Two-tank suspended growth process for accelerating the detoxification kinetics of hydrocarbons requiring initial monooxygenation reactions

An experimental evaluation demonstrated that suspended growth systems operated in a two-tank accelerator/aerator configuration significantly increased the overall removal rates for phenol and 2,4-dichlorophenol (2,4-DCP), aromatic hydrocarbons that require initial monooxygenations. The accelerator tank is a small volume that receives the influent and recycled biomass. It has a high ratio of electrondonor (BOD) to electron acceptor (O₂). Biomass in the accelerator should be enriched in reduced nicotinamide adenine dinucleotide (NADH + H⁺) and have a very high specific growth rate, conditions that should accelerate the kinetics of monooxygenation reactions. For the more slowly degraded 2,4-DCP, the average percentage removal increased from 74% to 93%, even though the volume of the two-tank system was smaller than that of the one-tank system in most experiments. The average volumetric and biomass-specific removal rates increased by 50% and 100%, respectively, in the two-tank system, compared to a one-tank system. The greatest enhancement in 2,4-DCP removal occurred when the accelerator tank comprised approximately 20% of the system volume. Biomass in the accelerator tank was significantly enriched in NADH + H⁺ when its dissolved oxygen (DO) concentration was below 0.25 mg/L, a situation having a high ratio of donor to acceptor. The accelerator biomass had its highest NADH + H⁺ content for the experiments that had the highest rate of 2,4-DCP removal. Biomass in the accelerator also had a much higher specific growth rate than in the aerator or the system overall, and the specific growth rate in the accelerator was inverselycorrelated to the accelerator volume.     

A detailed analysis of the mechanisms controlling the acceleration of 2,4-DCP monooxygenation in the two-tank suspended growth process

The mechanisms underlying the observed acceleration of monooxygenationreactions in two-tank accelerator/aerator suspended growth system are evaluatedin detail. The accelerator tank is characterized by a very high electron flow throughreduced nicotinamide adenine dinucleotide (NADH + H⁺), particularly when the retention-time ratio is small. Only a small fraction of the electron flow wasdiverted to oxygenation reactions, and the major sinks of NADH + H⁺ were respiration and biomass synthesis. The main producer of NADH + H⁺ is oxidation of acetate, a rapidly degraded electron-donor substrate. The half-maximum-rate concentration for oxygen used in respiration was 0.03 mg/L, while the half-maximum-rate concentration for oxygen used as a cosubstrate in monooxygenation was 0.18 mg/L. Thus, monooxygenations were more sensitive to oxygen limitation than was respiration. The NADH + H⁺ concentration had a direct effect on the monooxygenation kinetics. Therate coefficients for both monooxygenation reactions were directly proportional to thespecific growth rate in the accelerator, which supports that the accelerator tank causedan up-regulation of the monooxygenase content. Because the rate coefficients in theaerator tank were much larger than in the one-tank system, even though the specificgrowth rates were nearly the same, monooxygenases may have carried over from theaccelerator tank to the aerator tank. Its higher concentration of 2,4-dichlorophenol(2,4-DCP) and the higher specific growth rate were the main reasons why the accelerator had faster kinetics for 2,4-DCP utilization than did the aerator tank. The apparently higher levels of monooxygenase in both tanks of the two-tank system also appears be a primary reason why its performance was substantially superior to that of the one-tank system in terms of 2,4-DCP removal.     

Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

Sodium ion-dependent hydrogen production in Acidaminococcus fermentans

Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO₂, acetate, butyrate, and H₂. The molecular hydrogen (approximately 10 kPa; E′ = –385 mV) stems from NADH generated in the 3-hydroxybutyryl-CoA dehydrogenase reaction (E°′ = –240 mV) of the hydroxyglutarate pathway. In contrast to growing cells, which require at least 5 mM Na⁺, a Na⁺-dependence of the H₂-formation was observed with washed cells. Whereas the optimal glutamate fermentation rate was achieved already at 1 mM Na⁺, H₂ formation commenced only at > 10 mM Na⁺ and reached maximum rates at 100 mM Na⁺. The acetate/butyrate ratio thereby increased from 2.0 at 1 mM Na⁺ to 3.0 at 100 mM Na⁺. A hydrogenase and an NADH dehydrogenase, both of which were detected in membrane fractions, are components of a model in which electrons, generated by NADH oxidation inside of the cytoplasmic membrane, reduce protons outside of the cytoplasmic membrane. The entire process can be driven by decarboxylation of glutaconyl-CoA, which consumes the protons released by NADH oxidation inside the cell. Hydrogen production commences exactly at those Na⁺ concentrations at which the electrogenic H⁺/Na⁺-antiporter glutaconyl-CoA decarboxylase is converted into a Na⁺/Na⁺ exchanger. Received: 3 May 1996 / Accepted: 12 August 1996     

