High-affinity NO(3-)-H+ cotransport in the fungus Neurospora:induction and control by pH and membrane voltage

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3(-)-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO3(-)-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 microM NO3-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 microM NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3(-)-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (-400 to +100 mV). Measurements of NO3- uptake using NO3(-)-selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K(m) and Jmax values around 25 microM and 75 pmol NO3- cm-2sec-1, respectively, and combined measurements of pHo and [NO3-]o showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pHo and [NO3-]o. Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pHo of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO3-. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H+]o. These, and additional results are consistent with a charge-coupling stoichiometry of 2(H+) per NO3- anion transported across the membrane, and implicate a carrier cycle in which NO3- binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO3- transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO3- transport; finally, they distinguish metabolite repression of NO3- transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

Effects of alcohols and compatible solutes on the activity of β-galactosidase

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell's macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme ß-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V (max), to fall and the Michaelis constant, K (m), to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited ß-galactosidase, the addition of compatible solutes was unable to restore the enzyme's kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme's structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.

Triose phosphate isomerase from the blood fluke Schistosoma mansoni:Biochemical characterisation of a potential drug and vaccine target

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0°C). The steady-state kinetic parameters are high (Km for dihydroxyacetone phosphate is 0.51mM; Km for glyceraldehyde 3-phosphate is 1.1mM; kcat for dihydroxyacetone phosphate is 7800s(-1) and kcat for glyceraldehyde 3-phosphate is 6.9s(-1)).

The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties

There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings. © 2014 Federation of European Biochemical Societies.

The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.

Release of β-galactosidase from poloxamine/α-cyclodextrin hydrogels

All mammals lose their ability to produce lactase (β-galactosidase), the enzyme that cleaves lactose into galactose and glucose, after weaning. The prevalence of lactase deficiency (LD) spans from 2 to 15% among northern Europeans, to nearly 100% among Asians. Following lactose consumption, people with LD often experience gastrointestinal symptoms such as abdominal pain, bowel distension, cramps and flatulence, or even systemic problems such as headache, loss of concentration and muscle pain. These symptoms vary depending on the amount of lactose ingested, type of food and degree of intolerance. Although those affected can avoid the uptake of dairy products, in doing so, they lose a readily available source of calcium and protein. In this work, gels obtained by complexation of Tetronic 90R4 with α-cyclodextrin loaded with β-galactosidase are proposed as a way to administer the enzyme immediately before or with the lactose-containing meal. Both molecules are biocompatible, can form gels in situ, and show sustained erosion kinetics in aqueous media. The complex was characterized by FTIR that evidenced an inclusion complex between the polyethylene oxide block and α-cyclodextrin. The release profiles of β-galactosidase from two different matrices (gels and tablets) of the in situ hydrogels have been obtained. The influence of the percentage of Tetronic in media of different pH was evaluated. No differences were observed regarding the release rate from the gel matrices at pH 6 (t50 = 105 min). However, in the case of the tablets, the kinetics were faster and they released a greater amount of 90R4 (25%, t50 = 40–50 min). Also, the amount of enzyme released was higher for mixtures with 25% Tetronic. Using suitable mathematical models, the corresponding kinetic parameters have been calculated. In all cases, the release data fit quite well to the Peppas–Sahlin model equation, indicating that the release of β-galactosidase is governed by a combination of diffusion and erosion processes. It has been observed that the diffusion mechanism prevails over erosion during the first 50 minutes, followed by continued release of the enzyme due to the disintegration of the matrix.

High-affinity phosphate/arsenate transport in white lupin (Lupinus albus) is relatively insensitive to phosphate status

The development of proteoid roots under phosphorus deficiency by white lupin (Lupinus albus) may result in increased arsenate uptake, as arsenate is a phosphate analogue. This, together with its high biomass production, rapid growth and ability to survive in soils with low phosphate and nitrogen contents, low pH and high metal contents make them an interesting species to investigate with respect to revegetation, and possibly also for long-term phytoremediation, of arsenic contaminated soils. Kinetic parameters for arsenate uptake for P-deficient and P-sufficient plants, as well as for proteoid and nonproteoid roots were obtained. Down-regulation of arsenate uptake by phosphate, as well as phosphate/arsenate competition for P-deficient and P-sufficient plants was studied. Arsenate uptake was reduced by phosphate, but small differences were found between P-deficient and P-sufficient plants. Arsenate uptake by proteoid roots was higher than for nonproteoid roots of P-deficient plants, with higher V_max and similar K_m values. Down-regulation of the high affinity phosphate/arsenate uptake system by phosphate does take place but seems to be slower than in other plants. This study suggests that the low sensitivity of the phosphate/arsenate uptake system to regulation by phosphate may be related to the adaptations of white lupin to low P available environments. Such adaptation are absent in plants unable to develop proteoid roots.

