H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.     

Dual effect of ADP on the activity of the plasma membrane H+‐ATPase of red beet

The effect of ADP on the activity of the plasma membrane (PM) H⁺‐ATPase of red beet ( Beta vulgaris L.) parenchyma discs was evaluated by analyzing the effect of increasing concentrations of ADP on the kinetics of the reaction. When the PM H⁺‐ATPase activity was assayed at pH 6.3, ADP behaved as a simple competitive inhibitor. When the activity was assayed at pH 7.1, ADP not only increased the apparent K_m for MgATP but also decreased the V_max of the reaction. When the C‐terminal domain of the PM H⁺‐ATPase was cleaved by controlled trypsin treatment or displaced by addition of lysophosphatidylcholine, only the competitive component of inhibition by ADP of the activity assayed at pH 7.1 was evident. The results are discussed in relation to the physiological relevance of the activation of the PM H⁺‐ATPase by displacement of the autoinhibitory C‐terminal domain.     

The plasma membrane H+‐ATPase from maize roots is phosphorylated in the C‐terminal domain by a calcium‐dependent protein kinase

A protein kinase activity associated with maize root plasma membranes was partially purified and characterized. Biochemical properties, such as calcium dependence, inhibition by calmodulin antagonists, and absence of calmodulin stimulation, indicated that the enzyme belongs to the calcium‐dependent protein kinase (CDPK) family. By means of an in‐gel phosphorylation assay the molecular mass of active polypeptides was determined: two bands of 55 and 51 kDa became labelled. The same proteins were also immunodecorated by monoclonal antibodies against soybean CDPK. Results from in vitro assays demonstrated that maize H⁺‐ATPase was a suitable substrate for this protein kinase and that the phosphorylation site was located at the C‐terminal domain of the enzyme. This result was confirmed by using as substrate in phosphorylation assays the isolated C‐terminal domain of the H⁺‐ATPase expressed in Escherichia coli as a glutathione‐transferase fusion protein.     

Identification and purification of the calcium‐regulated Ca2+‐ATPase from the endoplasmic reticulum of a higher plant mechanoreceptor organ

Erythrosin b, a potent inhibitor of the Ca²⁺‐ATPases and the Ca²⁺‐release channel (BCC1) in mechanosensitive tissue of Bryonia dioica Jacq., effectively suppresses a tendril's reaction to touch, suggesting that Ca²⁺‐transporters are involved in signal transduction in this organ. The Ca²⁺‐ATPase located in the endoplasmic reticulum (ER) represents a multiregulated enzyme that is stimulated by calmodulin (CaM), KCl and lysophospholipids. Limited proteolysis of ER‐membranes by trypsin results in an irreversible activation of the Ca²⁺‐ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domain where CaM binds. Mild trypsination mimics the effects of CaM on V_max and the affinity for Ca²⁺ and ATP. Irrespective of a trypsin treatment, the enzyme can be additionally stimulated by KCl and lysolipids, indicating that the sites of interaction for these effectors are not located in the domain removed by the protease. CaM‐stimulated ATPase activity was purified from microsomal and ER fractions using a combination of CaM‐affinity and anion‐exchange chromatography. The isolated polypeptide was enzymatically active, showed a calcium‐dependent mobility‐shift in SDS‐PAGE from 109 kDa in the absence of Ca²⁺ to 104 kDa in the presence of 10 m M CaCl₂ and could be radiolabeled with [³⁵S]‐CaM. The characteristics of the purified enzyme remained closely similar to those of the ER‐bound Ca²⁺‐transporting activity, including the enzymatic data, CaM stimulation, and the sensitivity towards a range of inhibitors.     

Aluminum impairs morphogenic transition and stimulates H(+) transport mediated by the plasma membrane ATPase of Yarrowia lipolytica

The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5–1.0 mM AlK(SO₄)₂) inhibits yeast–hypha transition. Both vanadate-sensitive H⁺ transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H⁺ pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H⁺ efflux in solid medium. The present results suggest that alterations in the plasma membrane H⁺ transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H⁺-ATPase as a key factor of this process.     

Blue light‐induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases

The phytohormone abscisic acid (ABA) inhibits blue light‐induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA‐insensitive ( abi ) mutants of Arabidopsis thaliana . Apoplastic acidification was monitored with a flat tipped pH‐electrode placed on epidermal strips, in which only guard cells were viable. Blue light‐induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane‐bound H⁺‐ATPases. In wild type epidermal strips, ABA reduced blue light‐induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca²⁺ concentration in guard cells, since factors that increase the Ca²⁺ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2 . The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K‐252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2 . The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.     