Formate Hydrogenlyase Is Needed for Proton-Potassium Exchange Through the F0F1-ATPase and the TrkA System in Anaerobically Grown and Glycolysing Escherichia coli

Anaerobically grown and glycolysing Escherichia coli produced H₂ and carried out H⁺-K⁺-exchange in two steps, the first of which had the fixed stoichiometry for DCCD-sensitive fluxes (2H⁺/K⁺), and the second one had a variable stoichiometry for DCCD-sensitive fluxes. H₂ production and the 2H⁺/K⁺-exchange were lost in the ΔfdhF or ΔhycA-H mutant. In the ΔfdhF mutant, H⁺-K⁺-exchange with K _m for K⁺-uptake of 2.3 mM and less K⁺-gradient between the cytoplasm and the medium were observed. H₂ production and H⁺-K⁺-exchange with a high K _m for K⁺-uptake were carried out in the uncD mutant; however, both H₂ production and H⁺-K⁺-exchange were lost in the Δunc or uncE mutant. H₂ production was observed in the trkA trkD kdpA mutant. It was displayed in protoplasts with increased membrane permeability when donor or acceptor of reducing equivalents—formate with DTT or NADH respectively—was added. The F₀F₁ and the TrkA(H) or the F₀ and the TrkA(G) had been assumed to form the united supercomplexes, functioning as a H⁺-K⁺-pump or antiporter respectively (for review see Bioelectrochem Bioenerg 33:1, 1994). Results allow the proposal that H₂ production by FHL has a relationship with the H⁺-K⁺-exchange through a H⁺-K⁺-pump and via an H⁺-K⁺-antiporter. Formate and NADH can serve as a donor and an acceptor of reducing equivalent respectively, for operation of such supercomplexes. Received: 12 December 1996 / Accepted: 19 March 1997     

Evidence for Involvement of a Zymogen Granule Na+/H+ Exchanger in Enzyme Secretion from Rat Pancreatic Acinar Cells

We have characterized a Na⁺/H⁺ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na⁺/H⁺ exchanger activity was estimated by measuring Na⁺ or Li⁺ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na⁺- or Li⁺-dependent ZG lysis was enhanced by increasing inward to outward directed H⁺ gradients. Na⁺-dependent ZG lysis was not prevented by an inside-positive K⁺ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na⁺ and H⁺ permeabilities and for coupled Na⁺/H⁺ exchange through an electroneutral carrier. Na⁺- and Li⁺-dependent ZG lysis was inhibited by EIPA (EC₅₀∼25 μm) and benzamil (EC₅₀∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na⁺ and Li⁺ salts only, and were inhibited by EIPA, suggesting the presence of a Na⁺/H⁺ exchanger in the membrane. Na⁺ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na⁺/H⁺ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na⁺ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na⁺ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na⁺ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P < 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na⁺ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P < 0.02). In the absence of Na⁺ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na⁺/H⁺ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini. Received: 7 December 1995/Revised: 2 April 1996     

Copper modifies the activity of sodium-transporting systems in erythrocyte membrane in patients with essential hypertension

The aim of the study was to verify the hypothesis if copper could influence the activity of sodium-transporting systems in erythrocyte membrane that could be related to essential hypertension. The examined group of patients consisted of 15 men with hypertension. The control group was 11 healthy male volunteers. The Na⁺/H⁺ exchanger (NHE) activity in erythrocytes was determined according to Orlov et al. The activity of transporting systems (ATP-Na⁺/K⁺; co-Na⁺/K⁺/Cl⁻; ex-Na⁺/Li⁺; free Na⁺ and K⁺ outflow [Na⁺, K⁺-outflow]) was determined according to Garay's method. The concentration of copper in plasma was assessed using atomic absorption spectrometry. The activity of ATP-Na⁺/K⁺ (μmol/L red blood cells [RBCs]/h) in hypertensive patients was 2231.5±657.6 vs 1750.5±291 in the control (p<0.05), the activity of co-Na⁺/K⁺/Cl⁻ (μmol/L RBCs/h) in hypertensives was 171.3±77.9 vs 150.7±53.9 in the control (NS). Na⁺-outflow (μmol/L RBCs/h) in hypertensives was 118.3±51.6 vs 113.3±24.4 in the control (NS). The K⁺-outflow (μmol/L RBCs/h) in hypertensives was 1361.7±545.4 vs 1035.6±188.3 in the control (NS). The activity of ex-Na⁺/Li⁺ (μmol/L RBCs/h) in hypertensive patients was 266.1±76.1 vs 204.1±71.6 in the control (p<0.05). NHE activity (mmol/L RBCs/h) in hypertensives was 9.7±2.96 vs 7.7±1.33 in the control (p<0.05). In hypertensive patients, negative correlation was found between the activity of Na⁺/K⁺/Cl⁻ co-transport and plasma copper concentration (R _s=−0.579, p <0.05) and between the activity of ex-Na⁺/Li⁺ and plasma copper concentration (R _s=−0.508, p<0.05). Plasma copper concentration significantly influences the activity of sodium transporting systems in erythrocyte membrane. Copper supplementation could be expected to provide therapeutic benefits for hypertensive patients.     

