Light-Induced Membrane Hyperpolarization and Adenine Nucleotide Levels in Perfused Characean Cells

This study examines the relationship between light-induced membranehyperpolarization and changes in adenine nucleotide levels intonoplast-free characean cells. When cells were perfused witha medium containing 1 mM ATP in the dark, the plasma membranedepolarized, the cytosolic ATP level decreased, and the ADPand AMP levels increased. Under light, the membrane hyperpolarized,the ATP level increased, and the ADP and AMP levels decreased.These changes in the adenine-nucleotide levels could partiallyexplain the membrane hyperpolarization. When cells were perfusedwith a medium containing an ATP-regenerating system consistingof phosphoenolpyruvate and pyruvate kinase, the membrane potentialremained in the hyperpolarized state, the ATP level remainedat a high level and no light-induced hyperpolarization was observed.The intracellular adenine nucleotide levels were also controlledby continuous perfusion. The membrane potential was determinedonly by the adenine nucleotide levels of perfusion media, irrespectiveof the light condition. Chloroplast-free Nitellopsis cells into which isolated Pisumchloroplasts were introduced also showed light-induced membranehyperpolarization. Pretreatment of chloroplasts with dicyclohexylcarbodiimide(DCCD) completely abolished the hyperpolarization with parallelinhibition of photophosphorylation. These results strongly suggestthat changes in adenine nucleotide levels caused by photophosphorylationare responsible for light-induced membrane hyperpolarizationin perfused cells. (Received August 17, 1985; Accepted December 13, 1985)     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Demonstration of Voltage Dependency of Light-induced Potential Change in Chara

The electromotive force (E_m) of the plasma membrane of the tonoplast-freecell of Chara australis decreased when the electrogenic pumpwas stopped by removing ATP or Mg²⁺ from the cell. Such a cellshowed a rapid light-induced potential change (rLPC). Threefactors were considered to be responsible for the generationof rLPC; removal of Mg-ATP, stoppage of the electrogenic pumpand membrane depolarization per se. Tonoplast-free cells having enough Mg-ATP occasionally showedsmall E_m (–87— –116 mV) due to stoppage ofthe electrogenic pump. Since the rLPC was induced in such cells,removal of Mg-ATP cannot be the factor. Cells having large E_mdue to active pump activity also showed rLPC when the potentialdifference across the plasma membrane (V_m) was depolarized byan outward electric current; evidence that not the stoppageof the pump but membrane depolarization is a necessary conditionfor the generation of rLPC. In the rLPC Vm always changed in the negative direction. However,calculation of E_m revealed the existence of a reversal potential[ (E_m)rev] toward which E_m converged from either more negativeor more positive values. The (E_m)rev approximately coincidedwith the equilibrium potential for K⁺ across the plasma membrane.Intracellular anions occupying lower positions in the lyotropicseries inhibited rLPC. (Received February 9, 1981; Accepted May 16, 1981)     

Cell physiological aspects of the plasma membrane electrogenic H<Superscript>+</Superscript> pump

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H⁺) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H⁺ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H⁺ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H⁺ efflux supports the notion that the ion carried by the electrogenic ion pump is H⁺. The role of the H⁺ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H⁺ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the K_m value for the ATP of H⁺-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H⁺-ATPase in guard cells is presented. However, there are cases where H⁺-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H⁺-ATPase activity. Finally, modulation of H⁺-pumping or H⁺-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H⁺ pump.     

Regulation of mammalian asparagine synthetase by adenine nucleotides.

Initial velocity kinetic data indicate that ADP and AMP are inhibitors of mammalian liver asparagine synthetase. The non-product nucleotide ADP is a much more potent inhibitor than AMP, although both apparently compete for the same site. This modifier site, however, does not overlap spatially with the substrate site for ATP. Both ADP and AMP are V_max inhibitors, but ADP also raises the K_m for ATP. Adenylate energy charge, calculated at various levels of ATP and ADP show typical correlations with activity, but with AMP these correlations are weak and atypical.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Transient change in the ATP Pool ofAnabaena cylindrica associated with ammonia assimilation

When N₂-grown cells ofAnabaena cylindrica were exposed to ammonia (50 M to 5 mM) in the dark, the size of the ATP pool was reduced by 40% within 1 min, but restored after 5 or 6 min. The decrease in ATP was accompanied by increases in ADP and AMP, while the total adenylate content remained unaltered. The ammonia-induced change in the ATP pool was completely eliminated when algal cells were treated withl-methionine-dl-sulfoximine, an inhibitor of glutamine synthesis. These results suggest that ammonia is rapidly assimilated through the pathway mediated by glutamine synthetase accompanied by reduction of the ATP pool.Abbreviations GS Glutamine synthetase - MSX l-methionine-dl-sulfoximine - CCCP carbonyl cyanidem-chlorophenyl-hydrazone     

