Modulation of water stress effects on photosynthesis by altered leaf k

Wheat irrigated with nutrient solutions containing 0, 0.2, 0.5, 1, 2, or 6 millimolar K⁺ had maximum photosynthetic rates at 1 to 2 millimolar K⁺ concentrations. Rates in the 6 millimolar K⁺-grown plants were not higher than the 2 millimolar K⁺-grown wheat, and rates were inhibited below 0.5 millimolar K⁺. Photosynthesis was measured by both attached whole leaf CO₂ uptake and by ¹⁴CO₂ fixation of leaf slices in solution. Exposure of leaf slices from 0.2, 2, and 6 millimolar K⁺-grown wheat to various assay media water potentials showed that photosynthesis of the 0.2 millimolar K⁺-grown wheat decreased from control (high water potential) rates by 35%, that of the 2 millimolar K⁺-grown wheat by 20.4%, and that of the 6 millimolar K⁺-grown wheat by only 8.3% at −3.11 megapascals. Also, photosynthesis of the 6 millimolar K⁺-grown wheat was enhanced by 28% over that of the 2 millimolar K⁺ wheat at the most severe water stress (−3.11 megapascals), indicating that the excess leaf K⁺ in the 6 millimolar K⁺-grown wheat partially reversed dehydration effects on photosynthesis. Oligomycin eliminated the protective effects of high K⁺ on photosynthesis in dehydrated leaf slices. These results suggest that the protective effect of high K⁺ under water stress may involve the exchange of K⁺ in the cytoplasm for stroma H⁺, thus altering stromal pH and restoring photosynthesis. The protective effect of high K⁺ was also observed in attached whole leaf photosynthesis of in situ water-stressed wheat grown on 0.2, 2, and 6 millimolar K⁺. Under water stress, rates of the 6 millimolar K⁺-grown wheat were enhanced by 66.2% and 113.9% over that of 2 millimolar K⁺-grown wheat in two separate experiments. Internal CO₂ concentration of the 6 millimolar K⁺-grown wheat was lower than that of the 0.2 and 2 millimolar K⁺-grown wheat. These results suggest that the high K⁺ effects on chloroplast photosynthesis seen in leaf slices also occur at the whole plant level.     

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells

Na⁺-ATPase of high-K⁺ and low-K⁺ sheep red cells was examined with respect to the sidedness of Na⁺ and K⁺ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na⁺ in the medium, i.e., at the cytoplasmic surface, Na_cyt⁺, the activation curves show that high-K⁺ Na⁺-ATPase has a higher affinity for Na_cyt⁺ compared to low-K⁺. The apparent affinity for Na_cyt⁺ is also increased by increasing the ATP concentrations in high-K⁺ but not low-K⁺. With Na_cyt⁺ present, Na⁺-ATPase is stimulated by intravesicular Na⁺, i.e., Na⁺ at the originally external surface, Na_ext⁺, to a greater extent in low-K⁺ than high-K⁺. Intravesicular K⁺ (K_ext⁺) activates Na⁺-ATPase in high-K⁺ but not in low-K⁺ vesicles and extravesicular K⁺ (K_cyt⁺) inhibits low-K⁺ but not high-K⁺ Na⁺-ATPase. Thus, the genetic difference between high-K⁺ and low-K⁺ is expressed as differences in apparent affinities for both Na⁺ and K⁺ and these differences are evident at both cytoplasmic and external membrane surfaces.     

Growth in high-K+ medium induces Friend cell differentiation.

Friend erythroleukemic cells can be induced to differentiate by growth in high-K⁺ medium. Growth of Friend cells in medium containing 60–90 mM K⁺ and 90-60 mM Na⁺ (keeping the osmotic pressure constant) induced differentiation as measured by iron-59 incorporation into heme, accumulation of globin mRNA, the appearance of benzidine-positive cells, and the expression of erythrocyte membrane antigens. In addition, these “high-K⁺, low-Na⁺” conditions were synergistic with low doses of dimethylsulfoxide (DMSO) for the induction of erythroid differentiation. Not all Friend cell clones examined could be induced to differentiate in high-K⁺, low-Na⁺ medium alone, but the synergism between DMSO and high-K⁺, low-Na⁺ was observed in all cases.     