Energization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> membrane vesicles increases catalytic activity of succinate: Menaquinone oxidoreductase

In this work, high ΔμH⁺-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells. Deenergization of the membrane vesicles with ionophores or alamethicin brings about an almost complete inhibition of succinate oxidation. An apparent K _m for succinate during the energy-dependent succinate oxidase activity of the vesicles (2.2 mM) is higher by an order of magnitude than the K _m value measured for the energy-independent reduction of 2,6-dichlorophenol indophenol. The data reveal critical importance of ΔμH⁺ for maintaining active electron transfer by succinate:menaquinone oxidoreductase. The role of ΔμH⁺ might consist in providing energy for thermodynamically unfavorable menaquinone reduction by succinate by virtue of transmembrane electron transport within the enzyme down the electric field; alternatively, ΔμH⁺ could play a regulatory role by maintaining the electroneutrally operating enzyme in a catalytically active conformation.     

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD⁺ to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K _d of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD⁺ (e.g., K _d of 87 μM for FurX-NAD⁺). The kinetic data suggest that the four enzymes are efficient “furfural reductases” with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°′) for ethanol-dependent reduction of furfural was determined to be −1.1 kJ mol⁻¹. The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.     

Kinetic Parameters and Lactate Dehydrogenase Isozyme Activities Support Possible Lactate Utilization by Neurons

Lactate is potentially a major energy source in brain, particularly following hypoxia/ischemia; however, the regulation of brain lactate metabolism is not well understood. Lactate dehydrogenase (LDH) isozymes in cytosol from primary cultures of neurons and astrocytes, and freshly isolated synaptic terminals (synaptosomes) from adult rat brain were separated by electrophoresis, visualized with an activity-based stain, and quantified. The activity and kinetics of LDH were determined in the same preparations. In synaptosomes, the forward reaction (pyruvate + NADH + H⁺ → lactate + NAD⁺), which had a V _max of 1,163 μmol/min/mg protein was 62% of the rate in astrocyte cytoplasm. In contrast, the reverse reaction (lactate + NAD⁺ → pyruvate + NADH + H⁺), which had a V _max of 268 μmol/min/mg protein was 237% of the rate in astrocytes. Although the relative distribution was different, all five isozymes of LDH were present in synaptosomes and primary cultures of cortical neurons and astrocytes from rat brain. LDH1 was 14.1% of the isozyme in synaptic terminals, but only 2.6% and 2.4% in neurons and astrocytes, respectively. LDH5 was considerably lower in synaptic terminals than in neurons and astrocytes, representing 20.4%, 37.3% and 34.8% of the isozyme in these preparations, respectively. The distribution of LDH isozymes in primary cultures of cortical neurons does not directly reflect the kinetics of LDH and the capacity for lactate oxidation. However, the kinetics of LDH in brain are consistent with the possible release of lactate by astrocytes and oxidative use of lactate for energy in synaptic terminals. Special issue dedicated to John P. Blass.     

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.     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000     

Evaluation of ammonium and phosphate release from intertidal and subtidal sediments of a shallow coastal lagoon (Ria Formosa – Portugal): a modelling approach