High-affinity NO3 --H+ cotransport in the fungus Neurospora:Induction and control by pH and membrane voltate

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO₃ ^- challenge and to quantify transport activity. The NO₃ ^--associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO₃ ^--free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μM NO₃ ^-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity mensurable approx. 100 min after first exposure to NO₃ ^-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO₃ ^- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 μM NO₃ ^-; and it was suppressed when NH₄ ⁺, was present during the first, inductive exposure to NO₃ ^-. Voltage clamp measurements carried out immediately before and following NO₃ ^- additions showed that the NO₃ ^--evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages -400 to +100 mV). Measurements of NO₃ ^- uptake using NO₃ ^--selective macroelectrodes indicated a charge stoichiometry for NO₃ ^- transport of 1(+):1(NO₃ ^-) with common K(m) and J(max) values around 25 μM and 75 pmol NO₃ ^- cm^-2sec^-1, respectively, and combined measurements of pH(o) and [NO₃ ^-](o) showed a net uptake of approx. 1 H⁺ with each NO₃ ^- anion. Analysis of the NO₃ ^- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pH(o) and [NO₃ ^-](o). Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH(o) of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO₃ ^-. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H⁺](o). These, and additional results are consistent with a charge-coupling stoichiometry of 2(H⁺) per NO anion transported across the membrane, and implicate a carrier cycle in which NO binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO₃ ^- transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO₃ ^- transport; finally, they distinguish metabolite repression of NO₃ ^- transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

Tyrosine phosphorylation and dephosphorylation in Burkholderia cenocepacia affect biofilm formation, growth under nutritional deprivation, and pathogenicity

Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important post-translational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF, and the low molecular weight protein tyrosine phosphatases BCAM0208, BceD and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208 and BceD contributed to biofilm formation, while BCAL2200 was required for growth in nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low molecular weight protein tyrosine phosphatases genes resulted in attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larvae infection model. Experimental evidence indicates that BCAM1331 displays a reduced
tyrosine autophosphorylation activity compared to BceF. Using the artificial substrate p-nitrophenyl phosphate, the phosphatase activity of the three low molecular weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 becomes tyrosine phosphorylated in vivo and catalyzes its auto-dephosphorylation. Together, our data suggest that despite having similar biochemical activities low molecular weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.

Optimising power take-off of an oscillating wave surge converter using high fidelity numerical simulations

Oscillating wave surge converters are a promising technology to harvest ocean wave energy in the near shore region. Although research has been going on for many years, the characteristics of the wave action on the structure and especially the phase relation between the driving force and wave quantities like velocity or surface elevation have not been investigated in detail. The main reason for this is the lack of suitable methods. Experimental investigations using tank tests do not give direct access to overall hydrodynamic loads, only damping torque of a power take off system can be measured directly. Non-linear computational fluid dynamics methods have only recently been applied in the research of this type of devices. This paper presents a new metric named wave torque, which is the total hydrodynamic torque minus the still water pitch stiffness at any given angle of rotation. Changes in characteristics of that metric over a wave cycle and for different power take off settings are investigated using computational fluid dynamics methods. Firstly, it is shown that linearised methods cannot predict optimum damping in typical operating states of OWSCs. We then present phase relationships between main kinetic parameters for different damping levels. Although the flap seems to operate close to resonance, as predicted by linear theory, no obvious condition defining optimum damping is found.

Kinetic Monte Carlo study of electrochemical growth in a heteroepitaxial system

Structural and kinetic aspects of 2-D irreversible metal deposition under potentiostatic conditions are analyzed by means of dynamic Monte Carlo simulations employing embedded atom potentials for a model system. Three limiting models, all considering adatom diffusion, were employed to describe adatom deposition. The first model (A) considers adatom deposition on any free substrate site on the surface at the same rate. The second model (B) considers adatom deposition only on substrate sites which exhibit no neighboring sites occupied by adatoms. The third model (C) allows deposition at higher rates on sites presenting neighboring sites occupied by adatoms. Under the proper conditions, the coverage (theta) versus time (t) relationship for the three cases can be heuristically fitted to the functional form theta = 1 - exp(-betat(alpha)), where alpha and beta are parameters. We suggest that the value of the parameter alpha can be employed to distinguish experimentally between the three cases. While model A trivially delivers a = 1, models B and C are characterized by alpha 1, respectively.