Active potassium transport in reticulocytes of high-K and low-K sheep

The kinetics of active K⁺ transport were studied in immature red blood cells cells from high-K⁺ and low-K⁺ sheep, particularly with respect to the effects of varying intracellular K⁺ concentration, [K]_i. Comparison was made with active transport, or pump, activity in mature high-K⁺ and low-K⁺ red cells. Reticulocytes from both types of sheep had much higher maximal active K⁺ influxes than did mature cells. In both types of reticulocytes, and in mature high-K⁺ cells as well, the pump was relatively insensitive to increasing [K]_i. In contrast, intracellular K⁺ markedly inhibited the pump in mature low-K⁺ cells. Active K⁺ transport in low-K⁺ reticulocytes, however, as in mature low-K⁺ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K⁺ pumps of high-K⁺ and low-K⁺ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K⁺ and low-K⁺ cells with K⁺ pumps of very different kinetic characteristics.     

Nicardipine-sensitive enhancement of high K+-evoked dopamine release in PC12 cells pretreated with 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on stimulus-evoked dopamine release were studied in PC12 cells. Pretreatment of the cells with TPA resulted in an enhancement of dopamine release which could be further stimulated by high concentrations of K⁺, A23187, but not with carbamylcholine. TPA-dependent, high-K⁺-evoked enhancement of dopamine release was studied in detail: a maximum release was observed (169% of control) in response to 50 mM KCl upon treatment with 10⁻⁷ M TPA for 5 min at 37°C. This enhancement of dopamine release was associated with the concomitant reduction of the concentration rise of intracellular Ca²⁺ ([Ca²⁺]_i) induced by a high concentration of K⁺ monitored by a fluorescent indicator, fura2. Thus, these data provide an example for alteration in the efficiency of stimulus-secretion coupling as pointed out in our previous paper. Moreover, we have shown that nicardipine, CdCl₂, and CoCl₂ inhibit high-K⁺-evoked dopamine release more effectively in TPA treated cells than that of untreated cells, and that the TPA-dependent, high-K⁺-evoked dopamine release observed in TPA treated cells is completely abolished by the presence of nicardipine, Cd²⁺ or Co²⁺, but is only partially inhibited in the presence of verapamil. These relevant findings suggest the possible involvement of protein kinase C in regulating the efficiency of a high-K⁺-evoked dopamine release through the modification of nicardipine-sensitive Ca²⁺ channels.     

The role of Ca2+ in the activity of Mycobacterium tuberculosis DNA gyrase

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca²⁺-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca²⁺ cannot support topoisomerase reactions in the absence of Mg²⁺, but partial removal of Ca²⁺ from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca²⁺. More extensive removal of Ca²⁺ by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca²⁺-binding residues also leads to loss of activity. We propose that Ca²⁺ has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca²⁺ binding.     

Effects of haemoglobin O2 saturation on volume regulation in adrenergically stimulated red blood cells from the trout, Oncorhynchus mykiss

Adrenergic stimulation of trout red blood cells activates a Na⁺/H⁺-exchange. If unopposed, the ensuing increase in cell Na⁺ leads to an isosmotic cell swelling. In this study the effect of the level of haemoglobin O₂ saturation on volume regulation has been investigated in adrenergically stimulated red blood cells from trout: at full haemoglobin O₂ saturation, net influx of Na⁺ through the Na⁺/H⁺-exchanger was balanced by net efflux of K⁺ and no increases in cell volume took place. In contrast, at low O₂ saturation (8–14%) adrenergic stimulation led to a substantial increase in cell Na⁺, K⁺ and volume. Moreover, cell volume recovery after adrenergic swelling was incomplete at low O₂ saturation, whereas cells at high O₂ saturation exhibited a fast and complete cell volume recovery. In cells exposed to alternating high and low O₂ saturation, volume regulation was similar to the regulation found in cells maintained at high O₂ saturation. In cells at high O₂ saturation, extrusion of cellular Na⁺ by the Na⁺/K⁺-pump significantly contributed to the volume decrease. It is concluded that trout red blood cells at high or alternating O₂ saturations possess a powerful regulatory volume decrease response that is shut off at low O₂ saturation. The physiological implications of this regulation is discussed. Accepted: 30 September 1996     

