NaCl-free Solution Research Articles

Enhanced salt-tolerance of Bacillus subtilis glutaminase by fusing self-assembling amphipathic peptides at its N-terminus.

Glutaminase (EC 3.5.1.2) can catalyze the deamidation of glutamine, which has been used to improve umami taste in oriental fermented foods. However, a high salt concentration is still a fundamental challenge for glutaminase application, especially in soy sauce production. To improve the salt tolerance of glutaminase, the self-assembling amphiphilic peptides EAK16 and ELK16 were fused to the N-terminus of a mutant (E3C/E55F/D213T) derived from Bacillus subtilis glutaminase, yielding the fusion enzymes EAK16-E3C/E55F/D213T and ELK16-E3C/E55F/D213T, respectively. As ELK16-E3C/E55F/D213T was expressed as insoluble active inclusion bodies, only the purified EAK16-E3C/E55F/D213T was subjected to further analyses. After the incubation with 18% (w/v) NaCl for 200 min, the residual activities of EAK16-E3C/E55F/D213T in a NaCl-free solution reached 43.6%, while E3C/E55F/D213T was completely inactivated. When the enzyme reaction was conducted in the presence of 20% NaCl, the relative activity of EAK16-E3C/E55F/D213T was 0.47-fold higher than that of E3C/E55F/D213T. As protein surface hydrophobicity and protein particle size analysis suggested, oligomerization may play an important role in the salt-tolerance enhancement of the fusions. Furthermore, EAK16-E3C/E55F/D213T achieved a 0.88-fold increase in the titer of glutamic acid in a model system of soy sauce fermentation compared to E3C/E55F/D213T. Therefore, the fusion with self-assembling amphiphilic peptides is an efficient strategy to improve the salt-tolerance of glutaminase.

Effects of α2-adrenoceptor stimulation on luminal alkalinisation and net fluid flux in rat duodenum.

The sympathetic nervous system is highly involved in the regulation of gastrointestinal functions such as luminal alkalinisation and fluid absorption. However, the exact mechanisms are not clear. This study aimed to delineate how α2-adrenergic receptor stimulation reduces duodenal luminal alkalinisation and induces net fluid absorption. This was tested by perfusing the duodenum of anesthetized rats with isotonic solutions devoid of Cl- and/or Na+, in the absence and presence of the α2-adrenoceptor agonist clonidine. The clonidine was also studied in rats treated with dimethylamiloride (a Na+/H+ exchange inhibitor), vasoactive intestinal peptide, and the nicotinic receptor antagonist hexamethonium. Clonidine reduced luminal alkalinisation and induced net fluid absorption. The Cl--free solution decreased luminal alkalinisation and abolished net fluid absorption, but did not prevent clonidine from doing so. Both the Na+-free solution and luminal dimethylamiloride increased luminal alkalinisation and abolished net fluid absorption, effects counteracted by clonidine. The NaCl-free solution (D-mannitol) did not affect luminal alkalinisation, but reduced net fluid absorption. Clonidine reduced luminal alkalinisation and induced net fluid absorption in rats perfused luminally with mannitol. However, clonidine did not affect the vasoactive intestinal peptide-induced increase in luminal alkalinisation or fluid secretion. Pre-treatment with hexamethonium abolished the effects of clonidine on luminal alkalinisation and net fluid flux. In summary, our in vivo experiments showed that clonidine-induced reduction in luminal alkalinisation and induction of net fluid absorption was unrelated to luminal Na+ and Cl-, or to apical Na+/H+ or Cl-/HCO3- exchangers. Instead, clonidine seems to exert its effects via suppression of nicotinic receptor-activated acetylcholine secretomotor neurons.

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Abstract Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K m (15.6260 g/L) and the rate constant of deactivation, K de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.

Thermal Inactivation Kinetics and Secondary Structure Change of a Low Molecular Weight Halostable Exoglucanase from a Marine Aspergillus niger at High Salinities

Two kinds of exoglucanase were purified from a marine Aspergillus niger. Catalytic ability of halophilic exoglucanase with a lower molecular weight and secondary structure change was analyzed at different salinities. Activity of the low molecular weight exoglucanase in 10% NaCl solution (w/v) was 1.69-fold higher of that in NaCl-free solution. Half-life time in 10% NaCl solution (w/v) was over 1.27-fold longer of that in NaCl-free solution. Free energy change of the low molecular weight exoglucanase denaturation, △G, in 10% NaCl solution (w/v) was 0.54kJ/mol more than that in NaCl-free solution. Melt point in 10% NaCl solution (w/v), 52.01°C, was 4.21°C higher than that in NaCl-free solution, 47.80°C. K m value, 0.179mg/ml in 10% NaCl solution (w/v) was less 0.044mg/ml than that, 0.224mg/ml, in NaCl-free solution. High salinity made content of α-helix increased. Secondary structure change caused by high salinities improved exoglucanase thermostability and catalysis activity. The halophilic exoglucanase from a marine A. niger was valuable for hydrolyzing cellulose at high salinities.

