Removing heavy metals from synthetic effluents using “kamikaze” Saccharomyces cerevisiae cells

One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated waters. In this study, the heavy metal hypersensitive yeast strain pmr1∆ was investigated for the ability to remove Mn²⁺, Cu²⁺, Co²⁺, or Cd²⁺ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in removing Mn²⁺, Cu²⁺, or Co²⁺ from synthetic effluents containing 1–2 mM cations, with a selectivity $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} and also in removing Mn²⁺ and Cd²⁺ from synthetic effluents containing 20–50 μM cations, with a selectivity Mn²⁺ > Cd²⁺.     

Influence of {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ( \textM₇^\textIIMTs {\text{M}}_7^{\text{IIMTs}} ) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd₃-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd₃-thiolate cluster and the enfolding protein domain. Alterations in the \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological function.     

Mimosine Mitigates Oxidative Stress in Selenium Deficient Seedlings of Vigna radiata. Part II: Mitochondrial Uptake of 75Selenium and Mimosine

During the growth of selenium (Se)-deficient seedlings of Vigna radiata, exposure to mimosine [2-amino-3-(3-hydroxy-4-oxo-1H-pyridin-1-yl)-propanoic acid], a nonprotein plant amino acid, effectively mitigated stress at 0.1 mM, as reflected in enhancement of growth and efficiency of mitochondrial functions. Since the changes in the seedlings elicited by exposure to mimosine were similar to those effected by Se at an optimal exposure level of 0.75 ppm (Sreekala et al., Biol Trace Elem Res 70:193–207, 1999), the uptake of Se and that of mimosine itself was individually studied in the respiring mitochondria of Se-deficient seedlings (−Se-stressed group) in comparison with those exposed to mimosine during growth at 0.1 mM (Mim 0.1 group). In both groups, the mitochondrial uptake of ⁷⁵Se at 10 μM added increased linearly up to 2 min, attaining steady-state levels thereafter. Uptake levels were 2.3-fold higher in the Mim 0.1 group than in the −Se-stressed group. Double-reciprocal plots of mitochondrial ⁷⁵Se uptake against 2–20 μM in the medium were nonlinear and negative cooperative effects during the uptake were confirmed by Scatchard plots, whereas Hill coefficients were 0.8 and 0.85 for the two groups. Mitochondrial uptake of mimosine, at added levels of 25 or 50 μM, increased linearly up to 1 min and decelerated thereafter. Initial uptake levels of mimosine at 1 min were higher by 6.5-fold at 25 μM and 4-fold at 50 μM in the Mim 0.1 group than those in the −Se-stressed group. Initial uptake levels with added mimosine up to 50 or 100 μM yielded nonlinear double-reciprocal plots; and kinetic analyses at 5 to 50 μM revealed the prevalence of positive cooperativity in the −Se-stressed group and negative cooperativity in the Mim 0.1 group. Involvement of active thiol groups in the uptake of both Se and mimosine were indicated by inhibition studies. Evidence presented for mimosine mediated increase in mitochondrial Se uptake and cooperative interactions thereof underscores the metabolic significance of mimosine.     

Comparative study of effects of cadmium cations in free and chelated forms on activity of glutathione S-transferase, growth, and endocytosis in culture of the infusoriumTetrahymena pyriformis

Effects of cadmium cations in free (Cd²⁺) and chelated with EDTA (Cd²⁺-EDTA) forms were studied on growth, endocytosis, and activity of glutathione S-transferase (GT) in the free-living infusoriaTetrahymena pyriformis. It is shown that the cytotoxicity of Cd²⁺ in the free form at a concentration of 10 μM is much higher than of the Cd²⁺-EDTA complex at the equimolar concentration. Even at a low concentration (2 μM), Cd²⁺ produces an inhibition of the growth rate and endocytosis in theT. pyriformis culture, while the Cd²⁺-EDTA complex suppresses these functions insignificantly. Cd²⁺ in the free form at concentrations of 10 and 100 μM reduced activity of glutathione S-transferase by 39 and 61%. The chelated Cd²⁺-EDTA complex at these concentrations inhibited the GT activity by 5 and 55%, respectively.     