Exogenous ATP induces electrical membrane responses in fibroblasts

Mouse fibroblastic L cells responded to exogenous ATP (0.2 mM) with a transient hyperpolarization due to increased membrane permeability to K⁺. By contrast, intracellular injection of ATP (up to about 3 mM) produced no noticeable effects on the membrane potential. The effects of a non-hydrolysable analogue of ATP (AMP-PNP) were similar to those of ATP. After successive applications of ATP, the cell membrane became virtually unresponsive (desensitized). Extracellular ADP was also effective, but AMP or adenosine was not. Antazoline suppressed the ATP response. Thus, exogenous ATP and ADP appear to stimulate P₂-purinoceptors. Similar responses to ATP (or ADP) were also observed in human normal diploid fibroblasts (Flow 1000 line).     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Correlation of Adenosine Triphosphate Levels in Chara corallina with the Activity of the Electrogenic Pump

The effect of a number of inhibitors on the ATP level in single cells of Chara corallina has been measured using the luciferin-luciferase assay. The uncouplers of phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol, and the ATPase inhibitors, dicyclohexyl-carbodimide and diethylstilbestrol, all caused a marked reduction of the ATP level. These inhibitors also produced a large increase in the membrane resistance and a depolarization of the membrane potential to the diffusion potential. This is consistent with the plasmalemma containing an ATP-dependent electrogenic pump that provides the primary conductance through the membrane.     

Conditions for a backward-running Na+/K+ pump in Xenopus oocytes.

Current generated by the electrogenic Na+/K+ pump protein was determined in oocytes of Xenopus laevis as strophantidine-sensitive current measured under voltage clamp. Under conditions of reduced intracellular [Na+] and [ATP], both to values below 1 mM, and in extracellularly K(+)-free medium, the Na+/K+ pump seems to operate in a reversed mode pumping Na+ into the cell and K+ out of the cell. This is demonstrated by strophantidine-induced hyperpolarization of the membrane and inward-directed current mediated by the pump protein. In addition, strophantidine-sensitive uptake of 22Na+ can be demonstrated under these conditions. The pump current decreases with membrane depolarization as expected for a pump cycle that involves inward movement of positive charges during Na+ translocation.     

A kinetic analysis of the electrogenic pump ofChara corallina: III. Pump activity during action potential

Summary The current-voltage curve (I–V curve) of theChara membrane was obtained by applying a slow ramp hyper- and depolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50m DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steadyI–V curve within two hours, which gave thei _d -V curve of the passive diffusion channel. Thei _p -V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. The sigmoidali _p -V curve could be simulated satisfactorily with a simple reaction kinetic model which assumes a stoichiometric ratio of 2. The emf of the pump (E _p ) is given as the voltage at which the pump current changes its sign. The conductance of the pump (g _p ) can be calculated as the chord conductance from thei _p -V curve, which is highly voltage dependent having a peak at a definite voltage. The changes of emf and conductance during excitation were determined by use of the current clamp (I=0). Since theE _p andg _p (V) are known, the changes, during excitation, of emf (E _d ) and conductance (g _d ) of the passive diffusion channel can be calculated. The marked increase of the membrane conductance and the large depolarization during the action potential are caused by the marked increase of the conductance of the passive diffusion channel and the large depolarization of its emf. The conductance of the electrogenic pump decreases to about half at the peak of action potential, while the pump current increases almost to a saturated level.     

Dependence of the membrane potential of Chara cells on external pH in the presence or absence of internal adenosinetriphosphate

The dependence of the membrane potential (E_m) and the membrane resistance (R_m) of Chara australis R. Brown on the pH of the external medium (pH₀) was studied by controlling the activity of the plasmamembrane H⁺ pump under both light and dark conditions. The activity of the pump was controlled by regulating the internal ATP or Mg²⁺ concentration in tonoplast-free cells prepared by vacuolar perfusion. In these cells, which contained Mg · ATP (mgATP cells), E_m and R_m were very sensitive to pH₀, as in normal cells. E_m was more negative in light than in the dark at all pH₀ values tested. Tonoplast-free cells with very low [ATP]_i (-ATP cells) or [Mg²⁺]_i (-Mg cells) showed very weak dependence of E_m and R_m on pH₀. Thus, the active and not the passive component of E_m was sensitive to pH₀. At the same time, the high permeability of the plasma membrane to H⁺ was questioned. In both-ATP cells and-Mg cells, E_m was scarcely affected and R_m markedly decreased on illumination.Abbreviations CyDTA 1,2-cyclohexanediamine-N,N-tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)N,N-tetraacetic acid - HK hexokinase     

Nucleotides modulate the activity of aspartate racemase of Scapharca broughtonii

The activity of -aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in V_max and not in K_m. Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and -aspartate in energy metabolism under anoxic conditions are discussed.     

The relationship between ATP and an electrogenic pump in the plasma membrane ofNeurospora crassa

Summary Sudden respiratory blockade has been used to study rapid changes of the resting membrane potential, of intracellular adenosine 5-triphosphate (ATP) levels, and of pyridine nucleotide reduction inNeurospora crassa. Membrane depolarization occurs with a first-order rate constant of 0.167 sec^–1, following a lag period of about 4 sec, at 24°C (ambient temperature). This depolarization is several-fold too slow to be directly linked to electron transfer, as judged from the rate of pyridine nucleotide reduction, but has essentially the same rate constant as the decay of ATP. The latter process, however, shows no lag period after the respiratory inhibitor is introduced. Plots of membrane potential versus the intracellular ATP concentration yield saturation curves which are readily fitted by a Michaelis equation, to which is added a constant term representing the diffusion component of membrane potential. Parameters obtained from such fits indicate the maximal voltage which the pump can develop at high ATP levels to be 300 to 350 mV, with an apparentK _1/2 of 2.0mm. The data strongly suggest that an electrogenic ion pump in the plasma membrane ofNeurospora is fueled by ATP; comparison of the measured membrane potentials with the energy available from hydrolysis of ATP indicates that two ions could be pumped for each molecule of ATP split.     

Subthreshold changes in excitable membranes of Cucurbits pepo L. stem cells during cooling-induced action-potential generation

The nature of subthreshold changes in excitable plasma membranes has been investigated in stem parenchyma cells of Cucurbita pepo L. during action-potential generation induced by gradual cooling (from 23 to 10 ° C). The character of the subthreshold depolarization of excitable cells is shown to be mainly defined by a decrease in the activity of the plasma-membrane electrogenie pump (H⁺-ATPase). In its turn, the pump activity is controlled by thermal changes in the structure of the membrane lipid matrix. Based on the results obtained, a sequence of subthreshold changes has been suggested in which thermally induced structural rearrangements of membrane lipids play the role of trigger.Abbreviations AP action potential - DCCD N,N-dicyclohexil-carbodiimide - E_m membrane potential - I_e/I_m ratio of pyrene excimer/monomer fluorescence intensities     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

Adenine nucleotides are apparently involved in the light-dark modulation of spinach-leaf nitrate reductase

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L. cv. Polka F1) leaves showed reversible modulation, being activated in the light and inactivated in the dark (t/2 = 20–30 min). The large changes in enzyme activity during light-dark transients were observed only when assayed in buffers containing free Mg²⁺. In the presence of EDTA (5 mM), the enzyme activity was high and the light modulation was barely evident.The inactivation of NR in the dark could be totally prevented by anaerobiosis, or by feeding mannose or 2,4-dinitrophenol through the leaf petiole. All these treatments drastically decreased ATP levels and increased AMP levels in leaf extracts, thus pointing to a close correlation between adenine-nucleotide levels and NR activity. Treatment of leaves in the dark with 2,4-dinitrophenol or with anaerobiosis brought about an accumulation of nitrite, thus confirming that under these conditions NR remained active also in vivo. The in-vivo dark-inactivated enzyme was reactivated in vitro by preincubating a leaf extract with AMP in the presence of the myokinase inhibitor p¹,p⁵-di(adenosine 5)pentaphosphate. It is suggested that NR responds to artificially induced drastic changes in cytosolic adeninenucleotide levels, being active when ATP is low and AMP is high. Adenine nucleotides also appear to participate in the light-dark modulation of NR, but additional regulatory factors have to be postulated.     

Day-night changes in the levels of adenine nucleotides,phosphoenolpyruvate and inorganic pyrophosphate in leaves of plants having Crassulacean acid metabolism

The levels of phosphorylated compounds studied during the dark period of Crassulacean acid metabolism (CAM) in Kalanchoë leaves showed increases for ATP and pyrophosphate and decreases for ADP, AMP and phosphenolpyruvate; levels of inorganic phosphate remained constant. Changes in adenylate levels and the correlated nocturnal increase in adenylate-energycharge were closely related to changes in malate levels. The increase in ATP levels was much inhibited in CO₂-free air and stimulated after induction of CAM in short-day-treated plants of K. blossfeldiana cv. Tom Thumb. Changes in levels of phosphoenolpyruvate and pyrophosphate were independent of the presence of CO₂. The results show the operation of complex regulatory mechanisms in the energy metabolism of CAM plants during nocturnal malic-acid accumulation.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - OAA oxaloacetic acia - PEP phosphoenol pyruvate - PP_i pyrophosphate