Effects of competition and N and P supply on carbon isotope discrimination and <Superscript>15</Superscript>N-natural abundance in four grassland species

The effect of interspecific competition and element additions (N and P) on four grassland species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale) grown under field conditions was studied. Two grasses (L. perenne, F. valida) grown in monoculture (absence of competition) showed lower carbon isotope discrimination (¹³C) and enriched ¹⁵N values. Nitrogen addition (as urea) had inconsistent effects on species ¹³C while caused enrichment of ¹⁵N of P. pratensis and F. valida but strong depletion of ¹⁵N of T. officinale. Phosphorous had no significant effect on ¹³C but depleted ¹⁵N of all species.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Immunolocalization of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in the branchial cavity during the early development of the crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> (Crustacea,Decapoda)

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium–potassium adenosine triphosphatase (Na⁺,K⁺-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430–450 m at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgG5 raised against the avian -subunit of the Na⁺,K⁺-ATPase. Immunoreactivity staining, indicating the presence of Na⁺, K⁺-ATPase appeared in the gills of late embryos (EI400 m), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410–420 m and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na⁺,K⁺-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na⁺active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.     

Effect of Extracellular pH on Presteady-State and Steady-State Current Mediated bythe Na<Superscript>+</Superscript>/K<Superscript>+</Superscript> Pump

A ouabain sensitive inward current occurs in Xenopus oocytes in Na⁺ and K⁺ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pH_o) produces a conducting pathway through the Na⁺/K⁺ pump that is permeable to H⁺ and blocked by [Na⁺]_o. An alternative suggestion is that the current is mediated by an electrogenic H⁺-ATPase. Here we investigate the effect of pH_o and [Na⁺]_o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pH_o the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pH_o, as predicted by a model in which protonation of the Na⁺/K⁺ pump reduces the energy barrier between the internal solution and the Na⁺ occluded state. The model also predicts that acidic pH_o increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. ²²Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pH_o suggest that both Na⁺ and H⁺ are permeant. The acid-induced inward current is reduced by high [Na⁺]_o, consistent with block by Na⁺. A least squares analysis predicts that H⁺ is four orders of magnitude more permeant than Na⁺, and that block occurs when 3 Na⁺ ions occupy a low affinity binding site (K _0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na⁺ and H⁺ through the Na⁺/K⁺ pump.     

Use of foliar <Superscript>15</Superscript>N and <Superscript>13</Superscript>C abundance to evaluate effects of microbiotic crust on nitrogen and water utilization in <Emphasis Type="Italic">Pinus massoniana</Emphasis> in deteriorated pine stands of south China

We compared the foliar ¹⁵N and ¹³C values of Pinus massoniana growing on soils with and without microbiotic crust to examine the influence of the microbiotic crust on N and water use in plants in deteriorated watersheds in southern China. At our study site, litterfall and undergrowth had been intensively removed for fuel and soil N concentration was extremely low. Microbiotic crust covered the lower slope within the watersheds and pine trees were taller here than on the middle and upper slopes, although the crust reduced the amount of rainfall that could penetrate the soil. The foliar ¹⁵N values were greater (closer to zero) in pine trees growing on soil covered with microbiotic crust on the lower slope than on the middle and upper slopes, which lacked the microbiotic crust. These data suggest that P.massoniana may depend on N fixed by the microbiotic crust on the lower slope, and on N carried by precipitation on the middle and upper slopes. The microbiotic crust did not influence foliar ¹³C, an index for water use efficiency, in P.massoniana. The fact that P.massoniana biomass was greater on the lower slope, which is less permeable to rainfall, suggests that P.massoniana growth may be limited by the amount of available N rather than by water. The microbiotic crust may improve plant productivity by increasing N availability, despite its negative effect on water availability.     

