Shuttles for translocation of NADH in isolated liver cells from fed rats during oxidation of xylitol.

Translocation of xylitol-derived NADH via malate-aspartate and alpha-glycerophosphate shuttles was studied in liver cells isolated from fed rats. In bicarbonate medium amino-oxyacetate, rotenone and antimycin A, were equally efficient in depressing the xylitol removal. Incubation of cells in nonbicarbonate medium did not affect the rate of xylitol removal. In this medium amino-oxyacetate and antimycin A, but not rotenone, inhibited xylitol removal. Xylitol inhibited the lactate accumulation found when the cells were incubated without any exogenous substrates. Glucose was the main end product of xylitol oxidation. In nonbicarbonate medium ketogenesis was high, whereas in bicarbonate medium a low rate of ketone body formation was found. Xylitol had no effect on the rate of ketone body formation in either medium tested. Xylitol markedly decreased the ATP and Pi contents of the cells, but no change in the ATP/ADP x Pi ratio or the rate of oxygen consumption was found. The results suggest that NADH formed during xylitol oxidation is translocated to the mitochondria mainly through the malate-aspartate shuttle and only when this shuttle is inhibited does the alpha-glycerophosphate shuttle transfer NADH. Intramitochondrial reactions which form NADH and FADH2 are also suggested to be important regulators of the activity of the alpha-glycerophosphate shuttle.     

Metabolic flux analysis of clostridium thermosuccinogenes

Clostridium thermosuccinogenes are anaerobic thermophilic bacteria that ferment various carbohydrates to succinate and acetate as major products and formate, lactate, and ethanol as minor products. Metabolic carbon flux analysis was used to evaluate the effect of pH and redox potential on the batch fermentation of C. thermosuccinogenes. In a first study, the effects of four pH values (6.50, 6.75, 7.00, and 7.25) on intracellular carbon flux at a constant redox potential of -275 mV were compared. The flux of carbon toward succinate and formate increased whereas the flux to lactate decreased significantly with a pH increase from 6.50 to 7.25. Both specific growth rate and specific rate of glucose consumption were unaffected by changes in pH. The fraction of carbon flux at the phosphoenolpyruvate (PEP) node flowing to oxaloacetate increased with an increase in pH. At the pyruvate node, the fraction of flux to formate increased with increasing pH. At the acetyl CoA node, the fraction of flux to acetate increased significantly with an increase in pH. A second study elucidated the effect of four controlled culture redox potentials (-225, -250, -275, and -310 mV) on metabolic carbon flux at a constant pH of 7.25. Lower values of culture redox potential were correlated with increased succinate, acetate, and formate fluxes and decreased ethanol and hydrogen fluxes in C. thermosuccinogenes. Lactate formation was not significantly influenced by redox potential. At the PEP node, the fraction of carbon to oxaloacetate increased with a decrease in redox potential. At the pyruvate node, the fraction of carbon to formate increased, while at the acetyl CoA node, the fraction of carbon flux to acetate increased with reduced redox potential. The presence of hydrogen in the headspace or the addition of nicotinic acid to the growth media resulted in increased hydrogen and ethanol fluxes and decreased succinate, acetate, formate, and lactate fluxes.     

Effect of Pyruvate on the Redox Behavior of the Iron (III)-(II) Couple

Abstract

Evidence for the formation of Fe(III) and Fe(II) complexes with pyruvate ion is presented. Complexes with a 1:2 ratio of Fe(II) to pyruvate and 1:1 ratio of Fe(III) to pyruvate were identified by spectrophotometry. The complexation results in partial kinetic control of the electrochemical oxidation of Fe(II) in citrate buffer. In addition, Fe(III) was found to be chemically reduced by pyruvate. The apparent first order rate constant at 25[ddot]C is 7.12 × 10^?2 s ^?1in pH 4.0 pyruvate buffer and 1.24 × 10^?1 s ^?1 in pH 3.2 pyruvate buffer. In pH 4.0 citrate buffer the reaction is not first order and is significantly slower.     