Catechol Formation and Melanization by Na+ -Dependent Azotobacter chroococcum: a Protective Mechanism for Aeroadaptation?

Aeroadaptive microaerophilic Azotobacter chroococcum 184 produced a cell-associated black pigment when grown at high aeration rates under nitrogen-fixing conditions. This pigment was shown to be a catechol melanin. Polyphenol oxidase activity was detected in cell extracts of cells grown for 72 h. Melanin formation was optimal in the later stages of growth, and there was no correlation between nitrogenase activity and melanization. Nitrogenase activity in strain 184 was optimal at 10% O₂, and melanin formation was suppressed by O₂ limitation. In the presence of charcoal, an adsorbent of toxic oxygen intermediates, and benzoic acid, a scavenger of hydroxyl radicals, melanization was inhibited. However, in the presence of copper, the intensity of pigment color increased and melanization was accelerated. Copper also eliminated catalase and peroxidase activities of the organism but still permitted aerobic growth. In the presence of low levels of iron, melanization was accelerated under high aeration rates, and under low rates of aeration, melanization was observed only at higher levels of iron. Hydroxamate-siderophore production was detectable in the presence of soluble iron under high rates of aeration but was repressed by the same levels of iron under low aeration rates. Unlike melanization and hydroxamate formation, catechol formation was observed under both low and high rates of aeration under nitrogen-fixing conditions. Catechol formation and melanization were repressed by 14 mM NH₄⁺, at which level nitrogenase activity was also repressed. Copper reversed the repressive effect of NH₄⁺. A role for catechol formation and melanization in aeroadaptation is proposed.     

(Na+,K+)-ATPase of mammalian brain: differential effects on cation affinities of phosphorylation by ATP and acetylphosphate

Because of their differing concentration dependencies, the Na⁺ interactions required for the phosphorylation of (Na⁺,K⁺)-ATPase ([Na⁺]_0.5 = 1.5 mm) and those required for the transformation of (Na⁺,K⁺-ATPase into its high-K⁺affinity form ([Na⁺]_0.5 = 6 mm with ATP and 28 mm without ATP) appear to be distinct. This distribution is not attributable to modulation by either nucleotide or K⁺ binding. In the absence of Na⁺, acetylphosphate reacts to form a phosphorylenzyme the hydrolysis of which is only slightly accelerated by K⁺. Phosphorylenzyme formed under similar conditions except for the presence of Na⁺ is highly sensitive to the addition of K⁺. ATP and acetylphosphate both act synergistically with sodium to favor the existence of the ATPase in its high-K⁺-affinity form. Acetylphosphate, however, acts only by increasing the proportion of enzyme in this form, whereas, ATP also causes a reduction in [Na⁺]_0.5. Previous studies have shown that this ATP effect is a consequence of formation of phosphorylenzyme. Results presented here suggest that Na⁺ binding may be necessary to produce K⁺-sensitive phosphorylenzyme and that nucleotide binding increases the Na⁺ affinity of phosphorylenzyme.     