An alternative non-cytochrome containing branch in the respiratory system of free-living Rhizobium phaseoli

The existence of a NADH oxidase catalyzed by a non-cytochrome containing pathway in membranes of free-living Rhizobium phaseoli was explored. This alternative electron transport route was distinguished from the cytochrome-oxidase linked pathway by its low affinity towards O₂ (K , higher K _m for NADH (75 μM), a hundred-fold lower sensitivity to quinarine inhibition, and resistance to UV (360 nm) photoinactivation. In addition to NADH, tetramethyl-p-phenylenediamine (TMPD) donates electrons to this low-O₂ affinity pathway, causing reduction bleaching of a flavoprotein absorption band at 455 nm. Ascorbate-TMPD dependent respiration was partially (25%) inhibited by 200 μM quinacrine. The low O₂-affinity oxidase activity promoted by NADH, or ascorbate plus TMPD was present in aerobic and microaerophilic grown cells and absent in anaerobic and bacteroid cells. Thus, a NADH linked flavoprotein type oxidase is suggested.     

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 μM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 μM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 μM but decreased at 50 μM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O₂ ^•−) accumulation, and H₂O₂ content at 5 and 10 μM Cu-treated roots were not increased but strongly increased at 50 μM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 μM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 μM Cu, but both G-POD and CAT activities were inhibited at 50 μM Cu. Glutathione metabolism enzymes such as γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 μM Cu but were strongly inhibited at 50 μM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 μM Cu was associated to the strong induction of γ-glutamyltranspeptidase (γ-GGT) activity. These results indicate that plant could grow under Cu stress (5–25 μM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.     

Soil temperature and biotic factors drive the seasonal variation of soil respiration in a maize (Zea mays L.) agricultural ecosystem

The diurnal and seasonal variation of soil respiration (SR) and their driving environmental factors were studied in a maize ecosystem during the growing season 2005. The diurnal variation of SR showed asymmetric patterns, with the minimum occurring around early morning and the maximum around 13:00 h. SR fluctuated greatly during the growing season. The mean SR rate was 3.16 μmol CO₂ m⁻² s⁻¹, with a maximum of 4.87 μmol CO₂ m⁻² s⁻¹ on July 28 and a minimum of 1.32 μmol CO₂ m⁻² s⁻¹ on May 4. During the diurnal variation of SR, there was a significant exponential relationship between SR and soil temperature (T) at 10 cm depth: . At a seasonal scale, the coefficient α and β fluctuated because the biomass (B) increased α, and the net primary productivity (NPP) of maize markedly increased β of the exponential equation. Based on this, we developed the equation to estimate the magnitude of SR and to simulate its temporal variation during the growth season of maize. Most of the temporal variability (93%) in SR could be explained by the variations in soil temperature, biomass and NPP of maize. This model clearly demonstrated that soil temperature, biomass and NPP of maize combined to drive the seasonal variation of SR during the growing season. However, only taking into account the influence of soil temperature on SR, an exponential equation over- or underestimated the magnitude of SR and resulted in an erroneous representation of the seasonal variation in SR. Our results highlighted the importance of biotic factors for the estimation of SR during the growing season. It is suggested that the models of SR on agricultural sites should not only take into account the influence of soil temperature, but also incorporate biotic factors as they affect SR during the growing season.     

