Effects of coenzyme Q<Subscript>0</Subscript>, ascorbic acid,and its glycoside on the radiation-induced dephosphorylation of organic phosphates in aqueous solutions

The effects of coenzyme Q₀, ascorbic acid, and 2-O-α-D-glucopyranosylascorbic acid on the radiation-induced dephosphorylation of 1-glycerophosphate, glucose-1-phosphate, and glucose-6-phosphate in deaerated aqueous solutions at pH 7 were studied by means of continuous radiolysis. It was found that the test compounds efficiently suppressed the radiation-induced dephosphorylation of organic phosphates in equimolar concentrations by interactions with water radiolysis products. At an organic phosphate to additive concentration ratio of 100: 1, ascorbic acid and coenzyme Q₀ can suppress the radiation-induced dephosphorylation of the initial substances in aqueous solutions by the reduction and oxidation of hydroxyl-containing carbon-centered radicals, respectively.     

Simple enzymatic in situ generation of dihydroxyacetone phosphate and its use in a cascade reaction for the production of carbohydrates: increased efficiency by phosphate cycling

A new enzymatic method for the generation of dihydroxyacetone phosphate (DHAP) using the acid phosphatase from Shigella flexneri (PhoN-Sf) and the cheap phosphate donor pyrophosphate (PPi) is described. The utility of this method was demonstrated in an aldolase-catalyzed condensation carried out in one pot in which DHAP was generated and coupled to propionaldehyde to give a yield of 53% of the isolated dephosphorylated end product.     

A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol

A total of four enzymatic steps were combined, in a one-pot reaction, to synthesize carbohydrates starting from glycerol. First, phosphorylation of glycerol by reaction with pyrophosphate in the presence of phytase at pH 4.0 in 95% glycerol afforded racemic glycerol-3-phosphate in 100% yield. The L-enantiomer of the latter underwent selective aerobic oxidation to dihydroxyacetone phosphate (DHAP) at pH 7.5 in the presence of glycerolphosphate oxidase (GPO) and catalase. Subsequently, fructose-1,6-bisphosphate aldolase catalyzed the aldol reaction of DHAP with butanal. Finally, dephosphorylation of the aldol adduct was mediated by phytase at pH 4 affording 5-deoxy-5-ethyl-D-xylulose in 57% yield from L-glycerol-3-phosphate. The phytase on/off-switch by pH was the key to controlling phosphorylation and dephosphorylation.     

Electrode Hybride Pour la Determination de Phosphates et de Polyphosphates. Application aux Problemes Lies a l'Environnement

Abstract

The determination of phosphates and polyphosphates has been effected using an hybrid electrode, which consists of a glucose oxydase enzymic membrane and a Solanum tuberosum tissue slice. This membrane, rich in acid phosphatase, catalyses the glucose-6-phosphate hydrolysis. This reaction is quantitatively inhibited by phosphate. Several factors involved in the electrode response, like substrate concentration, pH, ionic strength and type of buffer are discussed in detail. At a 4.10^?4 M glucose-6-phosphate concentration, the linear ranges of phosphates and polyphosphates are, respectively, 6.10^?5 M to 1.6.10^?3 M and 3.10^?5 M to 1.10^?3 M. The urinary phosphate contents determinated by this biosensor are in good agrement with those obtained by usual spectrophotometric techniques.     

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.     

Erythrocyte spectrin alteration induced by low-density lipoprotein.

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylation-dephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.     

Quantum mechanical calculations on phosphate hydrolysis reactions

Multiple biological processes are regulated by kinases and phosphatases. This study aims to provide nonenzymatic models for phosphorylation and dephosphorylation of serine, threonine, and tyrosine phosphate using ab initio guantum mechanical calculations. We reduce the problem to methyl phosphate hydrolysis to model serine/threonine, and the hydrolysis of phenyl phosphate to model the tyrosine. HF, B3LYP, and MP2 calculations with a 6‐31+G(d) basis set were employed. The effect of water as a catalyst was also analyzed. As expected, the activation energy barrier is lowered. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 43–51, 2000     

Der Anteil der Glykolyse und des Pentosephosphat-Weges am Glucoseumsatz in normalen und in Glucose-6-phosphat-Dehydrogenase-Mangel-Erythrocyten

