Showed Saturation Kinetics Research Articles

Kinetics and mechanism of the one‐step, two‐electron oxidation of diaquadichloro(1,10‐phenanthroline)chromium(III) chloride dihydrate by periodate to chromium(V) in aqueous acidic solutions

AbstractThe kinetics of oxidation of [CrIIICl2(phen)(H2O)2]Cl⋅2H2O (phen = 1,10‐phenanthroline) by periodate {I(VII)} have been studied in aqueous acidic solutions. The oxidation product has been isolated and characterized by elemental analysis, IR, and ESR as [CrVCl2(phen)(O)]Cl⋅1.5H2O. Under pseudo–first‐order conditions, the reaction showed first‐order dependence on the initial Cr(III) complex concentration. At fixed reaction conditions, the dependence of kobs on [I(VII)] showed saturation kinetics. The rate constant, kobs, increased with increasing pH, indicating that the hydroxo form of the chromium(III) complex is the most reactive species. A plot of kobs([H+] + K1) versus [H+] is linear with an intercept, indicating that the reaction proceeds via two parallel pathways. One pathway is independent of [H+] and the other on [H+]–1. Since water is known to be a very weak bridging ligand, the pathway involving the aqua form of the complex, almost certainly, is bridged to periodate via the chloro ligand of the chromium(III) complex. The thermodynamic parameters of the processes involved are reported. A one‐step two‐electron transfer via an inner‐sphere mechanism has been assigned to the two pathways based on the form of the rate law

Developing in vitro models to assess fish gill excretion of emerging contaminants.

Advances in analytical methods have enabled the detection of emerging contaminants at ever lower concentrations in freshwaters. However, such measurements need to be linked to effect-based assays to identify risks. The bioconcentration factor (BCF) forms part of a chemical's environmental risk assessment (ERA), and current regulatory testing guidelines to calculate fish BCFs use hundreds of fish per chemical. Due to ethical concerns a reduction in the numbers of animals used is desired, and there is a need to identify in vitro or in silico alternatives which meet regulatory acceptance. This study describes the successful demonstration of a FIsh Gill Cell culture System (FIGCS) to assess an often overlooked parameter in pharmacokinetics: the excretion of drugs across the gill. The FIGCS tolerates the application of natural waters on its apical surface, mimicking the situation of the live fish, and thus in combination with advanced analytical methods, offers an opportunity to take lab-based testing used for ERA, such as compound uptake, biotransformation or excretion directly into field for validation with natural waters. Here we used the basic drug propranolol and the acidic ibuprofen as a demonstration of the FIGCS utility in three separate experiments. Excretion across the apical membrane showed saturation kinetics, suggesting the involvement of carrier-mediated processes. Both propranolol and ibuprofen were excreted across the epithelium from the media (internal blood equivalent) to the water, with ibuprofen excretion being considerably slower than propranolol excretion. Further studies indicate that ibuprofen may be complexing with fetal bovine serum (FBS) reducing bioavailability; in contrast propranolol efflux rate was unaffected, indicating that drugs behave differently in the presence of FBS and other plasma proteins. A key issue in future ERA is to better understand the effects of mixtures of different pollutant classes found in environmental samples, and this model offers an ethical path to do this.

Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell.

Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Epimagnolin A, a tetrahydrofurofuranoid lignan from Magnolia fargesii, reverses ABCB1-mediated drug resistance

BackgroundEpimagnolin A is an ingredient of the Chinese crude drug Shin-i, derived from the dried flower buds of Magnolia fargesii and Magnolia flos, which has been traditionally used for the treatment of allergic rhinitis and nasal congestion, empyema, and sinusitis. The pharmacokinetic activity of epimagnolin A remains to be evaluated. PurposeIn this study, we examined the possible interactions of epimagnolin A with human ATP-binding cassette (ABC) transporter ABCB1, a membrane protein vital in regulating the pharmacokinetics of drugs and xenobiotics. Study design/methodsThe interaction of epimagnolin A with ABCB1 was evaluated in calcein, ATPase, and MTT assays by using Flp-In-293/ABCB1 cells and purified ABCB1 and simulated in molecular docking studies. ResultsEpimagnolin A inhibited calcein export by Flp-In-293/ABCB1 cells in a concentration-dependent manner in a calcein assay. ATPase assay revealed a concentration-dependent stimulation of the ATPase activity of ABCB1 by epimagnolin A. Epimagnolin A also showed saturation kinetics in the relationship between the compound-stimulated ATPase activity and the compound concentration, suggesting Michaelis–Menten kinetics similar to those of the control drug, verapamil. Km and Vmax values were calculated from Hanes–Woolf plots of (compound concentration) × (compound-stimulated ATPase activity)−1 vs. (compound concentration); the Km of epimagnolin and verapamil was 42.9 ± 7.53 μM and 12.3 ± 4.79 μM, respectively, and the corresponding Vmax values were 156 ± 15.0 μM and 109 ± 3.18 μM. Molecular docking studies on human ABCB1 showed that epimagnolin A docked to the same binding pocket as verapamil, and 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed that the sensitivities of Flp-In-293/ABCB1 cells against anti-cancer drugs were enhanced upon exposure to 10 μM epimagnolin A. ConclusionThese results strongly suggest that epimagnolin A affects the transport activity of ABCB1 as a substrate.