Valine inhibition of β-isopropylmalate dehydrogenase takes part in the regulation of leucine biosynthesis inCandida maltosa

The -isopropylmalate (IPM) dehydrogenase (EC 1.1.1.85) ofCandida maltosa, the third pathway-specific enzyme of leucine biosynthesis, was purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K_m values of the enzyme were estimated to be 0.42 mM for -IPM and 0.34 mM for NAD⁺. It is demonstrated that the enzyme can be regulated by L-valine. The inhibition was competitive with respect to -IPM (K_i=1.84 mM) and non-competitive with respect to NAD⁺ (K_i=5.67 mM). Exogenous addition of L-valine toC. maltosa cells increased the intracellular pool of some intermediates of leucine biosynthesis (-ketoisovalerate, -IPM, -IPM), but has hardly influence on the leucine pool.     

Functional identification of ATP-sensitive K<Superscript>+</Superscript> uniporter in mitochondria from sugar beet taproot

Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K⁺-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K⁺-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K⁺-dependent swelling of organelles in potassium acetate solutions. The presence of K⁺ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential () that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of . The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K⁺ uniporter. This system is likely to represent a K⁺ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K⁺/H⁺ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K⁺ in the presence of potassium acetate.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 209–215.Original Russian Text Copyright © 2005 by Shugaev, Andreev, Vyskrebentseva.This revised version was published online in April 2005 with a corrected cover date.     

Dual Effect of Isoprostanes on the Release of [<Superscript>3</Superscript>H]D-Aspartate from Isolated Bovine Retinae: Role of Arachidonic Acid Metabolites

The effect of 8-isoprostanes on potassium (K⁺)-depolarization-evoked release of [³H]D-aspartate from bovine isolated retinae was investigated. Isolated bovine retinae were prepared for studies of K⁺-evoked release of [³H]D-aspartate using the Superfusion Method. Low concentrations of 8-isoPGF₂(1–100 nM) inhibited whereas higher concentrations of this 8-isoprostane (100 nM–30 M) enhanced K⁺-induced [³H]D-aspartate overflow. The excitatory effect of 8-isoPGF₂ was mimicked by thromboxane receptor agonist, U-46619 and blocked by thromboxane receptor antagonist, SQ 29,548 (10 M). Pretreatment of tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen unmasked an inhibitory effect of high concentrations of 8-isoPGF₂(1–30 M) on [³H]D-aspartate release that was attenuated by AH 6809 (10 M). In conclusion, 8-isoPGF₂ exhibits a dual regulatory effect on K⁺-induced [³H]D-aspartate release in isolated bovine retinae. The inhibitory action caused by 8-isoPGF ₂ is due to the activation of EP₁/EP₂ receptors while the excitatory effects are due to the activation of thromboxane receptors.     

Study of the involvement of osmotic adjustment and H<Superscript>+</Superscript>-ATPase activity in the resistance of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> suspension cells to salt stress

The salt-induced H⁺-ATPase activity and osmotic adjustment responses of Catharanthus roseus (L.) G. Don suspension cultures were studied. Cells were treated with 0, 50 or 100mM NaCl for 7days or were maintained for 8 months with 50 mM NaCl (50T cells). Growth, osmotic potential (), ions content, soluble sugars, proline and total amino acids were determined in the sap of control and salt-treated cells. Salinity reduced cell growth and . The higher decrease in the in salt-treated cells was due to higher accumulation of Na⁺ and Cl^–. The levels of organic solutes, such as soluble sugars, free proline and total amino acids, increased with salt treatment. These results suggest that salt-tolerant cells are able to osmotically adjust. Salinity treatments stimulated H⁺-ATPase activity. Immunodetection of the enzyme showed that the increased activity was due to an increased amount of protein in the plasmalemma. The induction by NaCl, especially at 100 mM NaCl and for 50T cells, could account for the K⁺ and Cl^– uptake but not for higher or lower tolerance.     

