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PubMed1981

Control of ethanol utilization by rat hepatocytes.

Ethanol oxidation by hepatocytes from fasted rats was determined in the presence and absence of 0.2 mM ethyl hydrazinoacetate, a transaminase inhibitor which blocks the malate-aspartate cycle. 20 muM phenazine methosulfate caused the largest increase (nearly 150%) in ethanol utilization. 5 muM norepinephrine caused a 50% increase in ethanol oxidation, and most of this increase was caused by stimulation of the alpha-glycerophosphate shuttle, since it remained in the presence of ethyl hydrazinoacetate. 1 muM glucagon caused a 25% increase in ethanol uptake, and most of this increase was abolished by ethyl hydrazinoacetate, indicating that the malate-aspartate cycle was involved. 25 muM dinitrophenol increased ethanol use by 20% and this increase was nearly unaffected by ethyl hydrazinoacetate. The results indicate that ethanol utilization, under the conditions used, is primarily controlled by the capacity of the shuttle systems, and not by the capacity of the respiratory chain.

Animals↗
PubMed1992

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.

Alcohol Oxidoreductases↗
PubMed1978

Alcohol-oxidizing enzymes from various organisms.

1. Enzymes that catalyse the oxidation of aliphatic alcohols to aldehydes are reviewed. 2. Special attention is given to phenazine methosulphate-linked alcohol dehydrogenases from bacteria and to flavin-containing alcohol oxidases from yeasts, moulds and higher plants. 3. Some properties of the microsomal ethanol-oxidative system of rat liver are discussed.

Alcohol Oxidoreductases↗
PubMed1996

Phenothiazines are potent inhibitors of osteoclastic bone resorption.

1. We have previously shown that promethazine inhibits osteoclastic bone resorption in the in vitro bone slice assay (IC50 = 0.8 microM), but the mechanism(s) involved are unclear. 2. We have now tested the effects on osteoclast activity of five other structurally related compounds. Phenothiazine, chlorpromazine, amitriptyline, phenazine, and phenoxazine had IC50 values of 0.8, 0.9, 6, 7, and > 10 microM, respectively, in the bone slice assay, indicating that the basic phenothiazine structural element itself is important for osteoclast inhibitory activity. 3. The results are discussed in terms of the known effects of phenothiazines on plasma membrane fluidity, blocking of ion channels, and inhibition of calmodulin.

Animals↗
PubMed1990

A simple plate-assay for the screening of L-malic acid producing microorganisms.

A simple plate-assay has been developed to screen microorganisms for L-malic acid production. Acid producing organisms were identified, after microbial colony growth on media containing glucose or fumaric acid as sole carbons sources, by formation of a dark halo of formazan. The halo was observed when the plate was covered with a soft agar overlay containing NAD(+)-malate dehydrogenase, NAD+, phenazine methosulfate (PMS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay developed is simple, specific for L-malic acid and therefore can be used to identify L-malic acid producing filamentous fungi using glucose as carbon source (e.g. Aspergillus strains). The assay is also applicable for screening bacteria with high fumarase activity, able to convert fumaric acid to L-malic acid.

Aspergillus↗
PubMed1989

Superoxide anion radical-independent pathway for reduction of tetrazolium salts in aerobic mixtures consisting of NADH and 5-methylphenazinium methyl sulfate in the presence of aqueous micelles of nonionic and cationic detergents.

The effect of Triton X-100 and certain other nonionic as well as cationic detergents on 5-methyl-phenazinium methyl sulfate (PMS)-mediated reduction of tetrazolium salts was studied under aerobic conditions using an exogenous source of reducing equivalents, such as NADH or by generating NADPH through an enzymatic reaction. In the absence of detergents, 5,10-dihydro-5-methylphenazine (MPH), formed on reduction of 5-methylphenazinium cation (MP+) of PMS by NAD[P]H, was reoxidized allowing first the univalent reduction of molecular oxygen (O2) to the superoxide anion radical (O2-.) which, in turn, reduced tetrazolium salts. In the presence of detergents, however, a significant fraction of the PMS-mediated reduction of tetrazolium salts appeared to proceed without the intervention of O2-. The reasons for this were examined experimentally and it was suggested that the reduced phenazine (i.e., MPH), which is sparingly soluble in aqueous solutions, migrates into detergent micelles where tetrazolium salts are reduced in preference to O2. By lowering the pH and thereby facilitating the H+-mediated dismutation of O2-., it was possible to obtain the reduction of tetrazolium salts, mediated selectively and directly by MPH in the micellar pseudophase. Employing the technique of saturation analysis, further evidence was obtained that lends support for preferential reduction of tetrazolium salts (e.g., nitroblue tetrazolium chloride) to that of O2 by the micelle-bound MPH.