Synthesis, structure, and acid-base and redox properties of a family of new Ru(II) isomeric complexes containing the trpy and the dinucleating Hbpp ligands

Three pairs of mononuclear geometrical isomers containing the ligand 3,5-bis(2-pyridyl)pyrazole (Hbpp) of general formula in- and out-[RuII(Hbpp)(trpy)X](n+) (trpy=2,2':6',2' '-terpyridine; X=Cl, n=1, 2a,b; X=H2O, n=2, 3a,b; X=py (pyridine), n=2, 4a,b) have been prepared through two different synthetic routes, isolated, and structurally characterized. The solid state structural characterization was performed by X-ray diffraction analysis of four complexes: 2a-4a and 4b. The structural characterization in solution was performed by means of 1D and 2D NMR spectroscopy for complexes 2a,b and 4a,b and coincides with the structures found in the solid state. All complexes were also spectroscopically characterized by UV-vis which also allowed us to carry out spectrophotometric acid-base titrations. Thus, a number of species were spectroscopically characterized with the same oxidation state but with a different degree of protonation. As an example, for 3a three pKa values were obtained: pKa1(RuII)=2.13, pKa2(RuII)=6.88, and pKa3(RuII)=11.09. The redox properties were also studied, giving in all cases a number of electron transfers coupled to proton transfers. The pH dependency of the redox potentials allowed us to calculate the pKa of the complexes in the Ru(III) oxidation state. For complex 3a, these were found to be pKa1(RuIII)=0.01, pKa2(RuIII)=2.78, and pKa3(RuIII)=5.43. The oxidation state Ru(IV) was only reached from the Ru-OH2 type of complexes 3a or 3b. It has also been shown that the RuIV=O species derived from 3a is capable of electrocatalytically oxidizing benzyl alcohol with a second-order rate constant of kcat=17.1 M(-1) s(-1).     

Brominated Trihalomethylenones as Versatile Precursors to 3‐Ethoxy, ‐Formyl, ‐Azidomethyl, ‐Triazolyl,and 3‐Aminomethyl Pyrazoles

5‐Bromo[5,5‐dibromo]‐1,1,1‐trihalo‐4‐methoxy‐3‐penten[hexen]‐2‐ones are explored as precursors to the synthesis of 3‐ethoxymethyl‐5‐trifluoromethyl‐1H‐pyrazoles from a cyclocondensation reaction with hydrazine monohydrate in ethanol. 3‐Ethoxymethyl‐carboxyethyl ester pyrazoles were formed as a result of a substitution reaction of bromine and chlorine by ethanol. The dibrominated precursor furnished 3‐acetal‐pyrazole that was easily hydrolyzed to formyl group. In addition, brominated precursors were used in a nucleophilic substitution reaction with sodium azide to synthesize the 3‐azidomethyl‐5‐ethoxycarbonyl‐1H‐pyrazole from the reaction with hydrazine monohydrate. These products were submitted to a cycloaddition reaction with phenyl acetylene furnishing the 3‐[4(5)‐phenyl‐1,2,3‐triazolyl]5‐ ethoxycarbonyl‐1H‐pyrazoles and to reduction conditions resulting in 3‐aminomethyl‐1H‐pyrazole‐5‐carboxyethyl ester. The products were obtained by a simple methodology and in moderate to good yields.     

Site‐Specific Oxidation State Assignments of the Iron Atoms in the [4Fe:4S]2+/1+/0 States of the Nitrogenase Fe‐Protein

The nitrogenase iron protein (Fe‐protein) contains an unusual [4Fe:4S] iron‐sulphur cluster that is stable in three oxidation states: 2+, 1+, and 0. Here, we use spatially resolved anomalous dispersion (SpReAD) refinement to determine oxidation assignments for the individual irons for each state. Additionally, we report the 1.13‐Å resolution structure for the ADP bound Fe‐protein, the highest resolution Fe‐protein structure presently determined. In the dithionite‐reduced [4Fe:4S]¹⁺ state, our analysis identifies a solvent exposed, delocalized Fe^2.5+ pair and a buried Fe²⁺ pair. We propose that ATP binding by the Fe‐protein promotes an internal redox rearrangement such that the solvent‐exposed Fe pair becomes reduced, thereby facilitating electron transfer to the nitrogenase molybdenum iron‐protein. In the [4Fe:4S]⁰ and [4Fe:4S]²⁺ states, the SpReAD analysis supports oxidation states assignments for all irons in these clusters of Fe²⁺ and valence delocalized Fe^2.5+, respectively.     