Pre‐migratory differentiation of wild brown trout into migrant and resident individuals

In February to March, wild brown trout Salmo trutta were captured by electrofishing in a natural watercourse (tributaries of the River Lille Aa, Denmark), individually tagged (Passive Integrated Transponders), and released. Representatives of the tagged brown trout were recaptured on the release sites in April by electrofishing and eventually caught in downstream smolt traps ('migrants') placed in the main river or by electrofishing ('residents') on the initial sites in June. Upon each capture, smolt appearance and body size were evaluated, and a non‐lethal gill biopsy was taken and used for Na⁺,K⁺‐ATPase analysis. Based on repetitive gill enzyme analysis in individual fish, a retrospective analysis of the rate of development in individual brown trout ultimately classified as migrants or residents was performed. Two months prior to migration, a bimodal morphological and physiological (gill Na⁺,K⁺‐ATPase) development concurred and was related to the subsequent differentiation into resident and migratory fractions of each population. This differentiation was unrelated to growth rate and body size of individual fish but skewed in favour of migratory females. Individuals destined to become migrants developed a smolt‐like appearance before the onset of migration and had higher rate of change of gill Na⁺,K⁺‐ATPase activity than fish remaining residents. The rate of change of gill Na⁺,K⁺‐ATPase activity was independent of the distance migrated to the trap (3–28 km). Thus in bimodal wild brown trout populations a major increase in enzyme activity takes place before migration is initiated and is a characteristic of migratory individuals only.     

Effects of salt stress on H+- ATPase and H+-PPase activities of tonoplast-enriched vesicles isolated from sunflower roots

The control of ion concentration in the cytosol and the accumulation of ions in vacuoles are thought to be key factors in salt tolerance. These processes depend on the establishment in vacuolar membranes of an electrochemical H⁺ gradient generated by two distinct H⁺-translocating enzymes: a H⁺-PPase and a H⁺-ATPase. H⁺-lrans locating activities were characterized in tonoplast-enriched membrane fractions isolated by sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to different NaCl regimes. The 15/32% sucrose interface was enriched in membrane vesicles possessing a vacuolar-type H⁺-ATPase and a H⁺-PPase, as indicated by inhibitor sensitivity, pH optimum, substrate specificity, ion effects kinetic data and immunolabelling with specific antibodies. Mild and severe stress did not alter the pH profile, ion dependence, apparent K_m nor the amount of antigenic protein of either enzyme. Saline treatments slightly increased K⁺-stimulaied PPase activity with no change in ATPase activity, while both PP_i-dependent and NO₃-sensitive ATP-dependent H⁺ transport activities were strongly stimulated. These results are discussed in terms of an adaptative mechanism of the moderately tolerant sunflower plants to salt stress.     

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H⁺-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae , even after deletion of the enzyme's carboxy terminus. Functional chimerical H⁺-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H⁺-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H⁺-ATPase. The reverse Ala-598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H⁺-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K _m values for MgATP and decreased V _max compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H⁺-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Apoplastic and membrane‐associated Ca2+ in leaves and roots as affected by boron deficiency

A combination of fluorescein‐isothiocyanate (FITC), coumarin‐benzothiazol (BTC), and chlorotetracycline (CTC) fluorescence was used to simultaneously monitor apoplastic pH, apoplastic free Ca²⁺, and plasma membrane‐bound Ca²⁺. As early boron deficiency reactions supposedly include alterations of plasma membrane‐bound transport processes besides rapid effects on cell wall physical properties, the corresponding changes were followed in leaves and roots of Vicia faba L. cv. Troy.
Boron deficiency did not alter the apoplastic pH, but it reduced plasma membrane‐bound Ca²⁺ in roots at 4 h and leaves at 3 days after starting the deficiency treatment. The decrease in plasma membrane‐bound Ca²⁺ coincided with an increase in apoplastic free Ca²⁺ and K⁺, and occurred before the first visible symptoms were noticed.
It is proposed that less Ca²⁺ is bound to the plasma membrane due to a reduction of specific Ca²⁺‐binding sites (borate esters with vic ‐diols or polyhydroxy‐carboxylates) before plasma membrane integrity deteriorates.     