Na⁺-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L.) Delile.

Posidonia oceanica (L.) Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO3−, Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO3− and Pi were reduced by more than 70% in the absence of Na+. Micromolar concentrations of NO3− depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics (Km = 8.7 ± 1 μM NO3−), which were not observed in the absence of Na+. NO3− induced depolarizations at increasing Na+ also showed saturation kinetics (Km = 7.2 ± 2 mM Na+). Cytosolic Na+ measured in P. oceanica leaf cells (17 ± 2 mM Na+) increased by 0.4 ± 0.2 mM Na+ upon the addition of 100 μM NO3−. Na+-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO3−, amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na+-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

Nicotine transport in lung and non-lung epithelial cells

AimsNicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Materials and methodsCharacteristics of [3H]nicotine uptake was studied using these cell lines. Key findingsNicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. SignificanceThe present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed.

Record Broken: A Copper Peroxide Complex with Enhanced Stability and Faster Hydroxylation Catalysis

Tyrosinase model systems pinpoint pathways to translating Nature's synthetic abilities for useful synthetic catalysts. Mostly, they use N-donor ligands which mimic the histidine residues coordinating the two copper centres. Copper complexes with bis(pyrazolyl)methanes with pyridinyl or imidazolyl moieties are already reported as excellent tyrosinase models. Substitution of the pyridinyl donor results in the new ligand HC(3-tBuPz)2 (4-CO2 MePy) which stabilises a room-temperature stable μ-η2 :η2 -peroxide dicopper(II) species upon oxygenation. It reveals highly efficient catalytic activity as it hydroxylates 8-hydroxyquinoline in high yields (TONs of up to 20) and much faster than all other model systems (max. conversion within 7.5 min). Stoichiometric reactions with para-substituted sodium phenolates show saturation kinetics which are nearly linear for electron-rich substrates. The resulting Hammett correlation proves the electrophilic aromatic substitution mechanism. Furthermore, density functional theory (DFT) calculations elucidate the influence of the substituent at the pyridinyl donor: the carboxymethyl group adjusts the basicity and nucleophilicity without additional steric demand. This substitution opens up new pathways in reactivity tuning.

New metal complexes with diclofenac containing 2-pyridineethanol or 2-pyridinepropanol: synthesis, structural, spectroscopic, thermal properties, catechol oxidase and carbonic anhydrase activities

Four new neutral diclofenac-based complexes, [Co(dicl)2(2-pyet)2] 1, [Ni(dicl)2(2-pyet)2] 2, [Cu2(dicl)2(2-pyet)2] 3, and [Cu2(dicl)2(2-pypr)2] 4 have been synthesized and characterized by elemental analysis, FT-IR, thermal analysis. Complexes 1, 3, and 4 have also been characterized by X-ray single-crystal structural analysis. The compounds of Co(II) and Ni(II) have octahedral geometry with two diclofenac and two 2-pyridineethanol ligands in the coordination sphere. The compounds of Cu(II) have square-pyramidal geometry and Cu(II) ions are linked via oxygens to the bridging 2-pyridineethanol or 2-pyridinepropanol ligands. The Δν values acquired by FT-IR are in agreement with the single XRD data. Studies on the thermal properties are reported and the complexes are stable to 196, 216, 215, and 201 °C in air, respectively. Two dinuclear Cu(II) complexes have demonstrated catalytic activity on oxidation of 3,5-di-tert-butylcatechol to 3,5-di-tert-butylquinone showing saturation kinetics at high substrate concentrations. The diclofenac complexes are investigated as inhibitors of the human cytosolic isoforms hCA I and II. The complexes are good as hCA I inhibitors (Kis of 1.52–55.06 μM) but only moderately efficient as hCA II inhibitors (Kis of 0.23–5.61 μM).

