Nucleotidases in plants *1, , *2: III. Effect of metabolites on the enzyme hydrolyzing flavine adenine dinucleotide (FAD) from Phaseolus radiatus

The highly purified enzyme from mung bean seedlings hydrolyzing FAD at pH 9.4 and temperature 49 °, functioned with an initial fast rate followed by a second slower rate. The activity was linear with enzyme concentration over a small range of concentration and was dependent on the time of incubation. Inhibition of enzyme activity with increasing concentrations of AMP was sigmoid;concentrations less than 1 × 10−6 M were without effect, concentrations between 1 × 10−6 and 8 × 10−5 M inhibited by 20% and concentrations beyond 8 × 10−5 Image caused progressive inhibition. Concentrations beyond 1 × 10−3 Image inhibited the activity completely. Preincubation of the enzyme with PCMB or NEM, or aging, or reversible denaturation with urea abolished the inhibitory effect of AMP at concentrations lower than 8 × 10−6 Image . The aged enzyme could be reactivated by ADP.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

A simple route to incorporate redox mediator into carbon nanotubes/Nafion composite film and its application to determine NADH at low potential

Through a new and simple ion-exchange route, two-electron redox mediator thionine has been deliberately incorporated into the carbon nanotubes (CNTs)/Nafion composite film due to the fact that there is strong interaction between any of two among the three materials (ion-exchange process between thionine and Nafion, strong adsorption of thionine by CNTs, and wrapping and solubilizing of CNTs with Nation). The good homogenization of electron conductor CNTs in the integrated films provides the possibility of three-dimensional electron conductive network. The resulting integrated films exhibited high and stable electrocatalytic activity toward NADH oxidation with the significant decrease of high overpotential, which responds more sensitively more than those modified by thioine or CNTs alone. Such high electrocatalytic activity facilitated the low potential determination of NADH (as low as -0.1 V), which eliminated the interferences from other easily oxidizable species. In a word, the immobilization approach is very simple, timesaving and effective, which could be extended to the immobilization of other cationic redox mediators into the CNTs/Nafion composite film. And these features may offer potential promise for the design of amperometric biosensors.

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 muM.

Electrocatalytic oxidation of NADH by rutin in biomembrane-like films on glassy carbon electrode

Stable lipid film was made by casting dipalmitoylphosphatidylcholine (DPPC) and rutin onto the surface of a glassy carbon (GC) electrode. The electrochemical behavior of rutin in the DPPC film was studied. The modified electrode coated with rutin gave quasi-reversible reduction-oxidation peak on cyclic voltammogram in the phosphate buffer (pH 7.4). The peak current did not decrease apparently after stored at 4 degreesC for 8 hours in refrigerator. This model of biological membrane was used to investigate the oxidation of dihydronicotinamide adenine dinucleotide (NADH) by rutin. Rutin in the film acts as a mediator. The modified electrode shows a great enhancement and the anodic peak potential was reduced by about 220 mV in the oxidation of 5 X 10(-3) mol L-1 NADN compared with that obtained at a bare glassy carbon electrode. (C) 2000 Elsevier Science S.A. All rights reserved.

A COMPARATIVE-STUDY ON STM IMAGING AND ELECTROCATALYTIC ACTIVITY OF DIFFERENT SURFACES MODIFIED WITH FLAVIN ADENINE-DINUCLEOTIDE

Flavin adenine dinucleotide (FAD) was modified onto the highly oriented pyrolytic graphite (hopg) and glassy carbon electrode (gee) surfaces with three methods, respectively. Corresponding image analysis for FAD-modified hopg surfaces has been performed by scanning tunnelling microscope (STM) for the first time. The molecular resolution STM image of FAD adsorbed on the freshly-cleaved hopg was obtained, the quantitative size determination suggests that the FAD molecules adsorb side lying on the substrate surface. The anodization treatment of hopg surface yields many pits, which were clearly observed under STM. These pits provide active sites on the hopg surface for modification and the treated hopg can strongly adsorb FAD molecules, the latter exhibiting an irregular cluster structure on such a surface. When FAD was electrochemically deposited on the substrate surface, a chain structure was successfully observed. The adsorbed FAD on anodized glassy carbon electrode (gee) surface can effectively catalyze the reduction of glucose oxidase, hemoglobin and myoglobin, with a large decrease in the overvoltage, whereas the deposited FAD film exhibits excellent electrocatalysis towards dioxygen reduction.