Inward-rectifier K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Current in Guinea-pig Ventricular Myocytes Exposed to Hyperosmotic Solutions

Superfusion of heart cells with hyperosmotic solution causes cell shrinkage and inhibition of membrane ionic currents, including delayed-rectifer K⁺ currents. To determine whether osmotic shrinkage also inhibits inwardly-rectifying K⁺ current (I_K1), guinea-pig ventricular myocytes in the perforated-patch or ruptured-patch configuration were superfused with a Tyrodes solution whose osmolarity (T) relative to isosmotic (1T) solution was increased to 1.3–2.2T by addition of sucrose. Hyperosmotic superfusate caused a rapid shrinkage that was accompanied by a negative shift in the reversal potential of Ba²⁺-sensitive I_K1, an increase in the amplitude of outward I_K1, and a steepening of the slope of the inward I_K1-voltage (V) relation. The magnitude of these effects increased with external osmolarity. To evaluate the underlying changes in chord conductance (G_K1) and rectification, G_K1-V data were fitted with Boltzmann functions to determine maximal G_K1 (G_K1max) and voltage at one-half G_K1max (V_0.5). Superfusion with hyperosmotic sucrose solutions led to significant increases in G_K1max (e.g., 28±2% with 1.8T), and significant negative shifts in V_0.5 (e.g., –6.7±0.6 mV with 1.8T). Data from myocytes investigated under hyperosmotic conditions that do not induce shrinkage indicate that G_K1max and V_0.5 were insensitive to hyperosmotic stress per se but sensitive to elevation of intracellular K⁺. We conclude that the effects of hyperosmotic sucrose solutions on I_K1 are related to shrinkage-induced concentrating of intracellular K⁺.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Purification and characterization of mouse glucose 6-phosphate dehydrogenase

Summary Glucose-6-phosphate dehydrogenase was purified to homogeneity from testes and kidneys of the inbred strain of mice (DBA/2J) by a simple two-step affinity column procedure. This involved the sequential application of 8-(6-aminohexyl)-amino-AMP-and -2, 5-ADP-Sepharose columns and biospecific elution with NADP⁺ in both steps. The molecular and biochemical properties of the purified enzyme were studied in detail. These include the molecular weight determination, amino acid composition, steady-state kinetics, inactivation by high temperature, urea and iodoacetate, and immunology. The purified enzyme from mouse kidneys or testes was shown to be a tetramer with a molecular weight of 220,000. The enzyme is highly specific for glucose-6-phosphate, exhibits almost no activity with NAD⁺ as a coenzyme and is little inhibited by AMP or ATP. Michaelis constants for glucose-6-phosphate and NADP⁺ were determined to be 50 m and 10 m respectively. NADPH is a competitive inhibitor of NADP⁺ and has a K_i of 18 µm. Rabbit antisera against glucose-6-phosphate dehydrogenase were raised. The antisera also cross-react with the same enzyme from human and guinea pig.     

Identification of Ferredoxin-Dependent Cyclic Electron Transport around Photosystem I Using the Kinetics of Dark P700<Superscript>+</Superscript> Reduction

Kinetic curves of absorbance changes induced by far-red light (FR, 830 nm) (A ₈₃₀), which reflect redox transformations of PSI primary electron donor, P700, were examined in intact barley (Hordeum vulgare L.) leaves. In intact leaves, FR induced the biphasic increase in absorbance related to P700 photooxidation. Leaf treatment with methyl viologen or antimycin A suppressed the slow phase of P700 photooxidation, which was attained in such leaves within the first second of light exposure. With FR turned off, the previously increased absorbance at 830 nm dropped down to its initial level, thus reflecting P700⁺ reduction. In the control leaves, the kinetics of P700⁺ reduction consisted of three exponentially decaying components, with the corresponding half-times of 8.8 s (the slow component, with its magnitude comprising 24% of the total A ₈₃₀ signal), 0.73 s (the middle component, 49% of A ₈₃₀), and 0.092 s (the fast component, 26% of A ₈₃₀). The rate of the fast component of P700⁺ reduction, following FR irradiation of leaves, was about ten times lower than that of the noncyclic electron transfer from PSII to PSI computed from A ₈₃₀ relaxation after the abrupt offset of white light. The treatment of leaves with methyl viologen or antimycin A completely abolished the fast component of A ₈₃₀ relaxation after FR exposure. It was concluded that the fast component is determined by the operation of ferredoxin-dependent cyclic electron transport around PSI. This study represents the first report on the identification of this pathway of electron transport in vivo and the estimation of its rate.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 325–330.Original Russian Text Copyright © 2005 by Bukhov, Egorova.     