Zusammenfassung Der quantitative Anteil der Glykolyse und des Pentosephosphatweges am Glucoseumsatz in normalen Erythrocyten und solchen, die einen Mangel an Glucose-6-phosphat-Dehydrogenase haben, wurde durch Bestimmung des Glucoseverbrauchs, der Lactat- und Pyruvatbildung, der Änderung des 2,3-Diphosphoglycerinsäurespiegels sowie der ¹⁴CO₂-Freisetzung aus ¹⁴C-1-, ¹⁴C-6- und ¹⁴C-uniform-markierter Glucose ermittelt. In normalen Zellen werden bei pH 7,6 etwa 7% der umgesetzten Glucose über den Pentosephosphatweg, 93% über die Glykolyse zu Lactat verstoffwechselt. Bei pH 6,8 ist der Glucoseumsatz zu Pyruvat und Lactat um etwa 45% geringer als bei pH 7,6. Der Anteil des Pentosephosphatweges an diesem Umsatz ist jedoch kompensatorisch auf 13% erhöht. Die Menge des durch den oxydativen Glucoseabbau mittels der 2 Dehydrogenasereaktionen synthetisierten NADPH ist in beiden pH-Bereichen annähernd gleich. In Anwesenheit von Methylenblau wird Glucose fast vollständig oxydativ abgebaut. Eine nennenswerte Recyclisierung innerhalb des Pentosephosphatweges findet in normalen Erythrocyten nicht statt. In Erythrocyten mit einem Mangel an Glucose-6-phosphat-Dehydrogenase ist der Anteil des Pentosephosphatweges am Glucoseumsatz bei pH 7,6 nur 1,5%. Bei pH 6,8 erhöht sich dieser Anteil auf 5%, doch ist der Glucoseverbrauch entsprechend reduziert. Der glykolytische Glucosestoffwechsel in den Mangelzellen ist um etwa 20% geringer als in den normalen Zellen. Auch hier wird in beiden pH-Bereichen eine annähernd gleiche Menge NADPH synthetisiert. In Anwesenheit von Methylenblau kann in den Mangelerythrocyten der Anteil des Pentosephosphatweges am Glucoseumsatz nur unwesentlich von 1,5 auf 2,5% stimuliert werden. Die Kapazität des Glucose-6-phosphat-Dehydrogenase-Systems ist in den Mangelzellen um einen Faktor von 60 gegenüber der in normalen Zellen vermindert.

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Contribution of the glycolytic and the pentose phosphate pathway to the overall glucose metabolism in normal and glucose-6-phosphate dehydrogenase deficient erythrocytes

This has been measured by the glucose uptake, the formation of lactate and pyruvate, the change in the concentration of 2,3-diphosphoglyceric acid and the release of ¹⁴CO₂ from ¹⁴C-1-, ¹⁴C-6-, and ¹⁴C-uniformly labelled d-glucose.In normal cells at pH 7.6 about 7% of the glucose consumed has been metabolized via the oxidative pentose phosphate pathway, 93% via the glycolytic pathway to lactate. At pH 6.8 glucose metabolism to lactate and pyruvate is diminished by about 45% compared to that at pH 7.6. The amount of the pentose phosphate pathway is increased to 13%. Thus, the synthesis of NADPH is almost equal at both pH-ranges. In the presence of methylene-blue glucose is metabolized almost completely via the oxidative pathway. There is no significant recycling of glucose metabolites in the pentose phosphate pathway.In glucose-6-phosphate dehydrogenase deficient erythrocytes the contribution of the pentose phosphate pathway to glucose metabolism at pH 7.6 is only 1.5%, at pH 6.8 5%. In this pH-range, however, the glucose uptake is markedly decreased. The extent of NADPH synthesis therefore at both pH-ranges is again almost the same. In the presence of methylene-blue glucose metabolism via the pentose phosphate pathway can be stimulated only to a very small extent from 1.5% to 2.5%. The capacity of the glucose-6-phosphate dehydrogenase system in the deficient cells is by a factor of 60 smaller than that of the normal cells.

