In-vitro-cultured subclover root can develop Fe-deficiency stress response

The Fe-deficiency stress response is induced in most plants under Fe-deficient conditions, but whether the shoot and/or the root control development of the stress response is not known. The objectives of the present study were to determine whether in-vitro-cultured subclover roots can develop Fe-deficiency stress response and to examine this approach as a possible screening technique for Fe-deficiency resistance. One-cm long root tips of subclover seedlings were cultured in modified White's medium without (-Fe) or with (+Fe) 100 μM Fe³⁺EDTA. Root Fe³⁺ reduction and H⁺ release were evaluated. On the first day after transfer to the -Fe medium, the Fe-deficiency-resistant cultivar Koala (Trifolium brachycalycinum Katzn. and Morley) started to release H⁺, resulting in a decrease in pH of the culture medium, while the susceptible cultivar Karridale (T. subterraneum L.) did not release H⁺ until the second day. The H⁺-release rate of the -Fe Koala was approximately twice as high as that of the -Fe Karridale for the first 4 days of -Fe treatment. Both Koala and Karridale reached their highest H⁺-release rates on the fourth day after -Fe treatment initiation. The +Fe Koala released H⁺ after several days of culture, but the H⁺ release of the -Fe Koala was severalfold greater than that of the +Fe Koala. The implicit correlation between H⁺ release and Fe-deficiency resistance was substantiated by using a series of subclover cultivars with a range of susceptibilities to Fe deficiency. The pH of the -Fe culture media of the series of cultivars was positively correlated to their Fe-chlorosis scores reported in previous research. The results of the present study indicate that root itself has the full ability to develop Fe-deficiency stress response and the response is dependent on the root Fe status. The results also suggest that root culture could be used as a simple and efficient alternative technique for screening germplasm for Fe-deficiency resistance.     

SALINITY ADAPTATION BY DUNALIELLA TERTIOLECTA. I. INCREASES IN CARBONIC ANHYDRASE ACTIVITY AND EVIDENCE FOR A LIGHT-DEPENDENT Na+/H+ EXCHANGE1,2

When Dunaliella tertiolecta, previously adapted to medium containing 0.5 M NaCl, is transferred to higher salinities, there is a lag in growth, suggesting an adaptation period. Since there is no significant difference in the Na⁺ content of cells grown between 0.5 and 3.5 M NaCl, a mechanism for Na⁺ extrusion or exclusion is indicated. Increasing the salinity of cell suspensions stimulates an incorporation of H⁺ by the cells, suggesting an H⁺/Na⁺ exchange. Cells adapted to higher salinities have, increased carbonic anhydrase activity, suggesting that increased CO₂ or HCO₃^? transport may be required at higher salinities. Growth, of D. tertiolecta at salinities above 2.5 M requires continuous illumination; therefore a light-driven H⁺/Na⁺ exchange accompanied by a HCO₃^? influx is proposed.     

H+ uptake by chromatophores from Rhodopseudomonas spheroides; the relation between rapid H+ uptake and the H+ pump

1. H⁺ uptake induced by repeated flash excitation approached the full extent of H⁺ uptake induced by continuous light. At low repetition rates, the H⁺ uptake was seen to consist of repeated occurrences of rapid H⁺ uptake.2. The effects of ionophores and uncoupling agents on H⁺ uptake induced by continuous light could be adequately accounted for in terms of their effects on the flash induced changes. It is concluded that the reaction disclosed by rapid H⁺ uptake is an integral part of the process observed on continuous illumination, and therefore, in view of the association between rapid H⁺ uptake and the reduction of a hydrogen-carrying secondary acceptor, that the electron transport system is an integral part of the mechanism of the H⁺ pump.3. When the frequency of repetition of the flashes was increased, the full extent of H⁺ uptake or of the carotenoid change was seen only after the first few flashes. Thereafter, the extent decreased, and depended on the dark time between flashes. The full extent of the change could be restored even at high frequencies if uncoupling agents or valinomycin were present.4. It is concluded that the recovery of the extent of H⁺ uptake or the carotenoid change between flashes reflected the turnover of the electron transport chain, and that the increased recovery in the presence of uncoupling agents or valinomycin reflected the stimulation of electron flow under uncoupled conditions, or on dissipation of the membrane potential.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

Induction of the H+ Release from Thylakoid Membranes by Illumination in the Presence of Protonophores at High Concentrations