Limited proteolysis of myoglobin opens channel in ferrochelatase-globin complex for iron to zinc transmetallation

Recombinant ferrochelatase (BsFECH) from Bacillus subtilis expressed in Escherichia coli BL21(DE3) was found by UV-visible spectroscopy to bind the model substrate tetraphenylporphyrin-sulfonate, TPPS, with Ka=3.8 105mol/L in aqueous phosphate buffer pH 5.7 at 30°C, and to interact with metmyoglobin with Ka=1.07±0.13 105mol/L at 30°C. The iron/zinc exchange in myoglobin occurring during maturation of Parma hams seems to depend on such substrate binding to BsFECH and was facilitated by limited pepsin proteolysis of myoglobin to open a reaction channel for metal exchange still with BsFECH associated to globin. BsFECH increased rate of zinc insertion in TPPS significantly and showed saturation kinetics with an apparent binding constant of Zn(II) to the [enzyme-TPPS] complex of 1.3 104mol/L and a first-order rate constant of 6.6 10−1s−1 for dissociation of the tertiary complex, a similar pattern was found for zinc/iron transmetallation in myoglobin.

Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells

Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [3H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [3H]Nicotine uptake in these cells was not affected by Na+, but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [3H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [3H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

Functional Expression of PEPT2 in the Human Distal Lung Epithelial Cell Line NCl-H441.

The peptide transporter PEPT2 is expressed in alveolar type II epithelial cells. So far, however, no appropriate alveolar epithelial cell line for studying PEPT2 function has been known. In this study, we examined the functional expression of PEPT2 in the human distal lung epithelial cell line NCl-H441 (H441). Expression of PEPT2 mRNA and protein was examined in H441 cells. Transport function of PEPT2 was studied using glycylsarcosine (Gly-Sar) as a substrate. Lamellar bodies were well developed in H441 cells and mRNA expression of type II cell markers and PEPT2 increased during time in culture. PEPT2 protein expression was confirmed in H441 cells, but not in A549 cells, by immunostaining and Western blotting. The uptake of Gly-Sar in H441 cells was inhibited by cefadroxil, and the cefadroxil-sensitive uptake was pH-dependent and peaked at pH 6.5. Gly-Sar uptake in H441 cells showed saturation kinetics with a Km value of 112.5 μM. In addition, apical-to-basal, but not basal-to-apical, transport of cephalexin across H441 cell monolayers was sensitive to cefadroxil. PEPT2 is functionally expressed in H441 cells, making the cell line a good in vitro model to study PEPT2 function and its regulation in human distal lung.

Methotrexate influx via folate transporters into alveolar epithelial cell line A549

Methotrexate (MTX), a drug used for the treatment of certain cancers as well as rheumatoid arthritis, sometimes induces serious interstitial lung injury. Although lung toxicity of MTX is related to its accumulation, the information concerning MTX transport in the lungs is lacking. In this study, we investigated the mechanisms underlying MTX influx into human alveolar epithelial cell line A549. MTX influx into A549 cells was time-, pH-, and temperature-dependent and showed saturation kinetics. The influx was inhibited by folic acid with IC50 values of 256.1 μM at pH 7.4 and 1.6 μM at pH 5.5, indicating that the mechanisms underlying MTX influx would be different at these pHs. We then examined the role of two folate transporters in MTX influx, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). The expression of RFC and PCFT mRNAs in A549 cells was confirmed by reverse transcription polymerase chain reaction. In addition, MTX influx was inhibited by thiamine monophosphate, an RFC inhibitor, at pH 7.4, and by sulfasalazine, a PCFT inhibitor, at pH 5.5. These results indicated that RFC and PCFT are predominantly involved in MTX influx into A549 cells at pH 7.4 and pH 5.5, respectively.