Halostable catalytic properties of exoglucanase from a marine Aspergillus niger and secondary structure change caused by high salinities

Abstract Catalytic properties of halophilic exoglucanase from a marine Aspergillus niger and the secondary structure change caused by high salinity was analyzed under high salinities condition. Exoglucanase activity in 12% NaCl solution (w/v) was 1.32 folds higher of that in NaCl free solution. Half-life time in 12% NaCl solution (w/v) was over 1.41 folds higher of that in NaCl free solution. The exoglucanase had not endoglucanase activity. Gibbs free energy change of exoglucanase denaturation, △ G , in 12% NaCl solution (w/v) was about 1.0 kJ/mol more than that in NaCl free solution at 45 °C, 50 °C, 55 °C and 60 °C. Melt temperature in 12% NaCl solution (w/v), 48.02 °C, was 3.32 °C higher than that in NaCl free solution, 44.70 °C. High salinity made contents of β-sheet and α-helix increased. The secondary structure change caused by high salinities improved exoglucanase thermostability and activity. The halostable exoglucanase from a marine Aspergillus niger was valuable for cellulose hydrolysis at high salinities.

Enhancing the activity and thermostability of thermostable β-glucosidase from a marine Aspergillus niger at high salinity

To evaluate the effect of salinity on the catalyzing ability of β-glucosidase in the marine fungus Aspergillus niger, the thermodynamic parameters of the β-glucosidase were investigated at different salinities. At the optimum salinity of 6% NaCl (w/v) solution, the optimum temperature and pH of the β-glucosidase activity was 66°C and 5.0, respectively. Under these conditions, the β-glucosidase activity increased 1.46 fold. The half-life of denaturation in 6% NaCl (w/v) solution was approximately twice as long as that in NaCl free solution. The Gibb's free energy for denaturation, ΔG, was 2kJ/mol higher in 6% NaCl (w/v) solution than in NaCl free solution. The melting point (68.51°C) in 6% NaCl (w/v) solution was 1.71°C higher than that (66.80°C) in NaCl free solution. Similarly, the activity and thermostability of the pure β-glucosidase increased remarkably at high salinity. The thermostable β-glucosidase, of which the activity and the thermostability are remarkably enhanced at high salinity, is valuable for industrial hydrolyzation of cellulose in high salinity environments.

Responses of photosynthesis to NaCl in gametophytes of Acrostichum aureum.

Gametophytes of Acrostichum aureum were cultured in 0.0 to 1.0% NaCl solutions or in NaCl-free solution and then transferred to 1.0% NaCl solution. Photosynthetic light-response curves, efficiency of the primary photochemical reaction, relative electron transport rate, and photochemical and non-photochemical quenching at steady state were determined by photosynthetic O2 evolution and in vivo chlorophyll fluorescence. Results obtained showed that the chlorophyll fluorescence parameters, Fv /Fm and F'v /F'm and αO2 (the initial linear slope of the photosynthetic light-response curve) increased in gametophytes grown in NaCl. Linear electron transport rate was stimulated by NaCl. Based on the chlorophyll content, light-saturated photosynthesis in gametophytes grown in 0.2 to 0.7% NaCl increased slightly; it decreased in gametophytes grown in 1.0% NaCl. Photochemical quenching decreased in NaCl-grown gametophytes at all photosynthetic photon flux density (PPFD) levels measured, but there was no increase in non-photochemical quenching. The chlorophyll a/b ratio increased with increasing NaCl concentration in culture solutions. These results indicated that NaCl enhanced photochemical efficiency of photosystem II (PSII) and photosynthetic linear electron transport, thus resulting in the development of an excitation pressure in PSII. Such excitation pressure might act as a signal for photosynthetic acclimation to salt stress, thus allowing the gametophytes to grow in their natural habitats.

電気Zn-Fe合金めっき皮膜のアルカリ性水溶液中における電気化学的挙動

As a basis to elucidate the mechanism of underfilm corrosion of Zn-Fe alloy electroplated steel, the electrochemical behavior of electrodeposited Zn-Fe alloys was investigated in alkaline solution of pH ranging from 9.5 to 13.6.(1)In 5%NaCl solution of pH=9.5 and 11.7, low corrosion current density was observed in the range of low Fe content in the alloy due to the suppression action of oxygen reduction reaction caused by the initial oxide film formed on the alloys, which was observed also in neutral 5%NaCl solution.(2)In 5%NaCl solution of pH=13.6, the corrosion current density tended to increase with increasing Fe content in the alloy in the range of 50% or less Fe content and it significantly increased compared with that in the solution of pH=9.5 and 11.7 in the same Fe content range as above, presumably resulting from the change in both anodic and cathodic reactions.(3)In NaCl-free solution of pH=12, electrodeposited Zn and Zn-Fe alloys were passivated, thus leading to anodic control of corrosion. Furthermore, the corrosion current density decreased with increasing Fe content in the alloy in the solution. It was suggested that the decrease in the corrosion current density resulted from reinforcement of the surface film by iron in the alloys and passivation of iron in the alloys.