     

Determination of dehydrogenase substrates by Clark-type oxygen electrodes and photosensitized coenzyme oxidation

The photosensitized oxidation of NADPH by oxygen can be used for the determination of the reduced coenzymes by means of a Clark oxygen electrode. This method is suitable for coupling to enzyme-catalysed dehydrogenation reactions and thus for the determination of glucose-6-phosphate with glucose-6-phosphate dehydrogenase and of glucose with the combined ATP/hexokinase/glucose-6-phosphate dehydrogenase system, even with the use of immobilized mediators.     

Stereocomplementary bioreduction of alpha,beta-unsaturated dicarboxylic acids and dimethyl esters using enoate reductases: enzyme- and substrate-based stereocontrol

Asymmetric bioreduction of alpha,beta-unsaturated dicarboxylic acids, such as 2-methylmaleic/fumaric and 2-methylenesuccinic acid, as well as the corresponding dimethyl esters, using three cloned enoate reductases furnished 2-methylsuccinic acid or dimethyl 2-methylsuccinate, respectively. Opposite stereoisomeric products were obtained in up to >99% ee either by choice of the enzyme or by using E/Z-configurated substrates. Cofactor-recycling systems (NADH/FDH/formate, NADH/GDH/glucose or NADPH/G6PDH/glucose-6-phosphate) only worked in presence of a divalent metal ion, such as Ca2+, Mg2+, or Zn2+.     

In Vitro 31P MR Chemical Shifts of In Vivo-Detectable Metabolites at 3T as a Basis Set for a Pilot Evaluation of Skeletal Muscle and Liver 31P Spectra with LCModel Software

Most in vivo ³¹P MR studies are realized on 3T MR systems that provide sufficient signal intensity for prominent phosphorus metabolites. The identification of these metabolites in the in vivo spectra is performed by comparing their chemical shifts with the chemical shifts measured in vitro on high-field NMR spectrometers. To approach in vivo conditions at 3T, a set of phantoms with defined metabolite solutions were measured in a 3T whole-body MR system at 7.0 and 7.5 pH, at 37 °C. A free induction decay (FID) sequence with and without ¹H decoupling was used. Chemical shifts were obtained of phosphoenolpyruvate (PEP), phosphatidylcholine (PtdC), phosphocholine (PC), phosphoethanolamine (PE), glycerophosphocholine (GPC), glycerophosphoetanolamine (GPE), uridine diphosphoglucose (UDPG), glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), 2,3-diphosphoglycerate (2,3-DPG), nicotinamide adenine dinucleotide (NADH and NAD⁺), phosphocreatine (PCr), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and inorganic phosphate (Pi). The measured chemical shifts were used to construct a basis set of ³¹P MR spectra for the evaluation of ³¹P in vivo spectra of muscle and the liver using LCModel software (linear combination model). Prior knowledge was successfully employed in the analysis of previously acquired in vivo data.     

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phosphate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na(2)SO(4), pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively.     

Continuous‐Flow Reactor‐Based Enzymatic Synthesis of Phosphorylated Compounds on a Large Scale

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads‐150 or Sepabeads EC‐EP). After immobilization 70 % of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D ‐glucose‐6‐phosphate, N‐acetyl‐D ‐glucosamine‐6‐phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol‐1‐phosphate, and inosine‐5′‐monophosphate from the corresponding primary alcohol on gram scale using either a fed‐batch reactor or a continuous‐flow packed‐bed reactor.     

Selectivity for glucose,glucose-6-phosphate,and pyruvate in ternary mixtures from the multivariate analysis of near-infrared spectra

Near-infrared spectroscopy offers the potential for direct in situ analysis in complex biological systems. Chemical selectivity is a critical issue for such measurements given the extent of spectral overlap of overtone and combination spectra. In this work, the chemical basis of selectivity is investigated for a set of multivariate calibration models designed to quantify glucose, glucose-6-phosphate, and pyruvate independently in ternary mixtures. Near-infrared spectra are collected over the combination region (4,000–5,000 cm⁻¹) for a set of 60 standard solutions maintained at 37 °C. These standard solutions are composed of randomized concentrations (0.5–30 mM) of glucose, glucose-6-phosphate, and pyruvate. Individual calibration models are constructed for each solute by using the partial least-squares (PLS) algorithm with optimized spectral range and number of latent variables. The resulting standard errors are 0.90, 0.72, and 0.32 mM for glucose, glucose-6-phosphate, and pyruvate, respectively. A pure component selectivity analysis (PCSA) demonstrates selectivity for each solute in these ternary samples. The concentration of each solute is also predicted for each sample by using a set of net analyte signal (NAS) calibration models. A comparison of the PLS and NAS calibration vectors demonstrates the chemical basis of selectivity for these multivariate methods. Selectivity of each PLS and NAS calibration model originates from the unique spectral features associated with the targeted analyte. Overall, selectivity is demonstrated for each solute with an order of sensitivity of pyruvate > glucose-6-phosphate > glucose. Figure Combination near-infrared spectroscopy allows selective analytical measurements for glucose, glucose-6-phosphate, and pyruvate in ternary mixtures owing to the uniqueness of the individual absorption spectra for each solute     