During an annual cycle, overlying water and sediment cores were collected simultaneously at three sites (Tavira, Culatra and Ramalhete) of Ria Formosa’s intertidal muddy and subtidal sandy sediments to determine ammonium, nitrates plus nitrites and phosphate. Organic carbon, nitrogen and phosphorus were also determined in superficial sediments. Ammonium and phosphate dissolved in porewater were positively correlated with temperature (P < 0.01) in muddy and sandy sediments, while the nitrogen-oxidized forms had a negative correlation (P < 0.02) in muddy sediments probably because mineralization and nitrification/denitrification processes vary seasonally. Porewater ammonium profiles evidenced a peak in the top-most muddy sediment (380 μM) suggesting higher mineralization rate when oxygen is more available, while maximum phosphate concentration (113 μM) occurred in the sub-oxic layer probably due to phosphorus desorption under reduced conditions. In organically poor subtidal sandy sediments, nutrient porewater concentrations were always lower than in intertidal muddy sediments, ranging annually from 20 μM to 100 μM for ammonium and from 0.05 μM to 16 μM for phosphate. Nutrient diffusive fluxes predicted by a mathematical model were higher during summer, in both muddy (104 nmol cm⁻² d⁻¹––NH₄⁺; 8 nmol cm⁻² d⁻¹––HPO₄⁻²) and sandy sediments (26 nmol cm⁻² d⁻¹––NH₄⁺; 1 nmol cm⁻² d⁻¹––HPO₄⁻²), while during lower temperature periods these fluxes were 3–4 times lower. Based on simulated nutrient effluxes, the estimated annual amount of ammonium and phosphate exported from intertidal areas was three times higher than that released from subtidal areas (22 ton year⁻¹––NH₄⁺; 2 ton year⁻¹––HPO₄⁻²), emphasizing the importance of tidal flats to maintain the high productivity of the lagoon. Global warming scenarios simulated with the model, revealed that an increase in lagoon water temperature only produces significant variations (P < 0.05) for NH₄⁺ in porewater and consequent diffusive fluxes, what will probably affect the system productivity due to a N/P ratio unbalance.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H⁺-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H⁺-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V _max: 3.5 μmol mg⁻¹ min⁻¹, K _m for ATP: 67 μM, K _m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca²⁺ concentrations (K _d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H⁺-ATPase in a Ca²⁺-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998     

Concurrent and Independent HCO− 3 and Cl− Secretion in a Human Pancreatic Duct Cell Line (CAPAN-1)

The present study investigated both HCO⁻ ₃ and Cl⁻ secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (I _sc ) technique. In Cl⁻/HCO⁻ ₃-containing solution, secretin (1 μm) or forskolin (10 μm) stimulated a biphasic rise in the I _sc which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external Cl⁻ abolished the initial transient phase in the forskolin-induced I _sc while the plateau remained. In HCO⁻ ₃/CO₂-free solution, on the contrary, only the initial transient increase in I _sc was prominent. Summation of the current magnitudes observed in Cl⁻-free and HCO⁻ ₃-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the current observed in Cl⁻/HCO⁻ ₃-containing solution. Removal of external Na⁺ greatly reduced the initial transient rise in the forskolin-induced I _sc response, and the plateau level observed under this condition was similar to that obtained in Cl⁻-free solution, suggesting that Cl⁻-dependent I _sc was also Na⁺-dependent. Bumetanide (50 μm), an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, and Ba²⁺ (1 mm), a K⁺ channel blocker, could reduce the forskolin-induced I _sc obtained in Cl⁻/HCO⁻ ₃-containing or HCO⁻ ₃-free solution. However, they were found to be ineffective when external Cl⁻ was removed, indicating the involvement of these mechanisms in Cl⁻ secretion. On the contrary, the HCO⁻ ₃-dependent (in the absence of external Cl⁻) forskolin-induced I _sc could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 μm). Basolateral application of amiloride (100 μm) inhibited the I _sc ; however, a specific Na⁺-H⁺ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5–10 μm) was found to be ineffective, excluding the involvement of the Na⁺-H⁺ exchanger. However, an inhibitor of H⁺-ATPase, N-ethylmaleimide did suppress the I _sc (IC₅₀= 22 μm). Immunohistochemical studies also confirmed the presence of a vacuolar type of H⁺-ATPase in these cells. H₂DIDS (100 μm), an inhibitor of Na⁺-HCO⁻ ₃ cotransporter, was without effect. Apical addition of Cl⁻ channel blocker, diphenylamine-2,2′-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 μm) or SITS (100 μm), exerted an inhibitory effect on both Cl⁻ and HCO⁻ ₃-dependent forskolin-induced I _sc responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting that HCO⁻ ₃ secretion may be specialized to a certain population of cells. The present results suggest that both HCO⁻ ₃ and Cl⁻ secretion by the human pancreatic duct cells may occur concurrently and independently. Received: 17 October 1997/Revised: 3 April 1998     

The H+ Pump in Frog Skin (Rana esculenta): Identification and Localization of a V-ATPase

We here report on studies on the frog skin epithelium to identify the nature of its excretory H⁺ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A₁ (10 μm) blocked H⁺ excretion (69 ± 8% inhibition) and therefore Na⁺ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na₂SO₄ solution, ``low-Na<sup>+</sup> conditions' under which H<sup>+</sup> and Na<sup>+</sup> fluxes are coupled 1:1. The electrogenic outward H<sup>+</sup> current measured in absence of Na<sup>+</sup> transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A<sub>1</sub> or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na<sup>+</sup> transport (short-circuit current), which develops with apical solutions containing 115 mm Na<sup>+</sup> (``high-Na⁺ conditions'), demonstrating a specific action on H⁺ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H⁺ excretion is mediated by a V-ATPase being the electrogenic H⁺ pump in frog skin. Received: 21 May 1996/Revised: 24 December 1996     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.