Reactive oxygen species production and antioxidative defense system in pea root tissues treated with lead ions: the whole roots level

The lead absorbed by the roots induce oxidative stress conditions through the Reactive oxygen species (ROS) production for the pea plants cultivated hydroponically for 96 h on a Hoagland medium with the addition of 0.1 and 0.5 mM of Pb(NO₃)₂. The alterations in \textO₂ ^{- ·} {\text{O}}_{2}^{ - \cdot } and H₂O₂ concentrations were monitored spectrophotometrically which show a rapid increase in \textO₂ ^{- ·} {\text{O}}_{2}^{ - \cdot } production during the initial 2 h, and in case of H₂O₂, during the eighth hour of cultivation. The level of ROS remained higher at all the time points for the roots of the plants cultivated with Pb²⁺ and it was proportional to metal concentration. The production of \textO₂ ^{- ·} {\text{O}}_{2}^{ - \cdot } and H₂O₂ was visualized by means of fluorescence microscope technique. They are produced in nonenzymatic membrane lipid peroxidation and its final product is Malondialdehyde, the level of which increased together with the level of H₂O₂. As stress intensity raised (duration of treatment and Pb²⁺ concentration), so did the activities of superoxide dismutases, catalase and ascorbate peroxidase antioxidative enzymes and of low-molecular antioxidants, particularly glutathione (GSH), homoglutathione (h-GSH) and cysteine substrate toward their synthesis. The root cells redox state (GSH/GSSG) dropped proportionally to lead stress intensity.     

A quantitative appraisal of the ambivalent metal ion binding properties of cytidine in aqueous solution and an estimation of the<Emphasis Type="Italic"> anti</Emphasis>–<Emphasis Type="Italic">syn</Emphasis> energy barrier of cytidine derivatives

The recently defined versus straight-line plots for L = pyridine-type (PyN) and ortho-aminopyridine-type (oPyN) ligands now allow the evaluation in a quantitative manner of the stability of the 1:1 complexes formed between cytidine (Cyd) and Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Cd²⁺ (M²⁺); the corresponding stability constants, , including the acidity constant, , for the deprotonation of the (N3)H⁺ site had been determined previously under exactly the same conditions as the mentioned plots. Since the stabilities of the M(PyN)²⁺ and M(oPyN)²⁺ complexes of Ca²⁺ and Mg²⁺ are practically identical, it is concluded that complex formation occurs in an outer-sphere manner, and this is in accord with the fact that in the pK_a range 3–7 metal ion binding is independent of or . Ca(Cyd)²⁺ and Mg(Cyd)²⁺ are more stable than the corresponding (outer-sphere) M(PyN)²⁺ complexes and this means that the C2 carbonyl group of Cyd must participate, next to N3 which is most likely outer-sphere, in metal ion binding, leading thus to chelates; these have formation degrees of about 50% and 35%, respectively. Co(Cyd)²⁺ and Ni(Cyd)²⁺ show no increased stability based on the hence, the (C2)O group does not participate in metal ion binding, but the inner-sphere coordination to N3 is strongly inhibited by the (C4)NH₂ group. In the M(Cyd)²⁺ complexes of Mn²⁺, Cu²⁺, Zn²⁺ and Cd²⁺, this inhibiting effect on M²⁺ binding at N3 is partially compensated by participation of the (C2)O group in complex formation and the corresponding chelates have formation degrees between about 30% (Zn²⁺) and 83% (Cu²⁺). The different structures of the mentioned chelates are discussed in relation to available crystal structure analyses. (1) There is evidence (crystal structure studies: Cu²⁺, Zn²⁺, Cd²⁺) that four-membered rings form, i.e. there is a strong M²⁺ bond to N3 and a weak one to (C2)O. (2) By hydrogen bond formation to (C2)O of a metal ion-bound water molecule, six-membered rings, so-called semichelates, may form. (3) For Ca²⁺ and Mg²⁺, and possibly Mn²⁺, and their Cyd complexes, six-membered chelates are also likely with (C2)O being inner-sphere (crystal structure) and N3 outer-sphere. (4) Finally, for these metal ions also complexes with a sole outer-sphere interaction may occur. All these types of chelates are expected to be in equilibrium with each other in solution, but, depending on the metal ion, either the one or the other form will dominate. Clearly, the cytidine residue is an ambivalent binding site which adjusts well to the requirements of the metal ion to be bound and this observation is of relevance for single-stranded nucleic acids and their interactions with metal ions. In addition, the anti–syn energy barrier has been estimated as being in the order of 6–7.5 kJ/mol for cytidine derivatives in aqueous solution at 25 °C.Abbreviations ADP^3– adenosine 5-diphosphate - AMP^2– adenosine 5-monophosphate - ATP^4– adenosine 5-triphosphate - CDP^3– cytidine 5-diphosphate - cl closed - CMP^2– cytidine 5-monophosphate - 3-CMP^2– cytidine 3-monophosphate - CTP^4– cytidine 5-triphosphate - Cyd cytidine - DNA deoxyribonucleic acid - I ionic strength - K_a acidity constant - K_I intramolecular equilibrium constant - L general ligand - M²⁺ general divalent metal ion - NTP^4– nucleoside 5-triphosphate - op open - oPyN ortho-aminopyridine-type ligand - PyN pyridine-type ligand - t-RNA transfer ribonucleic acid - Tu tubercidin (7-deazaadenosine)In honor of Professor Liang-Nian Ji on the occasion of his 70th birthday in friendship and with best wishes.     