Aerobiosis↗
PubMed1990

Flavonoids as superoxide scavengers and antioxidants.

The superoxide anions scavenging activity and antioxidation of seven flavonoids--quercetin, rutin, morin, acacetin, hispidulin, hesperidin, and naringin--were studied. The superoxide anions were generated in a phenazin methosulphate-NADH system and were assayed by reduction of nitroblue tetrazolium. The scavenging activity ranked: rutin was the strongest, and quercetin and naringin the second, while morin and hispidulin were very weak. The concentration values yielding 50% inhibition of lipid peroxidation in mouse liver homogenate were in order of 10(-6) M for quercetin, rutin, and morin; and of 10(-5) M for acacetin and hispidulin, while naringin and hesperidin had no antioxidative action. In comparison with the antioxidative and scavenging activities of flavonoids, there are no correlations.

Animals↗
PubMed1992

Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode.

An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.

Biosensing Techniques↗
PubMed1995

An amperometric opiate assay.

Current techniques for the detection and measurement of diacetylmorphine (heroin), morphine and their principal metabolite morphine-3-glucuronide (M3G) are based mainly on chromatography or immunoassay. No enzymatic method for the detection of these compounds has yet been reported. Two novel microbial enzymes have been isolated and characterized in this laboratory: an acetylmorphine carboxyesterase (heroin esterase) and a morphine dehydrogenase (MDH). These highly specific enzymes have been incorporated in an amperometric assay for heroin and morphine using phenazine methosulphate as a mediator. The assay gives a rapid and sensitive response to heroin and morphine, with a detection limit for morphine of 6.8 micrograms ml-1 (23.7 microM).

Acetylesterase↗
PubMed1996

Mediated amperometric determination of ammonia with a methanol dehydrogenase from Pseudomonas sp. AM-1 immobilized carbon paste electrode.

PQQ-dependent methanol dehydrogenase (EC 1.1.99.8) was immobilized onto the surface of a carbon paste electrode by the cross-linking method and the electrode was covered with a porous polycarbonate membrane. An electrocatalytic steady-state current for the oxidation of methanol was observed using this electrode in the presence of phenazine methosulphate as an electron transfer mediator and NH4Cl as an activator for enzyme activity. The electrocatalytic current at +0.2 V vs. Ag/AgCl was analyzed. The properties of the electrode were examined for the determination of ammonia. The electrode responded linearly to ammonia in the range of 3 to 60 mM. The assay took 40 to 50 s. The electrode was used repeatedly at room temperature for 15 days and retained 50% of its initial activity.

Alcohol Oxidoreductases↗
PubMed1989

Pentose phosphate shunt metabolism by cells of the chick growth cartilage.

We have measured the activity of the pentose shunt pathway in the chick growth cartilage. Measurement of D-[1-14C] glucose and D-[6-14C] glucose metabolism by chondrocytes indicated that pentose phosphate shunt activity was low. However, when the cells were stimulated with phenazine methosulfate (PMS) and t-butyl hydroperoxide, a significant elevation in shunt activity was observed. This activity was further increased by dithiothreitol. Enzymatic and substrate requirements of the shunt pathway were examined and related to morphology of the tissue. It was found that as chondrocytes mature, there is increased glucose-6-phosphate dehydrogenase activity, and decreased quantities of glucose-6-phosphate and NADPH. While these investigations indicated that shunt activity was maximum in hypertrophic cartilage, the results of cytochemical studies suggested that the activity was greatest in those cells that were most removed from the O2 supply. Experiments were performed to examine O2 requirements of chondrocytes in relationship to the pentose phosphate shunt. First, using a phosphorescence quenching technique, total O2 uptake by these cells was found to be constant over a large part of the physiological range of O2 tensions. Over the same range, when stimulated by PMS, O2 uptake by CN- treated cells was increased. In the 1-5 microM O2 range, non-mitochondrial O2 consumption decreased more slowly than total respiration. Finally, the observation that NADPH directly stimulated chondrocyte O2 consumption suggest that cartilage cells may be able to form O2 metabolites.

Animals↗
PubMed2005

Estimating the number of viable animal cells in multiwell culture--a tetrazolium-based assay.

A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.

Animals↗
PubMed2006

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis.

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa.

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.

Biosensing Techniques↗
PubMed2005

Development of a commercial amperometric biosensor electrode for the ketone D-3-hydroxybutyrate.

Representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue (MB) 3, 4-methyl-1,2-benzoquinone (4-MBQ) 4, 1-methoxy phenazine methosulphate (1-MeO-PMS) 5 and 2,6-dichloroindophenol (DCIP) 6, are shown to inhibit the NAD-dependent enzyme D-3-hydroxybutyrate dehydrogenase (HBDH), severely limiting their utility in the construction of a stable biosensor electrode for the ketone body D-3-hydroxybutyrate (3-OHB). It is proposed that these mediators bind covalently to important thiol groups in the enzyme. This mode of inhibition is overcome through the use of mediators such as 1,10-phenanthroline quinone (1,10-PQ) 7, which avoid 1,4-nucleophilic addition with enzyme amino acid residues such as Cys. As a result, 1,10-PQ 7 was selected for incorporation in a biosensor electrode for 3-OHB. The resulting MediSense Optiumtrade mark beta-Ketone electrode is stable (<or=10% loss in response at 30 degrees C versus 4 degrees C) with a long shelf life of 18 months. Diabetics can determine their D-3-hydroxybutyrate level with good precision (0.43 mM 3-OHB, 10.5% CV; 1.08 mM, 5.9%; 3.55 mM, 3.2%; n=20 per level) and accuracy (versus reference assay: slope=0.98; intercept=0.02 mM, r=0.97, n=120) over the range 0.0-6.0 mM in 30 s using a small volume of blood (5 microl). The electrode has a low operating potential (+200 mV versus Ag/AgCl) such that the effect of electroactive agents in blood is minimised.

3-Hydroxybutyric Acid↗
PubMed2005

Strategies to develop malic acid biosensors based on malate quinone oxidoreductase (MQO).

An amperometric biosensor based on malate quinone oxidoreductase (MQO) was developed for monitoring of the malolactic fermentation of wines. Screen-printed electrodes coupled with appropriate mediators were used as transducers for this novel biosensor. MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) on the surface of the working electrode. Several electrochemical mediators were studied in order to lower the applied potential and minimise the matrix effects. Among them, 2,6-dichlorophenol indophenol (DPIP) and phenazine methosulfate (PMS) were chosen for further development. The working conditions (mediator concentration, applied potential and pH) were optimised for both DPIP and PMS. Detection limits for both types of biosensors were of 5 microM malic acid. Sensitivities obtained for the linear part of the calibration curve were 0.85 and 1.7 mA/M for the biosensors based on DPIP and PMS, respectively. Interferences due to non-specific oxidations were shown to be negligible when using PMS as mediator.

Biosensing Techniques↗
PubMed2006

A probe for NADH and H2O2 amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor applications.

Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.

Biosensing Techniques↗
PubMed2005

Effect of sulfur amino acids on stimulus-induced superoxide generation and translocation of p47phox and p67phox to cell membrane in human neutrophils and the scavenging of free radical.

BACKGROUND: Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among these metabolites, cystathionine ketimine significantly enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in parallel with tyrosyl phosphorylation of 45 kDa protein in human neutrophils. METHODS: We investigated the effect of various sulfur amino acids on fMLP-, phorbol-12-myristate-13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in human neutrophils. In addition, the effects of these sulfur amino acids on the membrane translocation of cytosolic compounds p47(phox) and p67(phox) and on the scavenging of superoxide anions were investigated. RESULTS: When the cells were preincubated with various sulfur amino acids, fMLP-induced superoxide generation was enhanced by D,L-homocysteine and D,L-homocysteine-thiolactone but was inhibited by other sulfur amino acids in a concentration-dependent manner. The AA-induced superoxide was enhanced by L-cysteine, N-acetyl-L-cysteine and D,L-homocysteine. The strength of enhancing effect was: L-cysteine>>N-acetyl-L-cysteine>D,L-homocysteine. On the other hand, the superoxide generation was weakly inhibited by L-cystathionine. The superoxide generation induced by PMA was weakly inhibited by L-cysteine, N-acetyl-L-cysteine and L-cystathionine. Homocysteine and D,L-homocysteine-thiolactone had no effect. In addition, D,L-homocysteine also enhanced translocation to the cell membrane of cytosolic compounds p47(phox) and p67(phox). Conversely, L-cystathionine and N-acetyl-L-cysteine inhibited the translocation to membrane of p47(phox) and p67(phox) in a concentration-dependent manner. N-acetyl-L-cysteine and L-cysteine revealed scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The sulfur amino acids tested also indicated radical scavenging activity on superoxide anion generated by phenazine methoxysulfate (PMS)-NADH system. CONCLUSION: D,L-homocysteine and D,L-homocysteine-thiolactone enhanced fMLP-induced superoxide generation by the increment of translocation to membrane of p47(phox) and p67(phox). L-cystathionine and N-acetyl-L-cysteine suppressed fMLP- and PMA-induced superoxide generation by the inhibition of translocation to membrane of p47(phox) and p67(phox). N-acetyl-L-cysteine also had scavenging activity against DPPH radicals and superoxide anion.

Amino Acids, Sulfur↗