The tonoplast H+-pyrophosphatase of radish seedlings: biochemical characteristics

The activity of the H⁺-pyrophosphatase (H⁺-PPase) was characterized in microsomes from 24-h-old radish ( Raphanus sativus L., ev. Tondo Rosso Quarantino) seedlings, which are virtually devoid of the tonoplast H⁺-ATPase. The H⁺-PPase was localized to membranes which roughly comigrated with the plasma membrane in a sucrose density gradient, but clearly separated from plasma membrane when microsomes were partitioned in an aqueous dextran-polyethylene glycol two-phase system. The H⁺-PPase activity was strictly dependent on Mg²⁺ and on the presence of a monovalent cation (K⁺=Rb⁺=NH₃⁺Cs⁺≫Na⁺Li⁺) and was insensitive to anions such as Cl−, Br−, NO₃− and SO₄^2-. It was inhibited by F−, imidodiphosphate and Ca²⁺. It had a pH optimum between pH 7.5 and 8.5 and was saturated by low concentrations of pyrophosphate (half saturation at 30 μ M pyrophosphate). All of these characteristics are identical to those reported for the tonoplast H⁺-PPase from various plant materials. The functional molecular weight of the H⁺-PPase, measured with the radiation-inactivation technique was 96 kDa.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock

Modulation of proton extrusion and ATP-dependent H⁺ transport through the plasma membrane in relation to the presence of 14-3-3 proteins in this membrane in response to osmotic shock was studied in tomato ( Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H⁺ extrusion by cells was activated rapidly and significantly after adding 100 m M NaCl, 100 m M KCl, 50 m M Na₂SO₄, 1.6% sorbitol or 2 µ M fusicoccin to the medium. The increase in H⁺ extrusion by cells treated with 100 m M NaCl was correlated with an increase of H⁺ transport by the plasma membrane H⁺-ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14-3-3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 m M NaCl as compared to control cells without changing the amount of plasma membrane H⁺-ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14-3-3/H⁺-ATPase complex.     

Electrical signalling and cytokinins mediate effects of light and root cutting on ion uptake in intact plants

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1–2 min) effects on net K⁺ and H⁺ transport at the root surface. H⁺ efflux decreased (from −18 to −12 nmol m⁻² s⁻¹) and K⁺ uptake (∼2 nmol m⁻² s⁻¹) reverted to efflux (∼−3 nmol m⁻² s⁻¹). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a ∼3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H⁺ efflux and stopped K⁺ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 m m NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (−50 nmol m⁻² s⁻¹). Kinetin (2–4  µ m ), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.     

Calcium transport and ATPase activity in a microsomal vesicle fraction from corn roots

Abstract. An investigation has been made of methods for isolating membrane vesicles from corn ( Zea mays L.) roots active in calcium transport and K⁺-stimulated ATPase. Pretreating and grinding the roots at room temperature with EGTA and fusicoccin increases basal ATPase activity. Improvement in Ca²⁺ uptake requires isolation of a scaled vesicle fraction by the method of Sze(1980). Sorbitol is superior to sucrose as an osmoticant. The pH optimum for Ca²⁺ uptake is 7.5. whereas that for associated ATPase activity is 6.5. Calmodulin strongly stimulates Ca²⁺ uptake in a process little affected by uncouplers and ATPase inhibitors, but blocked by chlorpromazine. Fusicoccin gives less stimulation of Ca²⁺ uptake which is sensitive to uncouplers, and is dependent upon isolation with fusicoccin present. It appears that the sealed vesicle fraction may possess two Ca²⁺ transport systems: a calmodulin-activated Ca²⁺-transporting ATPase, and a Ca²⁺/H⁺ antiport coupled through the protonmotive force to a fusicoccin-stimulated H⁺-ATPase.     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+-ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere-Derived Cells.

Embryos kept with omeprazole, a specific H⁺, K⁺-ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro-mere-derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole-sensitive H⁺, K⁺-ATPase, an H⁺pump, seems to be indispensable for CaCO₃ deposition (formation of spicule rod) in these spicule forming cells. H⁺, produced in overall reaction for CaCO₃ formation: Ca²⁺+ CO₂+H₂O°CaCO₃+2H⁺, is probably released from the cells by this H⁺pump and hence, this reaction tends to go to CaCO₃ production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H⁺, K⁺-ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H⁺ejection in H⁺, K⁺-ATPase reaction.