Light-Harvesting Artificial Cells for the Generation of ATP and NADPH†

The bottom-up construction of self-powered artificial cells is significant to understand the energy supply and metabolism of nature cells. Here, we demonstrate an efficient manner to build thylakoid-containing artificial cells, which continuously convert light energy into chemical energy to supply adenosine 5’-triphosphate (ATP) under light illumination. The production of ATP supplies energy to promote the biological enzyme cascade reactions, where glucose is transformed into glucose 6-phosphate (G6P) under the catalysis of hexokinase (HK). G6P was further converted to gluconolactone 6-phosphate (PG) in the presence of 6-phosphate dehydrogenase (G6PDH), meanwhile NADP⁺ was converted to nicotinamide adenine dinucleotide phosphate (NADPH). The self-powered artificial cells were demonstrated to generate ATP and NADPH successively, which provided a way for building more complicated artificial cells.      

Fluorometric assay of ultramicro quantities of glucose with somogyi filtrate and hexokinase

A procedure is described for the determination of glucose in 3 μl of serum or plasma. It is based on the phosphorylation of glucose with ATP mediated by hexokinase. The TPNH generated with glucose-6-phosphate dehydrogenase is measured fluorometrically.Sensitivity is greatly improved over the corresponding procedure using UV absorption for the determination of NADPH. Bilirubin does not interfere. Interference from hemolysis is eliminated by precipitation with Zn(OH)₂ Blood taken with fluoride can be utilized. Fluoride interference is removed with the Zn(OH)₂ and with excess Mg in the substrate. Positive interference by fructose is negligible with some enzyme preparations but is appreciable with others.The results correlate well with those obtained with an ortho toluidine method, after protein precipitation with trichloroacetic acid. The method is especially useful in the Pediatric Laboratory.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Enzyme Electrode for Fructose,Glucose-6-Phosphate and ATP Determination

Abstract

A bienzyme electrode for glucose-6-phosphate and ATP based on the sequential reactions of coimmobilized glucose-6-phosphate dehydrogenase and hexokinase and for fructose using the competition with glucose has been developed. Electrochemical indication is performed using air-oxidation of reduced N-methylphenazinium ion. The sensor responds linearly to fructose up to 3 mM with a lower detection limit of 0.3 mM.     

Current chemical biology tools for studying protein phosphorylation and dephosphorylation

Amongst different posttranslational events involved in cellular-signaling pathways, phosphorylation and dephosphorylation of proteins are the most prevalent. Aberrant regulations in the cellular phosphoproteome network are implicated in most major human diseases. Consequently, kinases and phosphatases are two of the most important groups of drug targets in medicinal research today. A major challenge in the understanding of protein phosphorylation and dephosphorylation is the sheer complexity of the phosphoproteome network and the lack of tools capable of studying protein phosphorylation and dephosphorylation as they occur in cells. We highlight herein various chemical biology tools that have emerged in the last decade for such studies. First, we discuss the use of small-molecule mimics of phosphoamino acids and their use in elucidating the function of protein phosphorylation and dephosphorylation. We also introduce recent advances in the field of activity-based protein profiling (ABPP) for proteome-wide detection of protein phosphorylation and dephosphorylation. We next discuss the key concepts in the design of peptide- and protein-based biosensors capable of real-time reporting of phosphorylation/dephosphorylation events. Finally, we highlight the application of peptide and small-molecule microarrays (SMMs), and their applications in high-throughput screening and discovery of new compounds related to phosphorylation/dephosphorylation.     

Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.