H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase

The H⁺/ATP stoichiometry was determined for an anion-sensitive H⁺-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A₃₄₀) or by monitoring the release of ³²P from [γ-³²P]ATP. Inasmuch as this anion-sensitive H⁺-ATPase is strongly inhibited by NO₃⁻, initial rates of H⁺ influx and ATP hydrolysis were measured in the absence and presence of NO₃⁻ to account for ATPase activity not involved in H⁺ transport. The NO₃⁻-sensitive activities were calculated and used to estimate the ratio of H⁺ transported to ATP hydrolyzed. These measurements resulted in an estimate of the H⁺/ATP stoichiometry of 1.96 ± 0.14 suggesting that the actual stoichiometry is 2 H⁺ transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H⁺ across the tonoplast measured in vivo, our result suggests that the H⁺-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample

A continuous spectrophotometric assay of H⁺-ATPase activity was developed by combining two well-known methods for measuring proton pumping and ATPase activity. Proton uptake into plasma membrane vesicles from Avena sativa L. (cv Rhiannon) was monitored as the absorbance decrease at 495 nm of the ΔpH probe acridine orange. Simultaneously, ATPase activity was measured by following the absorbance decrease at 340 nanometers by coupling ATP hydrolysis enzymatically to the oxidation of NADH. This H⁺-ATPase assay is convenient for determining the relative relationship between ATP hydrolysis and proton pumping.     

Regulation of the structure and catalytic properties of plasma membrane H<Superscript>+</Superscript>-ATPase involved in adaptation of two reed ecotypes to their different habitats

The properties and kinetics of ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activities of plasma membrane H⁺-ATPase from the two reed ecot ypes, swamp reed (SR) and dune reed (DR), were investigated. The pH optimum of the plasma membrane H⁺-ATPase in both reed ecotypes was similar but the sensitivity of the enzyme to the reaction medium pH seemed to be higher in DR than that in SR. Compared to SR, the DR exhibited a higher V_max value for ATP hydrolysis whereas the K_m value was almost similar in both reed ecotypes. The PNPP hydrolysis of the plasma membrane H⁺-ATPase was also studied in both reed ecotypes at increasing PNPP concentrations. K_m and V_max for PNPP hydrolysis showed great differences in the two reed ecotypes and in DR the K_m and V_max values were 2- and 10-fold, respectively, higher than those in SR. The ATP hydrolysis activity of the plasma membrane was markedly inhibited by hydroxylamine in both reed ecotypes, and the percentage inhibition of ATP hydrolysis rate seemed higher in DR than that in SR. In addition, the structure or property of the C-terminal end of the plasma membrane H⁺-ATPase were also different in the two reed ecotypes. These data suggest that different isoforms of the plasma membrane H⁺-ATPase might be developed and involved in the adaptation of the plant to the long-term drought-prone habitat.This research was supported by Natural Science Foundation of China (No. 30270238 & No. 30470274) and the National Key Basic Research Special Funds of China (G1999011705).     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma membrane vesicles

Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H⁺-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H⁺ was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H⁺-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, K_m for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H⁺-ATPase activity in vivo.     

Effects of freezing on plasma membrane H<Superscript>+</Superscript>-ATPase of the callus from <Emphasis Type="Italic">Chorispora bungeana</Emphasis>

The influence of freezing treatment on plasma membrane (PM) H⁺-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (−4 °C) for 5 d resulted in significant increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K_m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that PM H⁺-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana.     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.     

Drought stress stimulates p-nitrophenyl phosphate hydrolysis rate of the plasma membrane H+-ATPase from wheat leaves

The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H⁺-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The K_m for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H⁺-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H⁺-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H⁺-ATPase, therefore increasing the catalytic activity of the phosphatase domain.     

A novel method to quantify H-ATPase-dependent Na transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg^− 1 membrane protein min^− 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.     

Regulation of Glycolytic Oscillations by Mitochondrial and Plasma Membrane H-ATPases

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H⁺-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F₀F₁-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F₀F₁-ATPase and plasma membrane H⁺-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin

The effect of fusicoccin on Mg:ATP-dependent H⁺-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ΔpH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H⁺-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H⁺-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H⁺-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H⁺-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H⁺-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H⁺-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V_max of the plasma membrane H⁺-ATPase rather than a decrease in its apparent K_m for Mg:ATP.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

Simulation of the light-induced oscillations of the membrane potential in Potamogeton leaf cells

Summary An attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H⁺-ATPase with a variable H⁺/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H⁺ pump shows the characteristics of either a pump or a passive H⁺ conductance: the mode of operation of the H⁺ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H⁺ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.This research was supported by the Foundation of Biophysics, part of the Dutch Organization for Scientific Research (NWO) ECOTRANS publication No. 34.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H-ATPase

Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P₂-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H⁺-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H⁺ pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H⁺ transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H⁺ transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca²⁺ binding in the Ca²⁺-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H⁺-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H⁺-ATPase.