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe2+ transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe2+ or Cd2+ influx could be reliably monitored in calcium 5–loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe2+ and Cd2+, respectively. In addition, Cd2+ was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z′ factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

Potassium uptake in the halophyte Halimione portulacoides L. Aellen

The characteristics of root K+ uptake in the halophyte Halimione portulacoides have been studied using electrophysiological techniques. Plantlets grown in the absence of both K+ and Na+ are able to take up K+ at micromolar external concentrations and root cells of these plants depolarize in response to these K+ concentrations. Depolarizations show saturation kinetics with a Km around 300μM K+. Rubidium also induces membrane depolarizations and inhibits K+ transport at low micromolar concentrations whereas Cs+ hardly depolarizes the membrane. The addition of millimolar Na+ reduces both the affinity for K+ and the maximum transport rates, and membrane potentials become more positive only when Na+ concentration reaches 300mM. Plantlets grown at high Na+ show the same effects on K+ transport but membrane potentials are maintained at highly negative values. Plantlets grown in the absence or the presence of Na+ show similar responses to different inhibitors of K+ uptake. TEA+ does not affect K+ transport, Cs+ produces a partial inhibition whereas Ba2+ completely blocks the process. Besides, the external pH does not affect K+ transport. However, NH4+ inhibits partially this process in plants grown without Na+ but has no effect on plants grown at 300mM Na+. Altogether these results point out that the transport of K+ in this species is mediated mainly by channels, most probably of the AKT-1 type, both in plants grown with or without Na+; however, the operation of potassium transporters cannot be excluded in plants grown in the absence of Na+. Cytosolic K+ activities were also measured in plantlets grown without or with 300mM Na+, showing lower values at high Na+. Calculation of the electrochemical potential gradient for K+ in these plants indicates that the transport would be passive, i.e. mediated by channels, at concentrations higher than 20μM or 120μM, in the absence or the presence of 300mM Na+, respectively.

Investigating permeability related hurdles in oral delivery of 11-keto-β-boswellic acid

11-Keto-β-boswellic acid (KBA) is an important and potent boswellic acids responsible for anti-inflammatory action of Boswellia extract. However, its pharmaceutical development has been severely limited by its poor oral bioavailability. The present work aims to investigate the permeability related hurdles in oral delivery of KBA. Gastrointestinal stability, gastrointestinal metabolism, adsorption-desorption kinetics and Caco-2 permeability studies have been carried out. KBA was found poorly permeable with Papp value of 2.85 ± 0.14 × 10(-6)cm/s. Higher absorptive transport indicated role of carrier mediated transport. Moreover, KBA transport across monolayer showed saturation kinetics at higher concentrations. KBA exposed to 1α,25-(OH)2 vitamin D3 treated cell monolayer showed the lowest Papp value of 2.01×10(-6) ± 0.02 × 10(-6)cm/s indicating role of CYP3A4 mediated metabolism during KBA transport. Metabolic stability experiments in jejunum S9 fractions further confirmed this. KBA was found unstable in simulated gastrointestinal fluids and also got accumulated in the enterocytes. Sorption and desorption kinetic studies using Caco-2 cells further confirmed accumulation of KBA inside the enterocytes. KBA also showed pH dependent permeability with higher flux at gradient pH condition of pH 6.5 at apical and 7.4 at basolateral. Taken as whole, the major permeability related hurdles that hampered oral bioavailability of KBA included its gastrointestinal instability, CYP3A4 mediated intestinal metabolism, accumulation within the enterocytes and saturable kinetics. The present investigation may help in designing novel drug delivery system for KBA.

Glutathione Is a Low-Affinity Substrate of the Human Sodium-Dependent Dicarboxylate Transporter

Background/Aims: During a single pass through the kidneys, more than 80% of glutathione (GSH) is excreted, indicating not only glomerular filtration, but also tubular secretion. The first step in tubular secretion is the uptake of a substance across the basolateral membrane of proximal tubule cells by sodium-dependent and -independent transporters. Due to the dicarboxylate-like structure, we postulated that GSH uptake across the basolateral membrane is mediated by the sodium-dependent dicarboxylate transporter 3 (NaDC3). Methods: Tracer uptake and electrophysiologic measurements using a two-electrode voltage clamp device were performed in Xenopus laevis oocytes expressing the human (h)NaDC3. Results: Uptake of succinate, the reference substrate of hNaDC3, was inhibited by GSH in a dose-dependent manner with an IC₅₀ of 1.88 m<smlcap>M</smlcap>. GSH evoked potential-dependent inward currents, which were abolished under sodium-free conditions. At -60 mV, GSH currents showed saturation kinetics with a K_M of 1.65 m<smlcap>M</smlcap>. Conclusion: hNaDC3 present at the basolateral membrane of proximal tubule cells mediates sodium-dependent GSH uptake. The kinetic data show that NaDC3 is a low-affinity GSH transporter.