Stress response in two strains of the aquatic hyphomycete <Emphasis Type="BoldItalic">Heliscus lugdunensis</Emphasis> after exposure to cadmium and copper ions

Biochemical responses to cadmium (Cd²⁺) and copper (Cu²⁺) exposure were compared in two strains of the aquatic hyphomycete (AQH) Heliscus lugdunensis. One strain (H4-2-4) had been isolated from a heavy metal polluted site, the other (H8-2-1) from a moderately polluted habitat. Conidia of the two strains differed in shape and size. Intracellular accumulation of Cd²⁺ and Cu²⁺ was lower in H4-2-4 than in H8-2-1. Both␣strains synthesized significantly more glutathione (GSH), cysteine (Cys) and γ-glutamylcysteine (γ-EC) in the presence of 25 and 50 μM Cd²⁺, but quantities and rates of synthesis were different. In H4-2-4, exposure to 50 μM Cd²⁺ increased GSH levels to 262% of the control; in H8-2-1 it increased to 156%. Mycelia of the two strains were analysed for peroxidase, dehydroascorbate reductase, glutathione reductase and glucose-6-phosphate dehydrogenase. With Cd²⁺ exposure, peroxidase activity increased in both strains. Cu²⁺ stress increased dehydroascorbate reductase activity in H4-2-4 but not in H8-2-1. Dehydroascorbate reductase and glucose-6-phosphate dehydrogenase activities progressively declined in the presence of Cd²⁺, indicating a correlation with Cd²⁺ accumulation in both strains. Cd²⁺ and Cu²⁺ exposure decreased glutathione reductase activity.     

Different characteristics of roots in the cadmium-tolerance and Cd-binding complex formation between mono- and dicotyledonous plants

Effects of Cd²⁺ on growth and Cd-binding complex formation in roots were examined with various seedlings of mono- and dicotyledonous plants. Maize, oat, barley and rice exhibited the greater tolerance to Cd²⁺ (100 μM) than did azuki bean, cucumber, lettuce, pea, radish, sesame and tomato (10–30 μM). Azuki bean was the most sensitive to Cd²⁺ (<10 μM). Under these Cd-treatments, cereal roots accumulated Cd²⁺ in the cytoplasmic fractions and transported Cd²⁺ into the same fractions of shoot tissues, to larger extents than did dicotyledonous roots. Cereal roots synthesized a Cd-binding complex containing phytochelatins in the cytoplasmic fractions, depending upon Cd²⁺ concentrations applied (30–100 μM). Such a complex was not detected from the same fractions of dicotyledonous roots treated with Cd²⁺. These results suggest that the Cd-binding complex formation has an important role in the tolerance of cereal roots against Cd²⁺.     