Novel Multiplexed Biosensor System for the Determination of Lactate and Pyruvate in Blood Serum

Multiplexing is one of the main current trends in biosensors, especially important for clinical diagnosis. However, simultaneous determination of several substances in one sample is often difficult due to different performance and working conditions of separate biosensors. This work was aimed at the development of a multiplexed biosensor system for the determination of lactate and pyruvate concentrations in liquid samples (i. e., blood serum). The system consisted of two amperometric biosensors based on lactate oxidase and pyruvate oxidase, which worked simultaneously in a single measuring cell. Conditions for the biosensor system work were selected. Linear range of lactate determination was 5–1000 μM, pyruvate – 10–5000 μM. Steady‐state response time was 30 s and 50 s for the lactate and pyruvate biosensors, respectively. After 2 weeks of storage biosensor responses decreased to 95 % (lactate biosensor) or 82 % (pyruvate biosensor) of the initial value. A scheme of analysis of the concentrations of lactate and pyruvate in human blood serum was proposed. The lactate and pyruvate concentrations as well as their ratio in human blood serum samples were determined and compared with the control method. The proposed biosensor system is suitable for the rapid detection of lactate, pyruvate and their ratio and can be used for clinical diagnosis, e. g., evaluation of the reasons of lactic acidosis and prognosis of patient's recovery.     

Transketolase from human erythrocytes. Purification and properties

Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.     

Studies with Polyfunctionally Substituted Heterocycles: New Synthesis of Pyrido[5′, 4′:2,3][1,3,4]oxadiazolo[3,2-a]pyridine; Pyridazine; 1,3,4-Oxadiazote; and Pyrazolo[1,5-a]Pyrimidine Derivatives

Cyanoacetohydrazide 1a reacts with 2-arylhydrazoketons 2a,b and 3a,b in refluxing ethanol to yield pyrido[5′,4′:2,3][1,3,4]oxadiazolo[3,2-a]pyridine and pyridazine derivatives; in the absence of solvent pyrazolo[1,5-a]pyrimidine derivatives were obtained. The reaction of 2a,b and 3a,b will) benzoylhydrazine afforded 1,3,4-oxadiazole and pyrazole derivatives.     

Reactivity of tris(acetylacetonato) iron(III) with tridentate [ONO] donor Schiff base as an access to newer mixed-ligand iron(III) complexes

Two new mixed-ligand iron(III) complexes, [Fe(L(n))(acac)(C(2)H(5)OH)] incorporating coordinated ethanol from the reaction solvent were accessed from the reaction of [Fe(acac)(3)] with [ONO] donor dibasic tridentate unsymmetrical Schiff base ligands derived from condensation of 2-hydroxy-1-napthaldehyde with 2-aminophenol (H(2)L(1)) or 2-aminobenzoic acid (H(2)L(2)). The thermal study (TGA-DTA) provided evidence for weakly bound ethanol which is readily substituted by neutral N-donor molecule imidazole, benzimidazole or pyridine to produce an array of newer complexes, [Fe(L(n))(acac)X] (n=1, 2; X=Im, Bim, Py). The compounds were characterized by elemental analyses, FT-IR, UV-vis, solution electrical conductivity, FAB mass, (1)H and (13)C NMR spectroscopy. Room temperature magnetic susceptibility measurements (μ(eff)～5.8 B.M.) are consistent with spin-free octahedral iron(III) complexes. Cyclic voltammetry of ethanol complexes revealed a quasi-reversible one electron redox response (ΔE(p)>100 mV) for the Fe(III)/Fe(II) couple. Low half wave redox potential (E(1/2)) values suggested easy redox susceptibility. The ground state geometries of the ethanol and imidazole complexes have been ascertained to be distorted octahedral by density functional theory using DMol3 program at BLYP/DNP level.     