Nicardipine actions on smooth muscle cells and neuromuscular transmission in the guinea pig basilar artery.

The effects of nicardipine on smooth muscle cells of the guinea pig basilar artery were investigated by means of microelectrode and isometric tension recording methods. Nicardipine (1×10−8−3×10−6M) did not modify the membrane potential and resistance of smooth muscle cells. The spike evoked by application of outward current pulse in the presence of tetraethylammonium (>1×10−3M) was inhibited by 1x10−9M and was almost blocked by 3×10−7M nicardipine. Perivascular nerve stimulation evoked the excitatory junction potential which was slightly suppressed by 3×10−6M nicardipine. The contraction evoked by excess concentration of [K], NaCl-free solution or ATP was abolished and by 5-hydroxytryptamine (5-HTJ was markedly inhibited in Ca-free EGTA containing solution, but that induced by caffeine was only slightly inhibited. Nicardipine (>3×10−10M) markedly inhibited the K-induced contraction. The ATP induced contractions were inhibited by nicardipine (>1×10−8M) to a lesser extent than the K-induced contractions. On the other hand, nicardipine (1×10−6M) had no effect on the NaCl-free- or 5-HT-induced contraction. When nicardipine (1×10−6M) was applied during 2.5mM Ca loading to muscle cells after depletion of the stored Ca, the subsequently generated caffeine-induced contraction was slightly depressed due to inhibition of the passive Ca influx. These results indicate that nicardipine possesses a selective inhibitory action on the Ca channel, but that the inhibition is limited to particular Ca influxes such as the voltage dependent ones, but not the receptor operated and NaCl-free induced Ca influxes. This agent acts predominantly on the post-junctional muscle rather than on the prejunctional nerve terminal.

Diltiazem-induced vasodilatation of smooth muscle cells of the canine basilar artery.

1 The effects of diltiazem on smooth muscle cells of the canine basilar artery were investigated by means of microelectrode, double sucrose gap and isometric tension recording methods. 2 The mean membrane potential of the smooth muscle cells was -49.8 mV and they were electrically quiescent. Diltiazem (over 10(-5) M) depolarized the membrane. After pretreatment with 5 and 10 mM tetraethylammonium (TEA), an outward current pulse (1 and 2 s in duration) produced a spike and this spike was abolished by application of 10(-5) M diltiazem. 3 The spike could also be generated by the excitatory junction potential (e.j.p.) evoked by perivascular nerve stimulation (0.05 ms in pulse duration) in the presence of 5 mM TEA. Diltiazem (greater than 10(-6) M) suppressed both the spike and the e.j.ps, the suppression being more apparent for spike generation. The amplitude of the e.j.ps was reduced by diltiazem in concentrations greater than 10(-6) M. The effects were dose-dependent: when the amplitude of e.j.ps was reduced by application of diltiazem, the resulting mechanical response was also proportionally smaller. 4 The contractions evoked by 128 mM [K]o, 10(-3) M adenosine 5'-triphosphate (ATP) or, NaCl-free solution were abolished in Ca-free solutions containing 2 mM EGTA, but the amplitude of caffeine-induced contraction (10 mM) was only slightly reduced. Diltiazem, in concentrations above 3 X 10(-7) M suppressed the contraction evoked by excess [K]o, ATP or caffeine, but the inhibitory action of diltiazem on the K-induced contraction was greatest. 5 Following pretreatment with 2.5 mM [Ca]o, a contraction was evoked by caffeine in Ca-free solution. The amplitude of the caffeine-induced contraction was increased by simultaneous application of 2.5 mM [Ca]o with 128 mM [K]o and to a lesser extent by simultaneous application of 2.5 mM [Ca]o with 5.9 mM [K]o. The amplitude of the caffeine-induced contraction generated in the presence of 5.9 mM or 128 mM [K]o was suppressed to the same extent by application of diltiazem [10(-5) M) during preincubation in [Ca]o. This result suggests that the Ca stored in cell is replenished by Ca-influx from [Ca]o during the resting and active states of the membrane, and that diltiazem has no effect on the mobilization of Ca stored in the cell. 6 Thus, diltiazem acts on the canine basilar artery suppressing the Ca-influx during the active condition as a Ca-spike suppressor and the voltage-dependent Ca-influx induced by excess [K]o or by chemical depolarization. Diltiazem has no effect on the Ca mobilization from the store site. This agent also suppresses the amplitude of e.j.ps due to inhibition of the release of chemical transmitter from nerve terminals following the suppression of the Ca-influx. Diltiazem appears to act as a vasodilator on the canine basilar artery.