REACTION KINETICS OF VERSATILE PEROXIDASE FOR THE DEGRADATION OF LIGNIN COMPOUNDS

The H2O2-dependent degradation of adlerol by a crude versatile peroxidase from Bjerkandera adusta, a new ligninolytic enzyme, was investigated. Adlerol (1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol)) is a non phenolic β-O-4 dimer whose structural architecture represents the most abundant unit (50-65%) of the valuable renewable biopolymer lignin. Lignin removel plays a key role in utilizing lignocellulosic biomass in biorefineries. Steady state analyses in the µL scale showed saturation kinetics for both, H2O2 and adlerol with quite sensitive response to H2O2. This was characterized through slow transient states (lag phases) prior steady state and were enhanced by increasing H2O2 concentration. The major reason for such phenomena was found to be an accumulation of compound III (EIII) via reaction of compound II (EII) with H2O2; instead with adlerol to the enzyme’s ground state E0 in order to restart another catalytic cycle. As result, the enzyme deviated from its normal catalytic cycle. A corresponding threshold was determined at ≥ 50 µM H2O2 and an adlerol to H2O2 ratio of 15:1 for the given conditions. Furthermore, EIII did not represent a catalytical dead end intermediate as it is generally described. By an additional decrease of the adlerol to H2O2 ratio of ca. 3 at the latest, considerable irreversible enzyme deactivations occurred promoted through reaction of EIII with H2O2. At a mL scale deactivation kinetics by H2O2 were further examined in dependence on adlerol presence. The course followed a time dependent irreversible deactivation (two step mechanism) and was diminished in the presence of adlerol. The deactivation could be sufficiently described by an equation similar to the Michaelis Menten type, competitive inhibited by adlerol. Finally, first estimates of the kinetic parameters vmax, KmS1 (S1: H2O2), KmS2 (S2: adlerol), kiapp and Kiapp were made. Moreover, the peroxidase reaction mechanism was reviewed and recommendations are given preventing permature enzyme losses.

Reaction Pathway and Rate-Determining Step of the Schmidt Rearrangement/Fragmentation: A Kinetic Study

The Schmidt rearrangement of substituted 3-phenyl-2-butanone with trimethylsilyl azide in 90% (v/v) aqueous TFA gave two types of product, fragmentation and rearrangement, the ratio of which depends on the substituent: more fragmentation for a more electron-donating substituent. Rate measurements by azotometry indicated the presence of an induction period, and the pseudo-first-order rate constants showed saturation kinetics with respect to the azide concentration. It was indicated that the reaction proceeds through pre-equilibrium in the formation of iminodiazonium (ID) ion and that the N(2) liberation from the ID ion is rate-determining. Under high azide concentration conditions, where the effective reactant is the ID ion, the reaction gave a linear Hammett plot with a ρ value of -0.50. The observed substituent effects on the rate and the product selectivity imply that path bifurcation on the way from the rate-determining TS to the product states occurs, as suggested by previous molecular dynamics simulations, in a similar manner to the analogous Beckmann rearrangement/fragmentation reactions.

Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K (m) and V (max) values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min(-1) mg(-1) protein, respectively. The enzyme's pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.

An analysis of the performance of an anaerobic dual anode-chambered microbial fuel cell

The performance of a dual anode-chambered microbial fuel cell (MFC) inoculated with Shewanella oneidesis MR-1 was evaluated. This reactor was constructed by incorporating two anode chambers flanking a shared air cathode chamber in an electrically parallel, geometrically stacked arrangement. The device was shown to have the same maximum power density (approximately 24 W m −3, normalized by the anode volume) as a single anode-, single cathode-chambered MFC. The dual anode-chambered unit generated a maximum current of 3.66 mA (at 50 Ω), twice the value of 1.69 mA (at 100 Ω) for the single anode-chambered device at approximately the same volumetric current density. Increasing the Pt-coated cathode surface area by 100% (12 to 24 cm 2) had no significant effect on the power generation of the dual anode-chambered MFC, indicating that the performance of the device was limited by the anode. The medium recirculation rate and substrate concentration in the anode were varied to determine their effect on the anode-limited power density. At the highest recirculation rate, 5 ml min −1, the power density was about 25% higher than at the lowest recirculation rate, 1 ml min −1. The dependence of the power density on the lactate concentration showed saturation kinetics with a half-saturation constant K s on the order of 4.4 mM.