces (2), Lipomyces (1), Willia (1), Nematospora (1), Kluyveromyces (2), Candida (5), Torulopsis (5), Cryptococcus (1). No anomeric specificity was shown by the following genera: Nadsonia (1), Dipodascus (2), Rhodotorula (5), Sporobolomyces (2), Bullera (1), Rhodosporidium (1). A parallel investigation of the concentration dependence of glucose uptake indicates that most yeasts possess a constitutive monosaccharide carrier characterized by the following features: a high maximum rate of uptake, a relatively low affinity, and preference for alpha-anomers. Besides this carrier the majority of these microorganisms possess a glucose-transporting carrier with a higher affinity and a lower capacity.

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.

14] Anomeric specificity of carbohydrate-utilizing enzymes

14] Anomeric specificity of carbohydrate-utilizing enzymes

N-acetylglucosamine and the substrate-site hypothesis for the control of insulin biosynthesis and secretion

1. Introduction The means whereby the beta-cell of the islets of Langerhans recognises signals for insulin biosynthesis and secretion and subsequently transduces such signals to activate the biosynthetic and secretory mechanisms are unknown. Two models have been proposed [I] to describe the process of signal recog- nition. One, the receptor-site model, envisages that the interaction of the signal with a membrane receptor, results in a change in the beta-cell initiating insulin release. The second model, the substrate-site model, proposes that the signal enters the beta-cell where it is metabolised and it is the formation of some metab- olite or cofactor which causes initiation of the secretory response. These models have been extended [2] with the development of a dual-site model to encompass the phenomenon of potentiation. There is-now an increasing body of evidence to support the substrate-site model. Such evidence includes close correlations between the rates of islet glucose metab- olism and the ability of glucose to cause insulin release [2,3] , the effects of mannoheptulose to inhibit both glucose phosphorylation and glucose-stimulated insulin release but not glyceraldehyde metabolism or glyceraldehyde-stimulated insulin release [2] and the observation that the anomeric specificity for glucose metabolism by the islet is the same as that for insulin release [4] . The present study is concerned with the metabolism and insulin-releasing activity of N-acetylglucosamine in isolated rat islets. The ability of N-acetylglucos-

Speculative studies on an anomeric specificity of inducers of D-lyxose isomerase

Speculative studies on an anomeric specificity of inducers of D-lyxose isomerase

Tautomeric and anomeric specificity of allosteric activation of yeast pyruvate kinase by D-fructose 1,6-bisphosphate and its relevance in D-glucose catabolism

Tautomeric and anomeric specificity of allosteric activation of yeast pyruvate kinase by D-fructose 1,6-bisphosphate and its relevance in D-glucose catabolism