Effects of sodium depletion on contractions evoked in intact and skinned muscles of the guinea-pig mesenteric artery.

In the guinea-pig mesenteric artery, reduction in [Na]o by 30 mM (substituted by choline or sucrose; 137 mM [Na]o in Krebs solution) generated contraction with no change in membrane potential. In NaCl-free solution (15 mM [Na]o), the amplitude of phasic contraction reached 0.8 times the contraction evoked by 118 mM [K]o with only a slight depolarization. In NaCl-free solution, the amplitude of phasic contraction evoked by noradrenaline (NA) 5 X 10(-5) M or caffeine 5 mM increased to roughly twice the amplitude of the contraction evoked in the control solution. In Ca-free solution, the K-, NaCl-free- or Na-free-induced contractions rapidly ceased, but NA-induced contraction ceased within 5 min and the caffeine-induced contraction persisted for more than 15 min. In a skinned fiber, increase of [Na]o from 10 to 60 mM suppressed the pCa-tension relationship in the ranges of 10(-7) and 10(-5) M free Ca but not with a dose of 30 mM [Na]o. NA (10(-5) M) had no effect on skinned fibers. Increase in Na concentration (60 mM) had no effect on Ca accumulation in the store site or on Ca release by caffeine. Possible Na-related mechanisms on the development of mechanical response are discussed in relation to Ca on the surface and in the internal membrane structure. The NaCl-free-induced contraction in smooth muscles of the guinea-pig mesenteric artery is postulated to be due to influx of Ca through the Na channel, rather than the Ca channel.

Luminal Na+ entry into Necturus proximal tubule cells

The dependence of intracellular Na+ activity on the electrical driving force across the luminal membrane and the presence of Cl- in the luminal perfusate was studied in Necturus proximal tubule. Intracellular Na+ and K+ activities were measured with microelectrodes filled with liquid ion exchanger. Perfusion of the tubule lumen with a NaCl-free solution caused cell Na+ activity to fall from the control value of 29.7 to 6.6 mM. In the absence of luminal driving force across the luminal membrane in accordance with simple diffusion of Na+ across this membrane. When the tubule lumen contained Na+ and Cl-, an electrically neutral component of Na+ entry into cells from the lumen appeared in addition to the diffusional component of Na+ entry.

Dependence of tRNA structure in solution upon ionic condition of the solvent. Fluorescence studies of Mg2+ binding to tRNAPhe from barley embryos.

Barley phenylalanine tRNA (tRNAphe) exhibits a 4.7‐fold enhancement of fluorescence intensity in response to the addition of MgCl2 in 0.01 M Tris‐HCl, pH 7.5, 0.001 M Na2 EDTA. In the case of 2.0 M NaCl about 60% of this effect was observed. The change of fluorescence intensity was used as a measure of bound ligand concentration. From fluorescence Scatchard plots a different character of Mg2+ binding to tRNA was revealed, depending upon the initial conformation of tRNA in solution submitted to titration. In NaCl‐free tRNAphe solution strong interacting (Ks= 1.5 × 105 M−1n= 2.4) and weak independent (Kw= 9 x 103 M−1) Mg2+ binding were observed. In solution containing 0.1 M NaCl both the strong and the weak Mg2+ binding demonstrated independent behaviour. Ks and Kw, in the presence of NaCl, had lower values than in NaCl‐free solution, since Na+ was found to be a competitive inhibitor of Mg2+ binding. Fluorescence titrations of tRNAphe solutions containing Mg2+ evidenced 2 Na+ binding sites/Mg2+ binding site (at least in the region of weak sites of Mg2+) characterized by inhibitory binding constants K̄i1 Na= 45 M−1 and K̄i 1 Na= 8.5 M−1 (mean values) for first and second Na+ bindings respectively. Na+ exerts a destabilizing effect on tRNA conformation induced by Mg2+. A model of Mg2+ binding and concomitant conformational changes of the tRNA molecule is discussed: one class of Mg2+ binding sites in unfolded tRNA and two classes of sites, strong and weak, in folded conformation are postulated.

CAT HEART MUSCLE IN VITRO. IV. INHIBITION OF TRANSPORT IN QUIESCENT MUSCLES.

Cellular concentrations, [K](i), [Na](i), and [Cl](i), and cell water contents were measured in vitro at 27 degrees C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O(2), and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2-3 degrees C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K](i), was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K](i) remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O(2), or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.