The generally observed light-induced uptake of protons intothe thylakoid lumen is diminished by adding protonophores. Insteadof the H⁺ uptake, the release of protons was observed duringillumination in the presence of various protonophores at highconcentrations, namely, 1 µM nigericin, 10 µM carbonylcyanidem-chlorophenylhydrazone or 30 µM gramicidin. An uncoupler,NH₄C1 (4 mM), and a detergent, Triton X-100 (0.02%), also inducedthe H⁺ release but a K⁺ ionophore, valinomycin, did not. Theamount of H⁺ released reached about 100 nmol H⁺ (mg Chl)^–1at pH 7.5 under continuous illumination. The rate of the H⁺release was similar to that of the conventional H⁺ uptake butits dark relaxation was much slower than that of the H⁺ uptake.We compared the H⁺ release in protonophore-added thylakoidswith the previously reported H⁺ release in coupling factor 1(CF₁-depleted thylakoids. The H⁺ release in thylakoids withnigericin showed similar characteristics to that in CF₁-depletedthylakoids in terms of their responses to pH, phenazine methosulfateand light intensity. Both types of H⁺ release were relativelyinsensitive to DCMU and were stimulated somewhat by DCMU atlow concentrations (around 200 nM). Nigericin did not inhibitthe superoxide dismutase activity of the membranes. These resultsindicate that the H⁺ release in protonophore-added thylakoidsand that in CF₁ depleted thylakoids involve the same mechanismand that water-derived protons from PS II that result from animpairment of the activity of superoxide dismutase, as previouslyproposed, are not involved. Judging from the rate of electronflow and the lumenal acidification under the illumination, weconclude that the H⁺ release is a light-dependent scalar processwhich can be observed in thylakoid membranes with high H⁺ permeability.The H⁺ release of this type was not observed in mitochondriafrom rat liver or in chromatophores from Rhodobacter sphaeroides. (Received November 29, 1990; Accepted June 27, 1991)     

Transient,specific and extremely rapid release of osmolytes from growing cells of Escherichia coli K-12 exposed to hypoosmotic shock

The influence of hypoosmotic shock on the solute content of growing Escherichia coli K-12 cells was investigated at 37°C. Within 20 s after the shock the cells had released most of their osmolytes K⁺, glutamate and trehalose. This release was specific and not due to rupture of the cell membrane, since under these conditions i) the cells neither lost protein nor ATP, ii)[¹⁴C]-labeled sucrose did not enter the cytoplasm from the periplasm, and iii) except for their glutamate and aspartate level, which decreased, the amino acid pool of alanine, lysine and arginine of the cells remained approximately constant. Within a minute after the shock the cells started to reaccumulate parts of their previously released glutamate, aspartate and K⁺, but not trehalose and resumed growth within 10 min after the shock. Experiments with K⁺-transport mutants showed that none of the genetically-identified K⁺ transport systems is involved in the K⁺-release process. Reaccumulation of K⁺ took place via the uptake systems TrkG and TrkH. The possibility is discussed that the exit of solutes after hypoosmotic shock occurs via several stretch-activated channels, which each allow the release of a specific osmolyte.Abbreviations OD₅₇₈ optical density at 578 nm - TEA triethylammonium - TMG 1,-S-methyl--thiogalactopyranoside     

Characteristics of Fe‐deficiency‐induced acidification in subterranean clover

Iron-deficiency-induced acidification is one of the important reactions of plant Fe-deficiency-stress response, but the overall understanding of this reaction is limited. The characteristics of Fe-deficiency-induced acidification of subterranean clover (subclover) (Trifolium brachycalycinum Katzn. and Morley cv. Koala) were studied in this paper. Plants were grown hydroponically under -Fe conditions, and Fe-deficiency-induced acidification was determined using pH-stat, back-titration and chemical equilibrium procedures. Fe-deficiency-induced acidification was undetectable during the first day after Fe-deficiency stress initiation, but the maximum acidification rate was attained by the second day, when plants exhibited visual chlorosis symptoms. The acidification rate was relatively constant with increasing Fe-deficiency chlorosis, suggesting that a critical level of Fe deficiency was needed to trigger acidification, but that once the acidification process was initiated, the intensity of acidification was independent of severity of Fe deficiency. Net H⁺-release (PR) rate determined using a chemical equilibrium method and net acidity release (AR) rate determined using a back-titration method were practically identical, indicating that Fe-deficiency-induced acidification involved almost entirely the release of free H⁺, not organic acid. In the assay temperature range of 5 to 35°C, PR rate was highest at about 20°C. Net acidity release rate was almost totally inhibited at pH values ≤4.5 and increased with increasing assay pH up to pH 9. The pH effect occurred within 30 min of incubation initiation, implying that the effect of pH is probably on the activity of H⁺ transport through the plasma membrane, not on the quantity of responsible protein(s). Cations were required in the incubation solution for Fe-deficiency-induced acidification. Divalent cations in the assay solution resulted in a higher AR rate than monovalent cations, and essential cations resulted in a higher AR rate than non-essential cations, indicating that the relative effectiveness of cations is related to the efficiency of their absorption by plant roots. These results are discussed in relation to their practical significance and the mechanisms of Fe-deficiency-induced acidification.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Light-promoted changes in apoplastic K+ activity in the Samanea saman pulvinus,monitored with liquid membrane microelectrodes