ELECTROCATALYTIC REDUCTION OF DIOXYGEN BY AN ELECTROCHEMICALLY POLYMERIZED FLAVIN ADENINE-DINUCLEOTIDE FILM

An electrochemically polymerized flavin adenine dinucleotide (FAD) film electrode is reported for the first time. The polymerized film was prepared by a two-step method. The electrocatalytic reduction of dioxygen at a glassy carbon electrode (GCE) modifie

Riboflavin, Flavin Mononucleotide and Flavin Adenine Dinucleotide in Human Plasma and Erythrocytes at Baseline and after Low-Dose Riboflavin Supplementation.

Background: Vitamin B2 exists in blood as riboflavin and its cofactors, flavin mononucleotide (FMN) and FAD. The erythrocyte glutathione reductase activation coefficient (EGRAC) has traditionally been used to assess vitamin B2 status in humans. We investigated the relationships of EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD in elderly volunteers and their responses to riboflavin administration. Methods: EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD were determined in 124 healthy individuals with a mean age of 69 years. The same measurements were made in a subgroup of 46 individuals with EGRAC 1.20 who participated in a randomized double-blind 12-week intervention study and received riboflavin (1.6 mg/day; n = 23) or placebo (n = 23). Results: Median plasma concentrations were 10.5 nmol/L for riboflavin, 6.6 nmol/L for FMN, and 74 nmol/L for FAD. In erythrocytes, there were only trace amounts of riboflavin, whereas median FMN and FAD concentrations were 44 and 469 nmol/L, respectively. Erythrocyte FMN and FAD correlated with each other and with EGRAC and plasma riboflavin (P

Selecting Room-Temperature Ionic Liquids to Optimize Voltammetric Responses: The Oxidation of NADH

The electrochemistry of nicotinamide adenine dinucleotide (NADH) in its reduced form was examined in two room-temperature ionic liquids (RTILs): 1-ethyl-3-methylimidazolium bis-(trifluoromethylsulfonyl)imide ([C(2)mim][NTf2]) and 1-butyl-3-methylimidazolium hexafluorophos-phate ([C(4)mim][PF6]). NADH oxidation has previously been studied in aqueous solution where it follows the pathway: one-electron oxidation to the NADH(center dot+) radical cation, deprotonation to produce the neutral NAD(center dot) radical, then oxidation to the NAD(+) cation. The electrochemistry of NADH was examined in [C(2)mim][NTf2] and [C(4)mim][PF6] at the bare Pt electrode (10 mu m diameter): In [C(2)mim][NTf2], no oxidation was observed; in [C(4)mim][PF6], an oxidative signal was observed, which likely followed the pathway described above, where upon formation of the NADH(center dot+) radical cation, the [PF6](-) anion (unlike the [NTf2](-) anion) reacts with the proton to form HPF6, which decomposes. This demonstrates the tunability of RTILs, whereby the choice of one anion in an RTIL over another can promote a reaction. Poly(vinylferrocene) (PVF) was studied as a mediator for the NADH detection in both RTILs to attempt to lower the potential of NADH detection. The Pt electrode was modified with PVF, and the oxidation of PVF to PVF+ was observed in [C(2)mim][NTf2] and [C(4)mim][PF6], but no mediation of the NADH oxidation was observed.

Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 mu M), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ+NADPH+TCBZ (15 mu g/ml), or KTZ+NADPH+triclabendazole sulphoxide (TCBZ. SO; 15 mu g/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ. SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ+TCBZ, but more particularly KTZ+TCBZ. SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Novel synthetic route to the C-nucleoside, 2-deoxy benzamide riboside

2-Deoxy-C-nucleosides are a subcategory of C-nucleosides that has not been explored extensively, largely because the synthesis is less facile. Flexible synthetic procedures giving access to 2-deoxy-C-nucleosides are therefore of interest. To exemplify the versatility and highlight the limitations of a synthetic route recently developed to that effect, the first synthesis of 2-deoxy benzamide riboside is reported. Biological properties of this novel C-nucleoside are also discussed. (c) 2012 Elsevier Ltd. All rights reserved.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440.

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.