New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling

Summary We isolated new gyrA and gyrB mutations in Escherichia coli which have a graded effect on DNA supercoiling. The mutants, selected respectively for resistance to nalidixic acid and coumermycin, were sorted by means of a rapid in vivo assay of DNA gyrase activity (Aleixandre and Blanco 1987). Cells carrying a gyrB (Cou^r) mutation usually showed a decrease in DNA supercoiling, which would indicate a reduction in gyrase activity. In contrast, most of the gyrA (Nal^r) mutations had no significant effect on DNA supercoiling. Moreover, they conferred a high level of resistance to nalidixic acid and other quinolones, thus being similar to the gyrA(Nal^r) mutants currently used. We also detected rare gyrA mutants showing a reduction in DNA gyrase activity. These mutants were, in addition, resistant to only low concentrations of quinolones, which allowed us to use the phenotype of partial quinolone resistance as an indicator to score gyrA mutations affecting DNA supercoiling. When gyrB mutations were introduced into the gyrA mutants, these became more sensitive to quinolones and a decrease in supercoiling was observed. Moreover, the topA10 mutation sensitized gyrA(Nal^r) cells to quinolones. We conclude therefore that the GyrA-dependent quinolone resistance is diminished as a consequence of the reduction either in topoisomerase I or gyrase activities.     

Sodium-Dependent Azotobacter chroococcum Strains Are Aeroadaptive, Microaerophilic, Nitrogen-Fixing Bacteria

Na⁺ -dependent strains of Azotobacter chroococcum were observed to have very low reactivities with the H₂O₂ spot test for catalase. The cell extract of the representative Na⁺ -dependent strain 184 contained a catalase specific activity that was 10-to 600-fold lower than those found in Na⁺ -independent strains of A. chroococcum. Peroxidase and superoxide dismutase activities existed in all strains, although only certain Na⁺ -dependent strains contained a peroxidase reactive with p-phenylenediamine. The activities of catalase and peroxidase in the Na⁺ -dependent strain 184 were dependent on iron availability, which helped to explain the iron-dependent growth characteristic of this strain. The activities of these enzymes were not increased by subjecting the cells to increased aeration, nitrogen-fixing conditions, or paraquat. Strain 184 was found to be very sensitive to H₂O₂ or paraquat, even under iron-sufficient conditions, and was difficult to recover quantitatively on solid plating media. Strain 184 was more susceptible to H₂O₂ when grown under low-aeration, nitrogen-fixing conditions than when it was grown in the presence of NH₄⁺. Low population densities of strain 184 grew in nitrogen-free medium under microaerophilic conditions, while more dense populations were able to fix nitrogen under aerobic conditions. Therefore, these bacteria appeared to be aeroadaptive, microaerophilic, nitrogen-fixing bacteria.     

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO₃⁻-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH₄⁺ have high NADPH-GDH activity, the levels of which increase with increasing NH₄⁺ supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH₄⁺-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH₄⁺.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

Effects of oxidative stress inhibitors, neurotoxins, and ganglioside GM1 on Na+,K+-ATPase activity in PC12 Cells and brain synaptosomes

To elucidate mechanism of ganglioside neuroprotection, it is important to study their metabolic effects, specifically of action on Na⁺,K⁺-ATPase. It has been shown that under effect of oxidative stress inductors and neurotoxins an oxidative inactivation of this enzyme takes place in PC12 cells and brain cortex synaptosomes, this inactivation being able to be prevented or decreased by ganglioside GM1. Thus, for instance, 24 h after action of 1 mM H₂O₂, activity of Na⁺,K⁺-ATPase in PC12 cells decreased more than twice. However, in the case of preincubation of the cells with ganglioside GM1 prior to the H₂O₂ action, this enzyme activity did not differ statistically significantly from control. Ganglioside GM1 also was able to increase statistically significantly the enzyme activity decreased by action on the PC12 cells of amyloid β-peptide (Aβ) causing lesion of neurons in Alzheimer’s disease and of low H₂O₂ concentrations. Experiments on brain cortex synaptosomes have established that not only antioxidants—α-tocopherol and superoxide dismutase (SOD)—but also ganglioside GM1 prevent the glutamate-produced Na⁺,K⁺-ATPase oxidative inactivation. The obtained data agree with a suggestion that the ganglioside neuroprotective effect at action on nerve cells of such toxins as Aβ, glutamate or reactive oxygen species is due to their ability to inhibit the free-radical reactions.     

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.     

Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.