The movement of Samanea saman (Jacq.) Merrill leaflets is a consequence of the re-distribution of K⁺ and anions between motor cells on opposite sides of the pulvinus. We used a K⁺-sensitive microelectrode to study dynamic changes in K⁺ transport through motor-cell membranes during and immediately after change in illumination. Potassium-ion-sensitive and reference microelectrodes were inserted into extensor or flexor tissue of a whole pulvinus in white light (WL). A brief pulse of red light (RL) followed by darkness (D) (a) increased K⁺ activity in the extensor apoplast, indicating K⁺ release by the protoplast; and (b) decreased K⁺ activity in the flexor apoplast, indicating K⁺ uptake by the protoplast. White light after 35–40 min D reversed K⁺ activity in the extensor apoplast to approximately its original value. Blue light substituted partially for WL in this regard. Potassium-ion activity in the flexor apoplast reverted to approximately its original value after 2 h, with or without white illumination. Our data support the hypothesis that K⁺ efflux from extensor cells and K⁺ uptake by flexor cells following a WLRLD transition occurs by way of K⁺ channels.Abbreviations L light - WL white light - RL red light - BL blue light - D darkness     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

CO-REQUIREMENT FOR CALCIUM AND POTASSIUM IN THE GERMINATION OF SPORES OF THE FERN ONOCLEA SENSIBILIS

Spores of Onoclea sensibilis L. do not germinate on distilled H₂O if they are pretreated for sufficient time with dilute NaClO solution. However, spores will germinate, after NaClO pretreatment, on a simple mineral medium containing the major and trace elements. Complete germination after pretreatment also is obtained on a solution containing only Ca²⁺ and K⁺ as the cations, but neither ion by itself is sufficient. Rb⁺, but not Li⁺ or Na⁺, can replace K⁺. Hypochlorite-treated spores do not require the continuous presence of Ca²⁺ and K⁺ to germinate; exposure during the first 4 hr of culture, with the remainder of the time on distilled H₂O, is sufficient. Extraction of spores with ethylene glycol bis(aminoethyl ether) tetraacetic acid [EGTA] makes their germination dependent on Ca²⁺, as reported by other workers, but it does not produce a co-requirement for K⁺. Colorimetric analysis with arsenazo III confirms that Ca²⁺ is extracted from Onoclea spores by NaClO. Extractable Ca²⁺ amounts to about 78 nmol/mg spore dry wt. Of this amount, 31% is contained in the perispore. The perispore comprises 13% of the total spore dry wt.     

Effect of Low Root Medium pH on Net Proton Release, Root Respiration, and Root Growth of Corn (Zea mays L.) and Broad Bean (Vicia faba L.)

The effect of low pH on net H⁺ release and root growth of corn (Zea mays L.) and broad bean (Vicia faba L.) seedlings was investigated in short-term experiments at constant pH. Broad bean was more sensitive to low pH than corn: the critical values (pH values below which net H⁺ release and root growth ceased) were pH 4.00 (broad bean) and pH 3.50 (corn) at 1 millimolar Ca²⁺. Both proton release and root growth were progressively inhibited as the medium pH declined. Additional Ca²⁺ in the root medium helped to overcome the limitations of low pH for net H⁺ release and root growth. Potassium (for corn) and abscisic acid (for broad bean) increased both net H⁺ release and root growth rate at the critical pH value. It is concluded that poor root growth at low pH is caused by a lack of net H⁺ release that may decrease cytoplasmic pH values. Inhibited net H⁺ release at high external H⁺ activity is not due to a shortage of energy supply to the H⁺ ATPase. Instead, a displacement of Ca²⁺ by H⁺ at the external side of the plasmalemma may enhance reentry of H⁺ into root cells.     

Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid

The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H₂O₂ content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC‐treated cells. This study, aimed to further characterize the inhibitor's features, shows that (1) FC‐induced H₂O₂ accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H⁺ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H⁺‐ATPase; (2) treatment with 10 µM erythrosin B (EB) early and totally inhibits net H⁺ and K⁺ fluxes across the PM, indicative of the H⁺ pump activity; nevertheless, also in these conditions a huge FC‐induced H₂O₂ accumulation occurs, confirming that this effect is not related to the FC‐induced PM H⁺‐ATPase activation; (3) the inhibitor's release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH₂Cl₂‐soluble fraction extracted from the incubation medium by sequential acid–base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC‐induced catalase inhibition belongs to the organic acid class.     