Evidence for the adaptive significance of enzyme activity levels: Interspecific variation in α-GPDH and ADH in Drosophila

The activity levels of alcohol dehydrogenase and -glycerophosphate dehydrogenase were compared among nine species of Drosophila representing three phylogenetic groups. For any given life stage, interspecific variability in activity level was much greater for ADH than for -GPDH. Patterns of ontogenetic expression of enzyme activity were also much more variable among species for ADH than for -GPDH. These results are consistent with the interpretation that -GPDH is involved with a relatively uniform adaptive function among species, whereas ADH levels may reflect variable adaptive capabilities. There is a significant correlation between ADH activities and survivorship on alcohol-treated media for these nine species.This research was supported by Contract AT(04-3)-34 200 with ERDA. The authors are supported by an NIH training grant in genetics.     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

Essential role of the small subunit of thermostable glucose dehydrogenase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The co-expression in Escherichia coli of the -subunit and the catalytic -subunit of the thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 produced 12.7 U GDH activity mg^–1 protein. A 47-amino acid, twin-arginine translocase signal peptide was identified at the amino terminus of the -subunit. The expression of the -subunit in the absence of the -subunit or the -subunit signal peptide failed to produce any detectable GDH protein or activity. The -subunit may be a chaperone-like component that assists folding of the -subunit polypeptide to the active form and its translocation to the periplasm.     

<Superscript>1</Superscript>H and <Superscript>15</Superscript>N resonance assignment,secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6

E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data. Recently, we described the expression and purification of E6-C 4C/4S, a stable, folded mutant of the C-terminal domain of HPV16 E6. Here, we have produced ¹⁵N-labelled samples of E6-C 4C/4S for structural studies by NMR. We have assigned most ¹H and ¹⁵N resonances and identified the elements of secondary structure of the domain. The domain displays an original / topology with roughly equal proportions of -helix and -sheet. The PDZ-binding region of E6, located at the extreme C-terminus of the domain, is in a random conformation. Mass spectrometry demonstrated the presence of one zinc ion per protein molecule. Kinetics of replacement of zinc by cadmium followed by ¹H,¹⁵N-HSQC experiments revealed specific frequency changes for the zinc-binding cysteines and their immediate neighbours. NMR spectra were affected by severe line-broadening effects which seriously hindered the assignment work. Investigation of these effects by ¹⁵N relaxation experiments showed that they are due to heterogeneous dynamic behaviour with s–ms time scale motions occurring in localised regions of the monomeric domain.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

External Ni<Superscript>2+</Superscript> and ENaC in A6 Cells: Na<Superscript>+</Superscript> Current Stimulation by Competition at a Binding Site for Amiloride and Na<Superscript>+</Superscript>

In cultured A6 monolayers from distal Xenopus kidney, external Ni²⁺ stimulated active Na⁺ uptake via the epithelial Na⁺ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni²⁺ effect. Na⁺ current noise analysis was performed using the neutral Na⁺-channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni²⁺ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni²⁺ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni²⁺ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na⁺—known to compete with amiloride and so producing the self-inhibition phenomenon—and Ni²⁺ exerted mutually exclusive analogous effects on amiloride kinetics. Na⁺ current kinetics revealed that Ni²⁺ prevents ENaC to be downregulated by self-inhibition. Co²⁺ behaved similarly to Ni²⁺, whereas Zn²⁺ did not. Attempts to disclose the chemical nature of the site reacting with Ni²⁺ suggested cysteine but not histidine as reaction partner.