PHOTOMINERALIZATION OF SALICYLIC-ACID - A KINETIC-STUDY

The kinetics of the photomineralization of salicylic acid (SA) sensitized by Degussa P25 titanium dioxide (TiO2) dispersions in oxygenated aqueous solution are reported as a function of the following experimental parameters: [TiO2], percentage of O-2, [SA], temperature (T) and light intensity (I). The kinetics of SA photomineralization conform to a Langmuir-Hinshelwood kinetic scheme with SA and O-2 adsorbed at different sites with apparent Langmuir adsorption coefficients of (6.1 +/- 1.2) x 10(4) mol(-1) dm(3) and 0.061 +/- 0.007 kPa(-1) respectively. The overall activation energy for the system was determined as 4.6 +/- 0.2 kJ mol(-1). Two major stable reaction intermediates are identified (dihydroxybenzoic acids (DHBA) and catechol (C)) and the existence of a further pathway involving one or more very unstable and, as yet, unidentified reaction intermediates is proposed. A kinetic model is presented which describes the temporal behaviour of the concentrations of SA, CO2 and the major photogenerated intermediates (DHBA and C). This model is used to predict successfully the temporal behaviour of the major intermediates in the photomineralization of SA under non-standard conditions.

Optical and near-infrared coverage of SN 2004et: physical parameters and comparison with other Type IIP supernovae

We present new optical and near-infrared (NIR) photometry and spectroscopy of the Type IIP supernova (SN), SN 2004et. In combination with already published data, this provides one of the most complete studies of optical and NIR data for any Type IIP SN from just after explosion to +500 d. The contribution of the NIR flux to the bolometric light curve is estimated to increase from 15 per cent at explosion to around 50 per cent at the end of the plateau and then declines to 40 per cent at 300 d. SN 2004et is one of the most luminous IIP SNe which has been well studied and characterized, and with a luminosity of log L = 42.3 erg s-1 and a 56Ni mass of 0.06 +/- 0.04 M-circle dot, it is two times brighter than SN 1999em. We provide parametrized bolometric corrections as a function of time since explosion for SN 2004et and three other IIP SNe that have extensive optical and NIR data. These can be used as templates for future events in optical and NIR surveys without full wavelength coverage. We compare the physical parameters of SN 2004et with those of other well-studied IIP SNe and find that the kinetic energies span a range of 1050-1051 erg. We compare the ejected masses calculated from hydrodynamic models with the progenitor masses and limits derived from pre-discovery images. Some of the ejected mass estimates are significantly higher than the progenitor mass estimates, with SN 2004et showing perhaps the most serious mass discrepancy. With the current models, it appears difficult to reconcile 100 d plateau lengths and high expansion velocities with the low ejected masses of 5-6 M-circle dot implied from 7-8 M-circle dot progenitors. The nebular phase is studied using very late-time Hubble Space Telescope photometry, along with optical and NIR spectroscopy. The light curve shows a clear flattening at 600 d in the optical and the NIR, which is likely due to the ejecta impacting on circumstellar material. We further show that the [O i] 6300, 6364 A line strengths in the nebular spectra of four Type IIP SNe imply ejected oxygen masses of 0.5-1.5 M-circle dot.

A curvilinear approach to the kinetic analysis of linoleate peroxidation in aqueous liposomes by 2,2'azobis(2-amidoinopropane) dihydrochloride

Lipid peroxidation is a common feature of many chemical and biological processes, and is governed by a complex kinetic scheme. A fundamental stage in kinetic investigations of lipid peroxidation is the accurate determination of the rate of peroxidation, which in many instances is heavily reliant on the method of finite differences. Such numerical approximations of the first derivative are commonly employed in commercially available software, despite suffering from considerable inaccuracy due to rounding and truncation errors. As a simple solution to this, we applied three empirical sigmoid functions (viz. the Prout-Tompkins, Richards & Gompertz functions) to data obtained from the AAPH-mediated peroxidation of aqueous linoleate liposomes in the presence of increasing concentrations of Trolox, evaluating the curve fitting parameters using the widely available Microsoft Excel Solver add-in. We have demonstrated that the five-parameter Richards' function provides an excellent model for this peroxidation, and when applied to the determination of fundamental rate constants, produces results in keeping with those available in the literature. Overall, we present a series of equations, derived from the Richards' function, which enables direct evaluation of the kinetic measures of peroxidation. This procedure has applicability not only to investigations of lipid peroxidation, but to any system exhibiting sigmoid kinetics.