Effect of water extracts of aloe and some herbs in decreasing blood ethanol concentration in rats. II

Oral administration of ethanol to rats at a dose of 3 g/kg decreased alcohol dehydrogenase (ADH) activity and metabolism of lactate to pyruvate in the liver. The effects of water extracts of Aloe and some other herbs on blood ethanol concentration and on ADH activity in liver cytosol were examined. The water extracts of these herbs caused a faster elimination of ethanol from blood of normal rats when administered orally 30 min before oral administration of ethanol. The rapid elimination of ethanol seems to be due to a protection of ADH activity and the supply of nicotinamide dinucleotide, both of which are reduced by high ethanol concentration. The effects of ethanol in decreasing the enzyme activities relating to its own metabolism occur when high concentrations of ethanol pass through the liver, and thus may primarily appear during the absorption of alcohol from the gastrointestinal tract, when portal concentration of ethanol are very high.     

Nanomolar Level Amperometric Determination of ATP Through Substrate Recycling in an Enzyme Reactor in a FIA System

Abstract

ATP, adenosine-5′-triphosphate, was determined by recycling in an enzyme reactor with co-immobilized pyruvate kinase and hexokinase in the presence of glucose, NAD⁺, and phosphoenolpyruvate, PEP. Recycling produces glucose-6-phosphate which is converted to an equivalent amount of NADH by glucose-6-phosphate dehydrogenase. The NADH is detected at a graphite flow-through electrode modified with an adsorbed 3,3′-bis(benzo[a]phenoxazin-7-ium, 5-amino-9-(diethylamino))1,4,N,N′-diamidobenzene, BPT. Oxidation of NADH takes place at 0 mM vs Ag/AgCl due to the adsorbed phenoxazine. The amplification factor is directly proportional to the residence time in the reactor and it is increased as the flow rate decreases; it becomes350 at a flow rate of 0.07 ml/min. The amplification factor can be increased further by a controlled stop-time recycling; it became 1200 at a stop-time of 12 min. A theoretical expression for the amplification factor was derived and it shows that the amplification depends o n the residence time and the pseudo-first order rate coefficients of the recycling enzyme systems. The response was linear over more than three decades, from 1 nM to 5 μM ATP. The detection limit, 1 nM ATP was set by cofactor impurities in the reagent rather than by system sensitivity or noise.     

An Efficient Four-Component Synthesis of Multisubstituted Pyrano[2,3-c]pyrazole

A series of substituted pyrano[2,3-c]pyrazole derivatives were synthesized by a one-pot reaction of methyl 4-methyl-3-oxovalerate, phenylhydrazine, aromatic aldehyde, and malononitrile in ethanol with catalysis by triethylamine. The title compounds were obtained in good to excellent yields. A possible mechanism for this reaction was proposed.     

Effect of lead poisoning on the thiamine status and function in liver and blood of rats

Three groups of Sprague-Dawley rats were fed a thiamine deficient diet, which was supplemented by daily subcutaneous injections of a minimum requirement of thiamine, and treated with lead(II) acetate in different molar ratios to thiamine (1:1, 2:1, 10:1) for 5 and 9 months, respectively. The prolonged administration of lead(II) acetate decreases the thiamine level in lead-treated rats and diminishes the enzymatic activity of pyruvate dehydrogenase as well as that of transketolase. The thiamine level in the liver decreased by 30 to 40% compared with a reference group and the activity of the erythrocyte transketolase diminished by 5 to 40%. The level of the blood pyruvate increased by about 20% and the rate of the oxidative decarboxylation of pyruvate by liver mitochondria decreased.     

Catalytic Action of Metallic Salts in Autoxidation and Polymerization. VII. Polymerization of Methyl Methacrylate by Cobalt(ll) or (III) Acetylacetonate-Dioxane Hydroperoxide

Abstract

Polymerization of methyl methacrylate by Co(II or III) acetylacetonate-dioxane hydroperoxide [abbreviated as Co(acac)2, Co(acac)₃, and DOX HPO, respectively] was carried out in dioxane solvent, and the differences in polymerization rate and the degree of polymerization between two initiating systems were compared. Co(acac)₂-DOX HPO for the initiation of the polymerization system was more effective than Co(acac)₃-DOX HPO. The polymerization rate equations for both initiating systems obtained from kinetic data were as follows. For Co(acac)₂-DOX HPO initiating system: R_p=k [M]^3/2[Co(acac)₂]^1/7[DOX HPO]^?     

Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound, cAMP-activated protein kinase.

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3',5'-cyclic monophosphate (8-N3 cAMP) and [gamma32P]ATP, the membrane-binding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [32P]8-N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [gamma32P]ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg.ATP concentration is below approximately 10 micrometer. The catalytic subunit is not required for the Mg.ATP inhibition of 8-N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied on other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg.ATP.     

Mechanism for activation of triosephosphate isomerase by phosphite dianion: the role of a ligand-driven conformational change

The L232A mutation in triosephosphate isomerase (TIM) from Trypanosoma brucei brucei results in a small 6-fold decrease in k(cat)/K(m) for the reversible enzyme-catalyzed isomerization of glyceraldehyde 3-phosphate to give dihydroxyacetone phosphate. In contrast, this mutation leads to a 17-fold increase in the second-order rate constant for the TIM-catalyzed proton transfer reaction of the truncated substrate piece [1-(13)C]glycolaldehyde ([1-(13)C]-GA) in D(2)O, a 25-fold increase in the third-order rate constant for the reaction of the substrate pieces GA and phosphite dianion (HPO(3)(2-)), and a 16-fold decrease in K(d) for binding of HPO(3)(2-) to the free enzyme. Most significantly, the mutation also results in an 11-fold decrease in the extent of activation of the enzyme toward turnover of GA by bound HPO(3)(2-). The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(c)) relative to an inactive open form (E(o)). We propose that this is due to the relief, in L232A mutant TIM, of unfavorable steric interactions between the bulky hydrophobic side chain of Leu-232 and the basic carboxylate side chain of Glu-167, the catalytic base, which destabilize E(c) relative to E(o).     

INTRACELLULAR DAMAGE IN YEAST CELLS CAUSED BY PHOTODYNAMIC TREATMENT WITH TOLUIDINE BLUE

Abstract— The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell-associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.     

Polyferredoxin-based electrode materials

Electrochemical oxidation of the hydrosulfide cluster [Fe4S4(SH)4]2- on gold, platinum or vitreous carbon in a methyl cyanide electrolyte leads to the growth of a conducting film. Spectroscopic and other evidence suggests that the film has cubane centres, predominately in the [4Fe4S]3+ oxidation state, which are linked by disulfide ligands to give an anionic array of [Fe4S4(S approximately)4]n- units. X-ray data suggests some long-range order in the electrode material. The polyferredoxin binds redox active cations consistent with an anionic array.     

Ligand K-edge X-ray absorption spectroscopy and DFT calculations on [Fe3S4]0,+ clusters: delocalization, redox, and effect of the protein environment

Ligand K-edge XAS of an [Fe3S4]0 model complex is reported. The pre-edge can be resolved into contributions from the mu(2)S(sulfide), mu(3)S(sulfide), and S(thiolate) ligands. The average ligand-metal bond covalencies obtained from these pre-edges are further distributed between Fe(3+) and Fe(2.5+) components using DFT calculations. The bridging ligand covalency in the [Fe2S2]+ subsite of the [Fe3S4]0 cluster is found to be significantly lower than its value in a reduced [Fe2S2] cluster (38% vs 61%, respectively). This lowered bridging ligand covalency reduces the superexchange coupling parameter J relative to its value in a reduced [Fe2S2]+ site (-146 cm(-1) vs -360 cm(-1), respectively). This decrease in J, along with estimates of the double exchange parameter B and vibronic coupling parameter lambda2/k(-), leads to an S = 2 delocalized ground state in the [Fe3S4]0 cluster. The S K-edge XAS of the protein ferredoxin II (Fd II) from the D. gigas active site shows a decrease in covalency compared to the model complex, in the same oxidation state, which correlates with the number of H-bonding interactions to specific sulfur ligands present in the active site. The changes in ligand-metal bond covalencies upon redox compared with DFT calculations indicate that the redox reaction involves a two-electron change (one-electron ionization plus a spin change of a second electron) with significant electronic relaxation. The presence of the redox inactive Fe(3+) center is found to decrease the barrier of the redox process in the [Fe3S4] cluster due to its strong antiferromagnetic coupling with the redox active Fe2S2 subsite.