Light-dependent Proton Excretion of Wheat (Triticum aestivum L.) and Maize (Zea mays L.) Roots

We investigated the change of root net proton excretion of seedlings of Triticum aestivum L. and Zea mays L. with daily variation of illumination using a multi-channel pH-stat system. We found an increase of net proton excretion during darkness and a drop after the beginning of illumination. Inhibition of carotenoid biosynthesis by norflurazone and photooxidation of chlorophylls did not change the periodicity or its induction. The induction of diurnal periodicity was possible with blue, green and red light. After induction the oscillation of net proton excretion continued for at least two cycles under constant light. We conclude that net H⁺ excretion of wheat and maize roots may be regulated by an endogenous clock or by a signal from the leaves. The nature of such a hypothetical signal remains unknown.     

Functional domains of the gastric HK ATPase

The gastric H⁺ + K⁺ ATPase is a member of the phosphorylating class of transport ATPase. Based on sequence homologies and CHO content, there may be ab subunit associated with the catalytic subunit of the H⁺ + K⁺ ATPase. Its function, if present, is unknown. The pump catalyzes a stoichiometric exchange of H⁺ for K⁺, but is also able to transport Na⁺ in the forward direction. This suggests that the transport step involves hydronium rather than protons. The initial binding site is likely to contain a histidine residue to account for the high affinity of the cellular site. The extracellular site probably lacks this histidine, so that a low affinity for hydronium allows release into a solution of pH 0.8. Labelling with positively charge, luminally reactive reagents that block ATPase and pump activity has shown that a region containing H5 and H6 and the intervening luminal loop is involved in necessary conformational changes for normal pump activity. The calculated structure of this loop shows the presence of ana helical,b turn, andb strand sector, with negative charges close to the membrane domain. This sector provides a possible site of interaction of drugs with the H⁺ + K⁺ ATPase, and may be part of the K⁺ pathway in the enzyme.Emory University, Atlanta, Georgia.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Pre‐steady state kinetics of ATP hydrolysis by Na,K‐ATPase

Fast reaction kinetics of ATP hydrolysis by Na,K‐ATPase has been investigated by following absorption pattern of pH sensitive dye in stopped flow spectrophotometer. Distinct pre‐steady state phase signal could be recorded with an initial decrease in acidity followed by increase in acidity. Average half time for H⁺ absorption and peak alkalinity was, respectively, 30 ms and 60 ms. Under optimal Na⁺ (120 mM) and K⁺ (30 mM) concentrations, magnitude of both H⁺ absorption and H⁺ release are found to be about 1.0 H⁺/ATPase molecule. H⁺ absorption and release decreased with decrease in Na⁺ concentration, H⁺ release was more affected. Both H⁺ absorption and H⁺ release are found to be independent of K⁺ concentration in the pre‐steady state phase. No H⁺ absorption or release was observed following mixing of either ADP, Na⁺ or K⁺ alone with ATPase. Effect of delayed mixing of Na⁺ or K⁺ on two phases of pre‐steady state cycle indicates that ATP hydrolytic cycle starts without K⁺ ions if optimal Na⁺ is present. ATP hydrolytic cycle does not start in the absence of Na⁺ ions. Results obtained have been interpreted in terms of an extended kinetic scheme for Na,K‐ATPase. Copyright © 2009 John Wiley & Sons, Ltd.     

Light-dependent hydrogen evolution by Scenedesmus

Summary The effect of glucose and the uncoupler Cl-CCP upon hydrogen production was studied in adapted cells of Scenedesmus obliquus D₃. Cl-CCP at 10^-5M concentration completely inhibited the evolution of H₂ in the dark and increased the apparent rate of H₂ evolution in the light. At 10^-5M Cl-CCP, photosynthesis and photoreduction by anaerobically adapted algae were only temporarily inhibited; O₂ evolution reappeared after approximately 1 hr of illumination if CO₂ was present. Increasing the Cl-CCP concentration to 5 x 10^-5M led to a maximum rate of photohydrogen production and fully inhibited H₂ evolution, photoreduction and dark H₂ evolution. H₂ evolution was accompanied by a release of varying amounts of CO₂ in the light, as well as in the dark. Dark CO₂ production was stimulated by Cl-CCP. H₂ evolution in the light was stimulated by adding glucose to autotrophically grown cells or by growing the cells heterotrophically with glucose; starvation had an opposite effect. Adapted cells released ¹⁴CO₂ from the 3 and/or 4 position of specifically labeled glucose, indicating that degradation occurred via the Embden-Meyerhof pathway. The amount of H₂ released by autotrophically grown cells was the same either with continuous illumination or with short periods of light, followed by darkness. Scenedesmus mutant No. 11, which is unable to evolve O₂ was not inhibited in its capacity to evolve H₂ in the light. These data indicate that the evolution of H₂ in the light by adapted Scenedesmus depends upon the degradation of organic material and does not require the production of free O₂ by photosystem II.The following abbreviations are used: Cl-CCP = carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DNP = 2,4-dinitrophenol.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission.