Stefan Hanstein
email: stefan.m.hanstein@ernaehrung.uni-giessen.de

Xiaozhi Wang

Xiaoqing Qian

Peter Friedhoff

Ammara Fatima

Yuhua Shan

Ke Feng

Sven Schubert

Changes in cytosolic Mg 2+ levels can regulate the activity of the plasma membrane H + -ATPase in maize

Keywords: P-type ATPases, Mg 2+ activity, free magnesium, nucleotide-binding, nucleotide-inhibition

Running head: ATP inhibition of maize plasma membrane H + -ATPase Plant plasma membrane (PM) H + -ATPase, a major consumer of cellular ATP, is driven by the magnesium ATP complex which may dissociate at low cytosolic Mg 2+ activity. We investigated whether hydrolytic activity of PM H + -ATPase is inhibited at ATP concentrations exceeding the Mg 2+ concentration. Activity in isolated maize plasma membranes was measured at pH 6.5 in the presence of 5 mM Mg 2+ (high) or 2 mM Mg 2+ (low), while K + was applied at concentrations of 155 mM (high) or 55 mM (low). In all experiments, with membrane vesicles either from roots or leaves, the enzyme activity decreased in the presence of Mg 2+ -free ATP. At inhibitory ATP concentrations, the activity was not influenced by the K + concentration. The activity was restored after increasing the Mg 2+ concentration. ATP inhibition also occurred at pH 7.5. Kinetic modeling shows that Mg 2+ -free ATP acted as a competitive inhibitor with a K i in the range of the K m . K i decreased by 75% at low K + concentration. K i was one order of magnitude lower at pH 7.5 compared to pH 6.5. The observed inhibition is consistent with a concept in which down-regulation of the cytosolic Mg 2+ activity is involved in (phyto)hormonal stress responses.

INTRODUCTION

Plant plasma membrane (PM) H + -ATPase is the "powerhouse" for nutrient uptake, turgor formation and cellular growth [START_REF] Palmgren | Plant plasma membrane H + -ATPases: Powerhouses for nutrient uptake[END_REF] pumping protons from the cytosol into the extracellular space (apoplast). Under resting conditions enzyme activity is regulated by an autoinhibitory domain [START_REF] Palmgren | Plant plasma membrane H + -ATPases: Powerhouses for nutrient uptake[END_REF][START_REF] Chevalier | 14-3-3 and FHA domains mediate phosphoprotein interactions[END_REF] and probably by reduced coupling efficiency [START_REF] Buch-Pedersen | Potassium as an intrinsic uncoupler of the plasma membrane H + -ATPase[END_REF]. The crucial physiological role of down-regulation is evident from fusicoccin effects. This phytotoxin binds to 14-3-3 proteins, inactivates the autoinhibitory domain and causes a persistent activation of PM H + -ATPase, leading to abnormal apoplastic acidification and stomatal opening [START_REF] Oecking | Association of 14-3-3 proteins with the C-terminal autoinhibitory domain of the plasma-membrane H + -ATPase generates a fusicoccin-binding complex[END_REF].

The enzyme is driven by the Mg-ATP complex. A simple type of down-regulation or inhibition which does not involve protein-protein interaction could arise from an imbalance between the molar concentrations of Mg 2+ and ATP: ATP in excess of Mg 2+ inhibits PM H + -ATPase of yeast [START_REF] Koland | Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase[END_REF] and of Acetabularia [START_REF] Smahel | Vanadate-sensitive ATPase in the plasmalemma of Acetabularia: Biochemical and kinetic characterization[END_REF]. ATP is considered to be the most abundant intracellular chelating component of Mg 2+ in mammalian and plant cells [START_REF] Romani | Regulation of cellular magnesium[END_REF][START_REF] Igamberdiev | Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments[END_REF]. The dissociation constant for (Mg-ATP) 2-, the prevailing Mg-ATP species at normal physiological pH, is 28 µM at 25°C [START_REF] Smith | Critical evaluation of stability constants for nucleotide complexes with protons and metal ions and the accompanying enthalpy changes[END_REF]. Taking into account that ATP species with lower Mg 2+ affinity (mainly (H-ATP) 3-) and monovalent cations competing with Mg 2+ for free ATP are also present, an apparent dissociation constant of 71 µM was determined at pH 7.4 [START_REF] Pilotelle-Bunner | ATP binding equilibria of the Na + ,K + -ATPase[END_REF]. If the latter value is correct, then the Mg 2+ activity that could regulate H + -ATPase activity would be higher than 30 µM. Direct measurements of cytosolic Mg 2+ activity in the plant cytosol are difficult.

However, for wheat protoplasts the cytosolic Mg 2+ activity was estimated from measured adenylate concentrations to be 400 µM in the dark and 200 µM in the light [START_REF] Igamberdiev | Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments[END_REF]. Poor Mg 2+ supply from the soil and intracellular processes such as Mg 2+ sequestration, eg. with phytic acid (myo-inositol hexakisphosphate), a common constituent in eukaryotic cells and probably a major fraction of the cellular phosphate pool [START_REF] Letcher | Do mammals make all their own inositol hexakisphosphate?[END_REF]12], or Mg 2+ complexation in chloroplasts [START_REF] Wasilewska | An update on ascisic acid signaling in plants and more[END_REF], may keep or force the cytosolic Mg 2+ activity to lower levels which then leave a major fraction of ATP uncomplexed. Under such conditions, an inhibition mechanism of PM H + -ATPase by Mg 2+ -free ATP with sufficiently low inhibition constant may play a physiological role. The motivation of this study was to prove ATP inhibition of PM H + -ATPase in higher plants and to determine the kinetic parameters of ATP inhibition.

Previous kinetic studies of PM H + -ATPase from oat roots have demonstrated that ATP hydrolysis drops to 7% when Mg 2+ is omitted from the assay [START_REF] Vara | Partial purification and properties of the proton-translocating ATPase of plant plasma membrane[END_REF]. With Mg 2+ in excess of ATP the kinetics were hyperbolic and a K m for Mg-ATP of 0.3 mM was derived from double-reciprocal plots. Our investigation follows the consequences of a moderate decrease of Mg 2+ concentration down to a level of 30% of the ATP concentration, but still several-fold higher than the K m in roots of oat and maize [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF]. We show the non-hyperbolic kinetics of maize H + -ATPase in plasma membrane vesicles [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF]16,17] under various ionic conditions. Because of non-hyperbolic kinetics, non-linear regression analysis based on mechanistic models [START_REF] Marangoni | Enzyme Kinetics: A Modern Approach[END_REF]19] was employed to determine K m values and constants of ATP inhibition. The discussion of physiological relevance includes a specification of important regulatory processes which may be accompanied by a decrease of cytosolic Mg 2+ activity.

EXPERIMENTAL

Plant cultivation

Maize seeds (Zea mays L. cv. Pioneer 3906) were germinated in the dark at 20°C for 10 d. Seedlings were transferred to nutrient solution [START_REF] Zörb | Does H + pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?[END_REF] which was half-strength for 2 d, then full-strength and replaced every 3 d. Cultivar Pioneer was cultivated in the growth chamber for 16 d at a light intensity of 130 W m -2 (Master HPI-T Plus 400W, Philips, Netherlands) at 50% rel. humidity, with a day/night cycle of 16 h/8 h at 21°C/16°C. For the experiment on discrimination between inhibition mechanisms Zea mays L. cv. AMADEO (KWS, Kleinwanzleben, Germany) was grown in hydroponics for 14 d at 26°C/18°C.

Plasma membrane isolation

Plasma membrane vesicles from the lower 10 cm of roots and from young leaves were prepared by aqueous two-phase partitioning [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF]16,[START_REF] Zörb | Does H + pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?[END_REF]. Shoots were cut above the fourth leaf and immediately placed in a beaker with the cut end immersed in cold water. The lower segment of 10 cm length was cut and the mid-rib was peeled top-down and discarded. Remaining blade tissue was homogenized in the presence of 250 mM KJ in order to remove peripheral membrane proteins [START_REF] Briskin | Characterization of a K + -stimulated adenosine triphosphatase associated with the plasma membrane of red beet[END_REF][START_REF] De Michelis | H + -pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots[END_REF][START_REF] Galtier | Preparation of corn root plasmalemma with low Mg-ATPase latency and high electrogenic H + pumping activity after phase partitioning[END_REF]. Potassium iodide was not used for preparation of root vesicles [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF]. Phase partitioning was performed with a polymer concentration of 6.2% for roots and 6.1% for leaves [START_REF] Zörb | Does H + pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?[END_REF]. Vesicle aliquots were stored in liquid N 2 . Protein was quantified according to Bradford [START_REF] Bradford | A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. The purity of vesicle preparation was confirmed using specific ATPase inhibitors [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF][START_REF] Gallagher | Effect of vanadate, molybdate and azide on membraneassociated ATPase and soluble phosphatase activities of corn roots[END_REF].

Kinetic assays

In order to suppress hydrolytic activity originating from traces of unspecific phosphatases, all assays were performed in the presence of 1 mM Na 2 MoO 4 . Interference from ATP hydrolysis originating from other membranes was eliminated with two complementary methods: (a) In the vanadate method, the decrease in ATPase activity upon addition of 300 µM Na 3 VO 4 was taken as PM ATPase activity [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF][START_REF] Galtier | Preparation of corn root plasmalemma with low Mg-ATPase latency and high electrogenic H + pumping activity after phase partitioning[END_REF][START_REF] Gallagher | Effect of vanadate, molybdate and azide on membraneassociated ATPase and soluble phosphatase activities of corn roots[END_REF]. This method has the advantage that unphysiologically high KNO 3 concentrations and azide are avoided. However, since experimental errors of two hydrolytic assays contribute to final experimental error, experimental variation is inherently larger. Moreover, a higher amount of vesicle sample is required. (b) In the cocktail method, ATPase activity in the presence of 1 mM Na 2 MoO 4 , 1 mM NaN 3 and 100 mM KNO 3 was considered as PM ATPase activity. Since there was no NO 3 ATPase hydrolytic activity was determined according to Yan et al. [START_REF] Yan | Adaptation of active proton pumping and plasmalemma ATPase activity of corn roots to low root medium pH[END_REF] at 30°C with 3 µg vesicle protein in 0.5 mL of 30 mM BTP-MES buffer with 0.02% (w/v) Brij 58 (Sigma), 2 or 5 mM MgSO 4 and K salts. Experiments at 155 mM K + were performed with 50 mM KCl + 100 mM KNO 3 + 5 mM K-PEP (root, some leaf experiments) or with 150 mM KCl + 5 mM K-PEP in a leaf experiment for discriminating between inhibitory ATP species. ATP was supplied as Na 2 -ATP or diTRIS-ATP (both from Sigma), adjusted with BTP to the assay pH. At maximum activity the increase in the concentration of inorganic phosphate throughout the incubation time did not exceed 3% of the ATP concentration. Unless otherwise stated, formation of ADP and decrease of ATP was prevented by an ATPregenerating system [START_REF] Sekler | Purification and properties of a plasma membrane H + -ATPase from the extremely acidophilic alga Dunaliella acidophila[END_REF], which included 5 units of pyruvate kinase (Fluka) and 5 mM K-PEP (Fluka) or 5 mM Na 3 -PEP (Serva). With the proton/cation exchanger gramicidin for preventing H + gradient across the plasma membrane, ATPase hydrolytic activity of root vesicles was 0.284 µmol mg -1 min -1 compared to 0.271 without gramicidin (n = 3). Unless otherwise indicated, ATPase assays were performed without gramicidin.

All enzyme assays were performed with two chemical parallels. Three or four vesicle preparations from parallel plant cultivations (biological replicates) were used to determine mean values of ATPase activity and standard errors (SE).

Normalisation of ATPase activities

The normalisation procedure applied for some data sets as indicated eliminates differences between replicates, if for all ATP concentrations activities are higher (or lower) by a constant factor. For example, such differences occur when enzyme concentrations slightly differ between stored vesicle "aliquots" for replicates. For each replicate normalised activities were determined as follows: (a) calculating the sum of activities over the whole range of ATP concentrations for each replicate (individual sum), (b) determining the mean sum of all replicates, (c) multiplying the activity values of a replicate with the ratio between the mean sum and the individual sum of that replicate.

Non-linear regression analysis with DynaFit

The principle of the analysis is to define a mechanistic model from which DynaFit derives a mathematical model, i.e. a set of simultaneous rate equations including the unknown rate constants [START_REF] Kuzmic | DynaFit Scripting Manual ver 3.28[END_REF] (Supplementary Annex 1). The mathematical model is then solved with numerical methods using iterative procedures. Mean values of non-normalised activity data were subjected to DynaFit regression analysis from which rate constants and their standard deviations were obtained. Regression curves are shown in all figures. If the legend does not specify the inhibition mechanism which was applied for curve fitting, curves did not differ for competitive, uncompetitive and non-competitive inhibition. The fit of different mechanistic models was compared (model discrimination) using the Akaike information criterion [28]. The most plausible model is one with the highest Akaike weight (Max = 1). The probability of one model in comparison to another model was calculated according to Mannervik [29].

DynaFit analysis was based on concentration values in µM and rate constants per s -1 (Supplementary Annex 1). Enzyme concentration (E) was set to 0.1 nM, assuming that 1 mg plasma membrane protein contains 1% H + -ATPases with a relative molecular mass of 100000. The start value for fitting the catalytic rate constant (turnover number, k r ) was 3500 min -1  58 s -1 . Koland and Hammes [START_REF] Koland | Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase[END_REF] determined a k r of 59 µmol min -1 mg -1 for reconstituted yeast PM H + -ATPase. One mg of a protein with a relative molecular mass of 100000 corresponds to 10 nmol. Thus, this rate constant can be expressed as 5900 min -1  100 s -1 . Using a higher enzyme concentration and correspondingly lower k r start value did not change calculated values of K m and K i . Activity data were processed with the dynamic method based on the simulation of pre-steady state dynamics of the biochemical system [START_REF] Kuzmic | DynaFit Scripting Manual ver 3.28[END_REF]. In contrast to the rapid-equilibrium method this method is also valid when equilibration between enzyme and enzyme-substrate complex is slow compared to catalytic turnover. Equilibration time was set to 3 s (Supplementary Annex 1, section [progress]).

Discrimination between ATP species

In order to investigate whether a single ATP species, e.g. (K-ATP) 3-, is more inhibitory compared to others, inhibition in the presence of 155 mM K + was compared to inhibition occurring at [START_REF] Sacher | Inositol and sugars in adaptation of tomato to salt stress[END_REF] A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

(K d for (K-ATP) 3-= 68 mM; [START_REF] Smith | Critical evaluation of stability constants for nucleotide complexes with protons and metal ions and the accompanying enthalpy changes[END_REF]). Non-linear regression analysis was performed considering three cases of inhibition which differed with respect to the assumed inhibitory ATP species: either (K-ATP) 3-, (H-ATP) 3-or ATP 4-(Supplementary Annex 2). For discriminating between these ATP species the set of rate equations was extended by two equilibrium reactions for ATP 4-association with H + and K + [START_REF] Smith | Critical evaluation of stability constants for nucleotide complexes with protons and metal ions and the accompanying enthalpy changes[END_REF].

Unless otherwise indicated, inhibition constants refer to the sum of all Mg 2+ -free ATP species. If inhibition is attributed to a single ATP species, K i (1) of this species will be according to the relationship:

(1) K i (1) / K i (A) = c(1) / c(A), where K i (A) is the K i assuming that all Mg 2+ -free ATP acts inhibitory, c(1) is the concentration of the ATP species and c(A) is the concentration sum of all Mg 2+ -free ATP (tested in Supplementary Annex 3).

RESULTS

H + -ATPase kinetics in root membranes

ATP was applied up to a concentration of 5 mM in the presence of 5 mM Mg 2+ . The kinetics showed a broad maximum between 1 and 3 mM with a slight decline at highest ATP concentrations (Figure 1A). After normalisation of data, standard errors at 1 to 2 mM ATP were reduced (Figure 1A, lower error bars compared to upper), but the decrease of activity at 5 mM ATP compared to 2 mM ATP remained insignificant (t-test). Four kinetic models were analysed with DynaFit with respect to their fit to the experimental data: three models of inhibition by Mg 2+ -free ATP (competitive, uncompetitive and non-competitive) and the Michaelis-Menten model (Supplementary Annex 1). The probability of any of the inhibition models compared to the Michaelis-Menten model was 99.9%. Regression curves were identical for all models of inhibition (Figure 1A). Akaike weights were 0.332 for each of the inhibition models (Supplementary Annex 4). In a second experiment the range of ATP concentrations was extended to 13 mM. At this concentration activity decreased to 57% of maximum activity (Figure 1B, non-normalised data in Supplementary Annex 5). Again, competitive, uncompetitive and non-competitive mechanisms all yielded the same fits of regression curves and identical Akaike weights of 0.333. Thus, it was not possible to derive the mechanism of inhibition. For this purpose a third experiment was conducted in which ATPase kinetics was measured at fixed concentration of the inhibitor, i.e. of Mg 2+ -free ATP (Figure 1C, thick line). In the case of competitive inhibition the relative decrease of activity caused by the inhibitor should become smaller with increasing concentration of the substrate, i.e. of Mg-ATP. Activity without inhibition was measured in the presence of 3 mM Mg 2+ in excess of total ATP (Figure 1C, thin line). With increasing concentration of Mg-ATP relative inhibition continuously decreased from 85% at 0.1 mM Mg-ATP to 23 % at 7 mM Mg-ATP. Indeed model discrimination with DynaFit yielded the highest possible Akaike weight of 1 for competitive inhibtion and Akaike weights of 0 for uncompetitive and non-competitive inhibition (DynaFit script and output for model discrimination in Supplementary Annex 6). The probability of competitive inhibition in comparison with each of the other mechanisms was 100%.

The shapes of the activity curves slightly differed at low ATP concentrations as indicated by the arrows in Figures 1A and1B. In the first experiment (Figure 1A) maximum activity was virtually attained at 1 mM ATP, while in the second experiment (Figure 1B) activity at 1 mM ATP clearly remained below maximum activity. The first experiment was performed at a K + concentration of 55 mM (vanadate method, see Experimental Procedures Kinetic assays), while the second experiment was conducted at 155 mM K + (cocktail method with additional 100 mM KNO 3 ).

H + -ATPase kinetics in leaf membranes

PM ATPase activity was measured with the cocktail method. Compared to roots a higher ATP concentration of 4.5 mM was required to reach maximum activity of PM ATPase (Figure 2A, nonnormalised data in Supplementary Annex 5). ATPase activity significantly decreased by 33% at 8.9 mM ATP. At 13.4 mM ATP activity was only 47% of the observed maximum activity. Thus, activity was the same at 13.4 mM ATP and 0.6 mM ATP.

Influence of cations on ATP inhibition

ATP in excess of Mg 2+ is partly protonated and partly bound to K + with a K d of 68 mM [START_REF] Smith | Critical evaluation of stability constants for nucleotide complexes with protons and metal ions and the accompanying enthalpy changes[END_REF]. In the search for the inhibitory ATP species it was investigated whether inhibition was modified when [K + ] was reduced from 155 mM to 55 mM. Figure 2B shows that the activities at high ATP concentrations were not changed by K + . However, the decrease of KCl concentration caused an increase of activity at low ATP concentrations (thick line vs. thin line). In this experiment, ATP was supplied as metal-free ATP (diTRIS-ATP), while in preceding experiments Na 2 -ATP was used. Therefore, the experiment also showed that sodium was not responsible for the inhibition at high [ATP]. Furthermore, the experiment demonstrated that inhibition was not related to high ionic strength imposed by increasing the ATP concentration.

Upon increasing Mg 2+ concentration from 5 mM to 15 mM, the decrease in ATPase activity at high [ATP] was prevented (Figure 3A). On the other hand, when Mg 2+ concentration was reduced to 2 mM, inhibition of PM ATPase already started at lower ATP concentrations (Figure 3B).

In the absence of free Mg 2+ the surplus of ATP may bind to other complexing agents present in the assay solution. Since complex formation between the divalent buffer cation BTP and ATP and inhibitory action of ATP-BTP could not be ruled out, we used the monovalent TRIS instead of BTP. Figure 4A shows that inhibition also occurred in the absence of BTP. Inhibition was also observed at pH 7.5 (Figure 4B).

Kinetic constants

Kinetic constants were derived from non-linear regression analysis (Table 1). For k r and K m only values based on the model of competitive inhibition are shown since these constants were very similar for all models (deviation less than 3%). The rate of ATP hydrolysis of PM H + -ATPase is known to increase with K + concentration. Usually, a test range of up to 50 mM K + is employed [START_REF] Buch-Pedersen | Potassium as an intrinsic uncoupler of the plasma membrane H + -ATPase[END_REF]. For our leaf preparations k r was 33% higher at 155 mM K + compared to 55 mM K + . However, this increase in K + concentration did not change k r of root preparations (Table 1). K m for leaf PM ATPase was similar with Na 2 -ATP and with metal-free ATP (977 ± 70 µM and 907 ± 155 µM; mean ± SD, n =3). When the concentration of Mg 2+ was decreased from 5 mM to 2 mM in leaf preparations at 155 mM K + , the K m decreased from 907 ± 155 µM (n = 3) to 340 ± 77 µM (n = 4). When the concentration of K + was decreased from 155 mM to 55 mM, at 5 mM Mg 2+ , the K m decreased from 907 ± 155 µM to 128 ± 30 µM in leaf preparations and from 410  46 µM to 203 ± 28 µM in root preparations (n = 3). For leaves the low K m at 55 mM K + was confirmed by measuring the activity at ATP concentrations of 50, 100, 200, 600, 1000 and 3000 µM using Na 2 -ATP. K m was 92 ± 20 µM (n = 3; data not shown).

Inhibition constants shown in Table 1 refer to the sum of Mg 2+ -free ATP species. As already illustrated (Fig. 1C), K i values assuming competitive inhibition are most probable. It is evident that in leaves the high K + concentration strongly increased K i (Table 1: Exp. 4 vs. 5). At high K + concentration, leaf K i exceeded root K i (Exp. 3 vs. 2). However, leaf K m also exceeded root K m . It is clear that this change of K m is also relevant to the degree of inhibition in a mechanism in which substrate and inhibitor compete for the same site at the enzyme: At given concentrations of substrate and inhibitor an increase of K m without a change in K i decreases the activity. Thus, for comparing leaves and roots the ratio between K i and K m is an additional instructive parameter: At saturating substrate concentration, the competitive mechanism of inhibition allows to relate the ratio between K i and K m to the Mg 2+ -activity at which 50% inhibition by Mg 2+ -free ATP would occur which will be explained below. For competitive inhibition the relationship between the inhibitor concentration for 50% inhibition [ATP] 50 and the substrate concentration [Mg-ATP] is given by Eqn. (9) of Cortes et al.

6) K i / K m ≈ K d / [Mg] 50
Thus, K i / K m is inversely related to the free Mg 2+ concentration at which 50% inhibition by Mg 2+ -free ATP occurs. A ratio of 1.92 was obtained for root preparations, while ratios were between 0.32 and 1.23 for leaf preparations. The ratio decreased by 50% when ATP was supplied as metal-free ATP instead of Na 2 -ATP. If inhibition were uncompetitive or non-competitive, K i values would be in the order of 5 to 10 mM at pH 6.5 with 5 mM Mg 2+ (Table 1).

DISCUSSION

All experiments in this investigation showed that ATP in excess of Mg 2+ causes a down-regulation of PM ATPase. A first explanation could be that the enzyme is inhibited by kinases. Kinase action is without doubt a crucial mechanism to regulate the activity of PM H + -ATPase in vivo under a variety of stress conditions such as osmotic stress or salt stress [START_REF] Wasilewska | An update on ascisic acid signaling in plants and more[END_REF][START_REF] Hrabak | The Arabidopsis CDPK-SnRK superfamiliy of protein kinases[END_REF].

Consideration of kinase action

Ca 2+ -dependent kinases [32] can be ruled out, since our assays were performed without Ca 2+ . The involvement of a PKS5-like kinase [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF], which may be present in the vesicle membrane in close vicinity to H + -ATPase [START_REF] Shahollari | Receptor kinases with leucine-rich repeats are enriched in Triton X-100 insoluble plasma membrane microdomains from plants[END_REF], is very unlikely because of three arguments:

(a) Usually kinase activity also requires Mg 2+ . Our data demonstrate that inhibition increased while the MgATP concentration was constant. This means that the kinase, e.g. PKS5 [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF], should utilise a Mg 2+ -free ATP species as substrate. It is generally accepted that the conserved catalytic domain of protein kinases binds MgATP or MnATP [START_REF] Zheng | Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complex with MgATP and peptide inhibitor[END_REF][START_REF] Gong | Biochemical and functional characterization of PKS11, a novel Arabidopsis protein kinase[END_REF]. Binding of the divalent cation neutralizes the negative charges of the nucleotide, in order to facilitate phosphoryl transfer [START_REF] Zheng | Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complex with MgATP and peptide inhibitor[END_REF]. Members of the SNF1-related kinase family, to which PKS5 belongs, require Mn 2+ or Mg 2+ for phosphorylation. PKS5 is not active in kinase assays without Mn 2+ or Mg 2+ [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF]. PKS5 activity was stopped by chelating Mg 2+ or Mn 2+ in the presence of 12.5 mM EDTA [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF]. When attributing H + -ATPase inhibition to PKS5-like kinases in the absence of the complexes MgATP or MnATP, one has to assume a high-affinity binding site of the kinase for the divalent cation. The K d of the Mg-kinase complex should be smaller than 30 µM, the K d of MgATP [START_REF] Smith | Critical evaluation of stability constants for nucleotide complexes with protons and metal ions and the accompanying enthalpy changes[END_REF], in order to maintain Mg 2+ binding in the presence of free ATP. This would be unexpected since neither free ATP nor free Mg 2+ bind well to the conserved catalytic domain of protein kinases [START_REF] Armstrong | Magnetic resonance and kinetic studies of the manganese(II) ion and substrate complexes of the catalytic subunit of adenosine 3´,5´-monophosphate dependent protein kinase from bovine heart[END_REF].

(b) Inhibition by a kinase of the PKS family in our measurements is also unlikely because ATPase activity decreased at high ATP concentrations above 5 mM. Therefore, the kinase should not be saturated with ATP at 5 mM ATP. A K m value in the milimolar range would be required. Gong et al. [START_REF] Gong | Biochemical and functional characterization of PKS11, a novel Arabidopsis protein kinase[END_REF] determined a K m for ATP of 0.83 µM for another member of the PKS family (PKS11). Accordingly for PKS5, kinase assays were conducted at 10 µM ATP [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF]. Fuglsang et al. [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF] performed H + -pumping assays with plasma membrane vesicles (Figure 8A in their publication) which for PKS5 support a K m for ATP below 0.5 mM. The initial rate of H + pumping was determined at various ATP concentrations up to 3 mM in vesicles from PKS5-free plant material and after adding PKS5 to the vesicle preparation. Inhibition was already detectable at ATP concentrations of 0.5 mM. Importantly, relative inhibition did not increase at higher ATP concentrations which is consistent with the low K m reported for PKS11.

(c) A third argument against a kinase-based interpretation of our results arises from the use of a chaotropic salt in our leaf vesicle preparation. PKS5 should associate with the plasma membrane in vivo and should remain associated with plasma membrane vesicles during vesicle preparation. Fuglsang et al. [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF] clearly showed that vesicles prepared from Arabidopsis expressing the PKS5 gene had lower H + -pumping activity compared to vesicles from loss-of-function pks5 mutants. These experiments prove the presence of inhibitory phosphorylated sites in vesicle preparations. However, they do not prove that phosphorylation occurred during the pumping assay. This may already have occurred before vesicle preparation. Up to now PKS5 was not identified in plasma membrane vesicles of Arabidopsis, although other members of this kinase family have been found in lipid rafts of vesicle preparations [START_REF] Shahollari | Receptor kinases with leucine-rich repeats are enriched in Triton X-100 insoluble plasma membrane microdomains from plants[END_REF]. The SNF1-like kinase family to which PKS5 belongs does not contain integral membrane proteins [START_REF] Hrabak | The Arabidopsis CDPK-SnRK superfamiliy of protein kinases[END_REF]. Therefore, PKS5 should have been anchored in the membrane by binding to another molecule with hydrophobic character prior to vesicle preparation; or PKS5 itself should have bound to the membrane surface as a peripheral membrane protein. Anchoring could have been achieved by interaction with a CBL protein, the N-terminal myristoyl group of which may immobilise PKS kinases at the plasma membrane. Indeed, interaction of PKS5 with a CBL protein was demonstrated in vivo. However, the physiological relevance of this interaction is not yet clear since PKS5 was active in vitro without CBL association [START_REF] Fuglsang | Arabidopsis protein kinase PKS5 inhibits the plasma membrane H + -ATPase by preventing interaction with 14-3-3 protein[END_REF]. After association of PKS5 with the plasma membrane this association should be stable enough to withstand our membrane isolation procedure. In contrast to the vesicle preparations from Shahollari et al. [START_REF] Shahollari | Receptor kinases with leucine-rich repeats are enriched in Triton X-100 insoluble plasma membrane microdomains from plants[END_REF], in which CBL proteins were proven, we used the chaotropic salt KJ, in order to remove peripheral membrane proteins as outlined in our method section. This treatment removes 30-40% of total membrane protein in maize root vesicles [START_REF] De Michelis | H + -pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots[END_REF]. Therefore, our investigation most likely reveals direct effects of Mg 2+ -free ATP on PM H + -ATPase activity.

Interaction of Mg 2+ -free ATP with H + -ATPase

If the Mg 2+ activity falls below the range of the dissociation constant of the Mg-ATP complex, then Mg 2+ -free ATP will occur. One possible interaction of Mg 2+ -free ATP with H + -ATPase could be that Mg 2+ -free ATP competes with the enzyme for Mg 2+ which is required as a cofactor in P-type ATPases. In fact, for Na + /K + -ATPase the dissociation constant of Mg 2+ interaction with the E1ATP(Na 3 ) state was identical to the dissociation constant of Mg-ATP [START_REF] Pilotelle-Bunner | Mechanism of Mg 2+ binding in the Na + ,K + -ATPase[END_REF] which would cause Mg 2+ withdrawal from the enzyme upon addition of Mg 2+ -free ATP. This would also occur in the normal functional cycle, in which ATP binds to the E2 or E2P state (see next section), if the dissociation constant of the Mg 2+ interaction with respect to these enzyme states were similar or even larger compared to the E1ATP(Na 3 ) state. Another mode of inhibition would be competition between Mg-ATP and Mg 2+ -free ATP for the same enzyme site. Inhibition by Mg 2+ -free ATP has been observed for many Mg 2+ dependent ATPases. For yeast mitochondrial F 1 -ATPase Jenkins [START_REF] Jenkins | Yeast mitochondrial F 1 -ATPaseeffects of metal ions[END_REF] suggested that excess ATP inhibits by complexing free Mg 2+ assuming that it is free Mg 2+ which binds to the enzyme-ATP complex. For crayfish mitochondrial F1-ATPase Li and Neufield [40] explained the kinetic data with a classical model of competitive inhibition by Mg 2+ -free ATP.

Binding site for Mg 2+ -free ATP

Our data on inhibition by Mg 2+ -free ATP are consistent with a model of competitive inhibition (Figure 1C). Competition between Mg-ATP and Mg 2+ -free ATP for the same binding site may occur in different states of the functional cycle of P-type ATPases. According to the most widely used model of the mechanism of P-type ATPases (Albers-Post model or E1-E2 model) ATP binds to the dephosphorylated E2 state [START_REF] Clarke | Mechanisms of allosteric effects of ATP on the kinetics of P-type ATPases[END_REF]. Indeed, ATP binding to this state dramatically accelerates the E2E1 transition of other structurally related P-type ATPases, i. e. PM Na + /K + -ATPase and sarcoplasmic reticulum Ca 2+ -ATPase. However, ATP binding to the E2P phosphoenzyme of Na + /K + -ATPase was recently demonstrated utilising a fluorescent dye which revealed changes of the E2P state at Mg-ATP concentrations above 100 µM [42]. Moreover, for sarcoplasmic reticulum Ca 2+ -ATPase X-ray crystallography shows binding of an ATP analogue to the transition state of dephosphorylation (E2-P*). The authors suggest that ATP binds in exchange for ADP already in the E2P state [START_REF] Olesen | The structural basis of calcium transport by the calcium pump[END_REF]. Khalid et al. [42] showed that in the absence of K + , Mg-ATP bound to the E2P state of PM Na + /K + -ATPase plays a modulatory role by decreasing the rate of dephosphorylation. For PM Na + /K + -ATPase it is now clear "that the enzyme only has a single ATP binding site which fulfills both allosteric and catalytic functions in different parts of the reaction cycle" [42].

Competitive inhibition of PM Na + /K + -ATPase by Mg 2+ -free ATP has been first described by Hexum et al. in 1970 [44]. Robinson [START_REF] Robinson | Nucleotide and divalent cation interactions with the (Na + + K + )-dependent ATPase[END_REF] confirmed this finding. His experiments were performed at pH 7.8, at which the concentration of (H-ATP) 3-is negligible. He did not discriminate between (K-ATP) 3-and ATP 4-and determined an apparent K i of 4.8 mM. For PM H + -ATPase in maize we obtained considerably lower values between 0.01 mM and 1.1 mM depending on the ionic conditions in the assay. Competitive inhibition requires that Mg 2+ -free ATP binds at or in the ATP-binding pocket of the nucleotide-binding domain (N-domain). For PM Na + /K + -ATPase site-directed mutagenesis was exploited to identify specific amino acid residues in the N-domain which contribute to high-affinity binding of Mg 2+ -free ATP [START_REF] Jacobsen | Importance of Na,K-ATPase residue α1-Arg 544 in the segment Arg 544 -Ap 567 for high-affinity binding of ATP, ADP, or MgATP[END_REF]. Indeed, an Arg residue at the mouth of the ATP-binding pocket was proven to be important for binding of Mg 2+ -free ATP as well as Mg-ATP.

Inhibitory ATP species

At inhibitory ATP concentrations changes of K + concentration did not influence PM ATPase activity (Figure 2B), although the concentration of all major Mg 2+ -free ATP species changed (Table 2). Does this observation rule out a model of inhibition by a single ATP species? The answer has to account for the important observation that the K m for Mg-ATP decreased at low [K + ] (Table 1: Exp. 5 compared to Exp. 4). This increase of substrate affinity should increase hydrolytic activity in the presence of the inhibitory ATP species. Because this did not occur, obviously affinities increased for Mg-ATP as well as for the inhibitor. Indeed, in any mechanism of competitive inhibition a change of K m is likely to affect K i as well. When regression analysis is not forced to use the same K m and K i for both K + concentrations, the K + -dependent concentration change of a given ATP species is completely compensated by a change in K i . As a consequence good fits are obtained for any ATP species and Akaike weights are similar for all of them (Table 2). Therefore, inhibition by a single ATP species cannot be ruled out.

Physiological relevance of inhibition by Mg 2+ -free ATP

Apparent K i values for competitive inhibition by Mg 2+ -free ATP are in the range of the K m values for Mg-ATP. This means that at a given Mg-ATP concentration activity declines by 50% upon withdrawal of half the Mg 2+ from Mg-ATP (in addition to the decrease due to smaller substrate concentration). It is evident that this mechanism is particularly useful for down-regulating PM H + -ATPase activity when Mg-ATP concentrations are in the saturating range in which changes in substrate concentration have little influence on activity. Moreover, this mechanism does not require disposal or removal of Mg 2+ -free ATP, which remains available as a regulator for other processes or for fast reactivation by Mg 2+ .

Our investigation revealed differences between leaves and roots with respect to K i and K m (Table 1: Exp. 2 and 3). The two parameters changed in the same direction, but K i /K m was smaller for leaves. As explained in the Results section and utilizing a K d for Mg-ATP of 71 µM a decrease of Mg 2+ activity to a level of 63 µM ([Mg 2+ ] 50 ) would cause 50% inhibition of PM H + -ATPase in leaves, while in roots a lower Mg 2+ activity of 37 µM would be required to achieve 50% inhibition. For the leaves the control potential of ATP inhibition is substantially higher at high K + concentration with a [Mg 2+ ] 50 of 127 µM (Exp. 4). It is particularly high at pH 7.5 with a [Mg 2+ ] 50 of 222 µM. This is well within the range of estimated Mg 2+ activities in leaf mesophyll cells [START_REF] Igamberdiev | Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments[END_REF]. Control by cellular Mg 2+ could play a physiological role in leaf cells once cellular turgor has been established. Inhibition by Mg 2+ -free ATP of E1 phosphorylation could act in concert with K + -stimulated dephosphorylation of the E1P state [START_REF] Buch-Pedersen | Potassium as an intrinsic uncoupler of the plasma membrane H + -ATPase[END_REF], both preventing E1P accumulation under conditions in which the membrane potential is close to the reversal potential of the pump. Since the E1P state can phosphorylate ADP (is ADP-sensitive), E1P accumulation could cause the pump to run backward [START_REF] Buch-Pedersen | Potassium as an intrinsic uncoupler of the plasma membrane H + -ATPase[END_REF]. We did not observe the stimulatory effect of K + on ATP hydrolysis of root vesicles. An explanation could be that hydrolytic stimulation of root isoforms is already saturated at 55 mM K + .

Mechanisms which decrease cytosolic Mg 2+ activity

ATP inhibition, in vivo, of PM ATPases by Mg 2+ -free ATP depends on the existence of mechanisms, which force the cytosolic Mg 2+ activity down to the range of 30 µM, the dissociation constant of (Mg-ATP) 2-. Two types of mechanisms have to be considered [START_REF] Romani | Regulation of cellular magnesium[END_REF]: (a) Mg 2+ transport into organelles or to the extracellular space through membrane transporters or channels and (b) Mg 2+ binding by other strong chelators or macromolecules.

For the small thin cytosolic compartment of a leaf mesophyll cell the voluminous chloroplasts with their ability to take up Mg 2+ appear to represent a competitive Mg 2+ sink. Ion translocation within the chloroplast is known to have a profound influence on ionic conditions in the cytosol and on PM H + -ATPase activity. After switching off the light or when the substomatal CO 2 concentration suddenly increases massive proton release from the thylakoid lumen to the chloroplast stroma occurs. Also within the cytosol proton activity transiently increases [START_REF] Felle | Light-induced cytoplasmic pH changes and their interrelation to the activity of the electrogenic proton pump in Riccia fluitans[END_REF]. At the plasma membrane both stimuli immediately invoke a transient hyperpolarisation [START_REF] Felle | Light-induced cytoplasmic pH changes and their interrelation to the activity of the electrogenic proton pump in Riccia fluitans[END_REF]48]. At the same time apoplastic pH decreases [49] consistent with a transient stimulation of PM H + -ATPase. While protons pass the thylakoid membrane, Mg 2+ ions move in the opposite direction for charge compensation. When the light is turned on, Mg 2+ is transported to the stroma and is bound to the N-carboxylated Rubisco thereby activating it. It is clear that upon light-off Mg 2+ must be released to be bound again upon the next exposure to light. Release probably causes a transient increase in stromal Mg 2+ activity [START_REF] Igamberdiev | Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments[END_REF]. Evidence indicates that after switching off the light the stromal Mg 2+ concentration eventually decreases by several mM. Taking into consideration that Mg 2+ can freely move through the outer chloroplast membrane, not only protons, but also Mg 2+ may leave the chloroplast upon light-off and may contribute to the transient stimulation of PM H + -ATPase by complexing Mg 2+ -free ATP.

For plant cells information on Mg 2+ transport, its affinity for Mg 2+ and its regulation is very limited [START_REF] Amtmann | Regulation of macronutrient transport[END_REF]. There is one report on a candidate Mg 2+ transporter of Arabidopsis thaliana which restores intramitochondrial Mg 2+ concentration in a yeast mutant. Membrane transporters for Mg 2+ are better investigated in mammalian cells. The channel kinase TRPM7 mediates Mg 2+ uptake from the extracellular space into the cytosol and is essential for cellular viability [51]. This channel is strongly down-regulated by intracellular Mg 2+ activity, MgATP and Mg-nucleotides thereby restricting cytosolic access of Mg 2+ . Massive, cAMP-mediated Mg 2+ release from mitochondria into the cytosol occurs upon catecholamine administration [START_REF] Romani | Regulation of cellular magnesium[END_REF]. Mg 2+ release from isolated mitochondria upon administration of cAMP is accompanied by an increase in respiration rate. The underlying activity changes of mitochondrial enzymes could be caused by a decrease of matrix Mg 2+ activity. In the intact cell, relief of ATP inhibition of the Na + /K + -ATPase, which may consume 20-30% of the cellular ATP [START_REF] Jacobsen | Importance of Na,K-ATPase residue α1-Arg 544 in the segment Arg 544 -Ap 567 for high-affinity binding of ATP, ADP, or MgATP[END_REF], by additional Mg 2+ could boost ATP hydrolysis and thus contribute to enhanced respiration. Besides ATP a number of Mg 2+ chelators are related to the regulation of metabolic activity. In chloroplasts Mg-chelatase activity and Mg-protoporphyrin concentration increase upon abscisic acid application [START_REF] Wasilewska | An update on ascisic acid signaling in plants and more[END_REF] suggesting a mechanism for hormone-stimulated Mg 2+ withdrawal from the cytosol. In non-phytosynthetic tissue the cytosolic Mg 2+ activity may be decreased by synthesis of phytic acid. Phytic acid probably exists mostly as a penta-Mg 2+ salt in vivo [START_REF] Torres | Solution behavior of myo-inositol hexakisphosphate in the presence of multivalent cations. Prediction of a neutral pentamagnesium species under cytosolic /nuclear conditions[END_REF]. Abscisic acid induces the enzyme inositol-3-phosphate synthase leading to the accumulation of phytic acid in duckweed [START_REF] Flores | Abscisic acid-induced changes in inositol metabolism in Spirodela polyrrhiza[END_REF]. For roots of maize seedlings it has been proven that exogeneous phosphate is readily incorporated into phytic acid [START_REF] Huebel | Maize root phytase[END_REF]. The level of the phytic acid precursor myo-inositol increased to 75% of soluble carbohydrates in roots of salt-adapted tomato plants [START_REF] Sacher | Inositol and sugars in adaptation of tomato to salt stress[END_REF]. Another physiological process involving Mg 2+ binding to phosphate groups is polyamine catabolism. Upon degradation of these abundant polycations their binding sites at DNA, RNA and proteins will adsorb free Mg 2+ [56].

There is evidence that ABA responsiveness of guard cells requires attenuation of H + pumping in addition to activation of anion channels [START_REF] Wasilewska | An update on ascisic acid signaling in plants and more[END_REF]. For activation of the guard cell anion channel GCAC1 ATP is required, but for this PM transporter the Mg 2+ -free nucleotide is sufficient for activation [START_REF] Schulz-Lessdorf | GCAC1 recognizes the pH gradient across the plasma membrane: a pH-sensitive and ATP-dependent anion channel links guard cell membrane potential to acid and energy metabolism[END_REF]. Because a large number of ABA responsive genes are triggered by Mg 2+ deficiency [START_REF] Hermans | Early transcriptomic changes induced by magnesium deficiency in Arabidopsis thaliana reveal the alteration of circadian clock gene expression in roots and the triggering of abscisic acid-responsive genes[END_REF], it is clear that ABA responses are not hampered by Mg 2+ deficiency. It is also conceivable that ABA responses may impose a physiological Mg 2+ deficiency on the cytosolic compartment.

For PM Na + /K + -ATPase not only inhibition by Mg 2+ -free ATP was found, but also inhibition by Mg-ADP in a competitive manner [START_REF] Robinson | Nucleotide and divalent cation interactions with the (Na + + K + )-dependent ATPase[END_REF]. In our assays ADP was also inhibitory (Fatima, unpublished). This is evident from assays which were performed with root vesicles at pH 6.5 in the presence of 5 mM ATP, 5 mM Mg 2+ and 155 mM K + , but without ATP-regenerating system. Derived from the increase in phosphate concentration, ADP accumulated to a concentration of 0.15 mM. Omission of the ATP-regenerating system decreased PM H + -ATPase activity by 15% ± 2 % (n = 4). Possibly, the inhibition mechanisms of Mg 2+ -free ATP and of Mg-ADP are closely related. Certainly, these mechanisms contribute to the complex metabolic and mineral requirements of PM H + -ATPase for maximum hydrolytic activity. With respect to mineral requirements, kinetic properties of PM H + -ATPase and emerging patterns of regulation of several intracellular Mg 2+ chelators indicate that ATP inhibition of PM H + -ATPase could be involved in regulatory mechanisms for reducing the activity of this "powerhouse" enzyme during stages of growth reduction, growth arrest or during the transition from active growth to dormancy.

ACKNOWLEDGEMENT

This work would not have been possible without the sound expertise of PD Dr. Feng Yan with respect to plasma membrane vesicle preparations from maize plants. Technical assistance of Ms. Anne Weber is gratefully acknowledged.

Figure 3 Influence of [Mg 2+ ]

A) Recovery of ATPase activity after addition of Mg 2+ . The assay was performed with vesicles from leaves at Mg 2+ concentrations of 5 mM (squares) and 15 mM (circles) with 150 mM K + at pH 6.5. ATP was provided as diTRIS-ATP. The two Mg 2+ concentrations were tested in the same assay. No ATP-regenerating system was used. B) Inhibition of plasma membrane ATPase activity at low [Mg 2+ ]. Assays were performed with vesicles from leaves at 2 mM Mg 2+ and 155 mM K + at pH 6.5. ATP was provided as diTRIS-ATP. Data are means  SE (n = 4).

Figure 4 Inhibitory effect of Mg 2+ -free ATP with different buffers

A) Inhibition in the presence of divalent (BTP, circles) or monovalent (TRIS, squares) amino buffer cations. The concentration of buffer cations was 30 mM. In both cases MES was used as acidic buffer component. The assay was performed with vesicles from leaves at 5 mM Mg 2+ , 55 mM K + with diTRIS-ATP at pH 6.5. The two buffer cations were tested in the same assay procedure. Regression curve refers to BTP. B) Inhibition at pH 7.5 (circles) in comparison with pH 6.5 (triangles, from Figure 2B). Assays were performed with vesicles from leaves in BTP buffer at 5 mM Mg 2+ and 55 mM K + with diTRIS-ATP. Data are means + SE (n = 2). A c c e p t e d M a n u s c r i p t

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TABLES Table 1 Kinetic constants of plasma membrane ATPase from maize roots and leaves

Assays were performed with different ATP reagents under the specified ionic conditions (number denotes ion concentration in mM). Catalytic rate constant k r (s -1 ), Michaelis-Menten constant K m (µM) and inhibition constant K i (µM) were calculated assuming competitive (c), uncompetitive (u) and noncompetitive (n) inhibition without discriminating between ATP species (mean ± SD). k r and K m refer to (c), but deviation with (u) and (n) was less than 3% (all data in Supplementary Annex 4). In lines "3sep" and "4sep" mean value and SD was calculated from kinetic constants determined separately for each replicate. n. d.: not determined because of small range of inhibitory ATP concentrations (see results). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Exp. ATP reagent Ionic conditions n

k r K m K i K i /K m Root 1 Na 2 -ATP 5

Table 2 Discrimination between different Mg 2+ -free ATP species

The enzyme activity data from the experiment at different [K + ] (Figure 2B) were analysed based on three models of competitive inhibition by a single ATP species. For each ATP species K i (µM) was calculated (Supplementary Annex 2). Percentage values denote ratio between K i of the species and the sum of K i values at the given [K + ]. This ratio reflects the molar composition of the Mg 2+ -free ATP pool. The fit of each of the models to the experimental data was expressed as Akaike weight. Model (ATP species) with highest Akaike weight is most plausible, but here differences are negligible.

ATP

  [START_REF] Cortes | Relationship between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data[END_REF]:[START_REF] Chevalier | 14-3-3 and FHA domains mediate phosphoprotein interactions[END_REF] [ATP] 50 = K i(1 + [Mg-ATP]/K m ) Dividing by [Mg-ATP] yields (3) [ATP] 50 / [Mg-ATP] = K i (1/[Mg-ATP] + 1/K m ) At saturating substrate concentrations, 1/[Mg-ATP] becomes negligible on the right side of the equation and (4) [ATP] 50 / [Mg-ATP] ≈ K i / K m Since the dissociation equilibrium of Mg-ATP is given by (5) [ATP] 50 [Mg] 50 / [Mg-ATP] = K d where all concentrations refer to the free species, it follows that

(

Biochemical

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FIGURE LEGENDS Figure 1

 1 FIGURE LEGENDSFigure 1 Effect of Mg 2+ -free ATP on plasma membrane ATPase activity from roots Assays were performed with vesicles (cytosolic side outside) at pH optimum (6.5) using Na 2 -ATP adjusted to pH 6.5. Released ADP was converted to ATP with pyruvate kinase and PEP. A) Experiment at ATP concentrations up to 5 mM at 5 mM Mg 2+ and 55 mM K + . Upper error bar denotes SE of original data, lower error bar SE of normalised data (n = 3). Regression curves by DynaFit assuming Michaelis-Menten kinetics (dashed line) or inhibition by Mg 2+ -free ATP (solid line, script in Supplementary Annex 1). Curves were identical for competitive, uncompetitive and non-competitive inhibition. B) Experiment at ATP concentrations up to 13 mM at 5 mM Mg 2+ and 155 mM K + . Gramicidin was used to prevent a pH gradient at the vesicle membrane. Data are means + SE of normalised data (n = 3). Regression curves calculated by DynaFit were identical for all tested inhibition types. C) Mg 2+ -free ATP is a competitive inhibitor. Thick line: activity in the presence of 2 mM Mg 2+ -free ATP. Thin line: activity with 3 mM Mg 2+ in excess of total ATP at all ATP concentrations (no inhibition). K + concentration was 155 mM. Data are means  SE of original data (n = 3). Regression curves by DynaFit assuming competitive inhibition.

Figure 2

 2 Figure 2Effect of Mg 2+ -free ATP on plasma membrane ATPase activity from leaves Assays were performed with ATP regeneration at pH 6.5 and at 5 mM Mg 2+ . A) Inhibition with two different ATP reagents: Na 2 -ATP (circles) and diTRIS-ATP (squares). K + concentration was 155 mM. Data are means + SE of normalised data (n = 3). For diTRIS-ATP SE was omitted for clarity and is shown in Figure2B. Regression curves calculated by DynaFit were identical for all tested inhibition types. B) Inhibition at different K + concentrations: 155 mM (squares) and 55 mM (triangles). ATP was provided as diTRIS-ATP. Data from experiment at 155 mM K + were already shown in Fig.2A. Data are means  SE (n = 3).

A c c e

  p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Publication. Published on 19 Jan 2011 as manuscript BJ20101414 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101414 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  Fig. 1

  Fig. 2

  Fig. 3

Fig. 4

 4 Fig. 4

  mM K +

	Biochemical Journal Immediate Publication. Published on 19 Jan 2011 as manuscript BJ20101414
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101414

  Biochemical Journal Immediate Publication. Published on 19 Jan 2011 as manuscript BJ20101414 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101414

				Mg, 55 K	3	72 ± 2,8	203 ± 28	n. d.
								c: 787 ± 108	c: 1.92
		2	Na 2 -ATP	5 Mg, 155 K	3	64 ± 2.2	410 ± 46	u: 9637 ± 1386
								n: 10410 ± 1464
								c: 1102 ± 80	c: 1.13
		3				179 ± 5.1	977± 70	u: 5666 ± 458
			Na 2 -ATP	5 Mg, 150 K	3			n: 6749 ± 507
		3sep				181 ± 18	1041 ± 268	c: 1133 ± 180
								c: 511 ± 68	c: 0.56
		4				144 ± 8.8	907 ± 155	u: 2843 ± 398
			diTRIS-ATP 5 Mg, 155 K	3			n: 3346 ± 419
	Leaf	4sep				147 ± 19	972 ± 426	c: 512 ± 37
								c: 129 ± 26	c: 1.01
		5	diTRIS-ATP 5 Mg, 55 K	3 108 ± 2.5	128 ± 30	u: 5085 ± 407
								n: 5207 ± 403
								c: 419 ± 95	c: 1.23
		6	diTRIS-ATP 2 Mg, 155 K	4 101 ± 7.7	340 ± 77	u: 2493 ± 670
								n: 2900 ± 730
								c: 8 ± 4	c: 0.32
		7	diTRIS-ATP 5 Mg, 55 K, pH 7.5 2	67 ± 3.1	24 ± 11	u: 1645 ± 457
								n: 1652 ± 458

  Biochemical Journal Immediate Publication. Published on 19 Jan 2011 as manuscript BJ20101414 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101414

	species	K i at 155 mM K +	Akaike weight at	K i at 55 mM K +	Akaike weight at
				155 mM K +			55 mM K +
	(H-ATP) 3-	150	28%	0.34380	60	41%	0.35495
	(K-ATP) 3-	269	50%	0.31140	38	26%	0.28737
	ATP 4-	119	22%	0.34481	47	33%	0.35768

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

FUNDING This work was supported by the National Basic Research Program of China (2007CB109303), by the Natural Science Foundation of China (30871588) and by the China Scholarship Council (2007832114).