A comparative study of antioxidative defense system in the copper and temperature acclimated strains of <Emphasis Type="Italic">Anabaena doliolum</Emphasis>

This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu²⁺ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu²⁺ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu²⁺ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu²⁺ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu²⁺ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu²⁺ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu²⁺ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu²⁺ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu²⁺ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu²⁺ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu²⁺ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu²⁺ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Hydrochemical fluctuations and crustacean community composition in an ephemeral saline lake (Sua Pan,Makgadikgadi Botswana)

Fluctuating hydrochemistry, as a result of extreme hydrological regimes, imposes major physiological constraints on the biota of ephemeral saline lakes. While the inverse relationship between salinity and zooplankton species richness is well-known across salinity gradients, few studies have documented closely the response of zooplankton to seasonal changes in salinity. Weekly sampling during two flood seasons at Sua Pan, an intermittent saline lake in central Botswana demonstrated the importance of spatial and temporal salinity gradients for crustacean community composition, associated with a decline in species richness, from 11 to three species. Conductivity ranged between 320 and 125,800 μS cm⁻¹ during seasonal flooding; changing from dominance by and , Ca²⁺ and Mg²⁺, at the beginning of the floods, to NaCl dominated waters as the lake dried out and salinities increased. pH estimates generally ranged between 8.6 and 10, with maximum values recorded during initial flooding. Crustaceans comprised mainly Branchinella spinosa, Moina belli, Lovenula africana and Limnocythere tudoranceai, all of which occurred across a wide range of salinities, while halotolerant freshwater species (Metadiaptomus transvaalensis, Leptestheria striatochonca and the ostracods Plesiocypridopsis aldabrae, Cypridopsis newtoni and a newly identified Potamocypris species) disappeared above conductivities of 1,500 μS cm⁻¹. A unique crustacean composition in southern Africa was attributed to Sua Pans’ rare chemical composition among southern African saline lakes; flood waters on Sua Pan contained a higher proportion of Na⁺ and , and less K⁺, Mg²⁺ and than over 80% of records from salt pans elsewhere in southern African. The freshwater species of crustaceans in Sua Pan were similar to those found in other southern Africa lakes, and these similarities decreased in lakes with higher pH and proportions of Na, and less SO₄ and Mg in their chemical composition. The predominant saline tolerant species on Sua Pan, however, showed a greater similarity to those in saline lakes in southern and East Africa with higher proportions of and, particularly, Mg²⁺ in their chemical composition. Handling editor: J. M. Melack     

Energetics of the growth of Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus on ammonium chloride and dinitrogen

Methanobacterium thermoautotrophicum was grown in continuous culture in a fermenter gassed with H₂ and CO₂ as sole carbon and energy sources, and in a medium which contained either NH₄Cl or gaseous N₂ as nitrogen source. Growth was possible with N₂. Steady states were obtained at various gas flow rates with NH₄Cl and with and the maintenance coefficient varied with the gas input and with the nitrogen source. Growth of Methanococcus thermolithotrophicus in continuous culture in a fermenter gassed with H₂, CO₂ as nitrogen, carbon and energy sources was also examined.Abbreviations molecular growth yield (g dry weight of cells per mol of CH₄ evolved) - growth rate (h^-1) - D dilution rate (h^-1) - rate (h_-1); relation of Neijssel and Tempest and of Stouthamer and Bettenhaussen - energy     

Growth yields and saturation constant of Desulfovibrio vulgaris in chemostat culture

Desulfovibrio vulgaris (strain Marburg) was grown on H₂ and sulfate as sole energy source in a chemostat limited by the sulfate supply. The biomass concentration and the sulfate concentration in the culture were determined as a function of the dilution rate. From the data a K _S (saturation constant) for sulfate of 10 M, a _max of 0.23 h^–1, and a of 13 g/mol were calculated. The organism was also grown in chemostat culture on H₂ and sulfite, H₂ and thiosulfate, and pyruvate (without sulfate). was found to be 35 g/mol, 36 g/mol, and Y _pyr ^max 10 g/mol. The growth yields are discussed with respect to ATP gains in dissimilatory sulfate reduction.     

Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain <Emphasis Type="Italic">Verticillium</Emphasis> sp. P9

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg²⁺and Br⁻ ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn²⁺, Cu²⁺, K⁺, Cd²⁺, Ag⁺, Fe³⁺, Mn²⁺, Co²⁺, Hg²⁺, Pb²⁺ and Sn²⁺), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E _a of these reactions and temperature dependence (at 0–30°C) of k _cat, k _cat/K _m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k _cat and k _cat/K _m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Minimal Functional Unit of Lactate Dehydrogenase

The tetrameric heart isozyme of lactate dehydrogenase (H₄) is modified by p-chloromercuribenzoate (PCMB) to produce the inactive tetramer and then hybridized with native tetrameric muscle isozyme (M₄). The hybrid mixture was isolated by polyacrylamide gel electrophoresis (PAGE) and then stained for enzyme activity and with Coomassie brilliant blue. Only three bands were found on the gels in either case. The hybrid enzymes as isolated by PAGE have half the specific activity of the native muscle enzyme. The electrophoresis properties of HM₃ are very similar to those of HM₃, while the electrophoresis properties of are very similar to those of H₂M₂. The above results strongly suggest that the tetramer having enzymatic activity contains at least two native subunits, and the di-subunit in the tetrameric enzyme is the minimal functional unit.     

Characterization of <Emphasis Type="Italic">Deinococcus radiophilus</Emphasis> thioredoxin reductase active with both NADH and NADPH

Thioredoxin reductase (TrxR, EC 1.6.4.5) of Deinococcus radiophilus was purified by steps of sonication, ammonium sulfate fractionation, 2′5′ ADP Sepharose 4B affinity chromatography, and Sephadex G-100 gel filtration. The purified TrxR, which was active with both NADPH and NADH, gave a 368 U/mg protein of specific activity with 478-fold purification and 18% recovery from the cell-free extract. An isoelectric point of the purified enzymes was ca. 4.5. The molecular weights of the purified TrxR estimated by PAGE and gel filtration were about 63.1 and 72.2 kDa, respectively. The molecular mass of a TrxR subunit is 37 kDa. This suggests that TrxR definitely belongs to low molecular weight TrxR (L-TrxR). The Km and Vmax of TrxR for NADPH are 12.5 μM and 25 μM/min, whereas those for NADH are 30.2 μM and 192 μM/min. The Km and Vmax for 5, 5′-dithio-bis-2-nitrobenzoic acid (DTNB, a substituted substrate for thioredoxin) are 463 μM and 756 μM/min, respectively. The presence of FAD in TrxR was confirmed with the absorbance peaks at 385 and 460 nm. The purified TrxR was quite stable from pH 3 to 9, and was thermo-stable up to 70°C. TrxR activity was drastically reduced (ca. 70%) by Cu²⁺, Zn²⁺, Hg²⁺, and Cd²⁺, but moderately reduced (ca. 50%) by Ag⁺. A significant inhibition of TrxR by N-ethylmaleimide suggests an occurrence of cysteine at its active sites. Amino acid sequences at the N-terminus of purified TrxR are H₂N-Ser-Glu-Gln-Ala-Gln-Met-Tyr-Asp-Val-Ile-Ile-Val-Gly-Gly-Gly-Pro-Ala-Gly-Leu-Thr-Ala-COOH. These sequences show high similarity with TrxRs reported in Archaea, such as Methanosarcina mazei, Archaeoglobus fulgidus etc.