Effect of abscisic acid on membrane-bound epidermal ATPase from tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase activity in epidermal stripsfrom tobacco leaves (Nicotiana tabacum L. Samsun NN) was stimulatedby abscisic acid (ABA) when the strips were floated on ABA solutionin light or in darkness. The optimum ABA concentrations in lightand in darkness were 10^–5 M and 10^–6 M, respectively.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) completely blocked the basal level membrane-bound epidermalATPase activity. ABAinduced membrane-bound epidermal ATPaseactivity was completely inhibited by CCCP, but only partly byDCCD. H⁺-influx into epidermal strips on a solution in light was lowerthan that in darkness. ABA stimulated H⁺-influx into epidermalstrips in light and in darkness. CCCP suppressed basal levelH⁺-influx, whereas DCCD did not. CCCP also suppressed ABA-inducedH⁺-influx, whereas DCCD did not. Interaction between H⁺-influxand membranebound epidermal ATPase activity is discussed. (Received May 23, 1978; )     

Presence of a Na+-activated ATPase in the Plasma Membrane of the Marine Raphidophycean Heterosigma akashiwo

Highly purified plasma membranes were isolated from Heterosigmaakashiwo cells, a marine raphidophycean unicellular biflagellate,by the silica microbead method, and the ATPase activity of themembranes was characterized. The ionic requirements and spectrumof effective inhibitors enable us to identify a novel Na⁺-activatedATPase in the plasma membrane of this organism. Furthermore,we detected two phosphorylated intermediate forms of ATPases,with molecular weights of 150 kDa and 95 kDa as judged by acidSDS-polyacrylamide gel electrophoresis of extracts of isolatedplasma membrane. The 150 kDa intermediate was phosphorylated in the presenceof both Mg²⁺ and Na⁺, while the 95 kDa intermediate was phosphorylatedin the presence of Mg²⁺ alone. Both were dephosphorylated inthe presence of monovalent cations. These results indicate thatthe former intermediate was a Na⁺-activated ATPase, similarto Na⁺,K⁺-ATPases from animals, and the latter was similar toH⁺,K⁺-ATPases from higher plants. The physiological significanceof the two kinds of ATPase in the plasma membrane of marinealgae. (Received March 15, 1989; Accepted June 23, 1989)     

Plasmalemma ATPase activity modifications induced by traumatisms in Bidens pilosa

It has previously been shown that cotyledonary pricks inducedmodifications of ion levels (H⁺ and K⁺) in hypocotyl cells ofBidens pilosa. These modifications differed according to thelight quality: H ⁺ levels increased and K⁺ levels decreasedin white light (WL), whereas H⁺ levels decreased and K⁺ levelsincreased in blue light (BL). In this study, in order to determinethe mechanism responsible for these ionic modifications, plasmamembrane vesicles have been isolated and characterized fromhypocotyl cells. The effects of light quality and cotyledonarypricks on plasma-lemma ATPase activity (EC 3.6.1.3 [EC] ) were studied.Cotyledonary pricks induced, in WL, rapid (5 min) and transient(restoration in 60 min) inhibition of plasmalemma ATPase activity.Conversely, in BL, a rapid and transient stimulation was observed.These results suggest that, in Bidens pilosa, plasmalemma ATPaseis involved in 'short-term' ionic level modifications inducedby traumatisms. Key words: ATPase activity, short-term ionic regulation, growth inhibition     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Effects of Fusicoccin and Abscisic Acid on Sugar and lon Transport from Plant Glands

Fusicoccin (FC) inhibited net excretion of Cl^– by theglands of the pitchers of the carnivorous plant Nepenthes hookeriana;of Na⁺ and Cl^– by the salt glands of the halophytes Limoniumvulgare and L. pectinatum and of K⁺ in the nectar of Acer platanoidesflowers. It had no effect on K⁺ elimination with nectar of Impatienswalleriana (extrafloral nectaries) and Abutilon striatum. Abscisicacid (ABA) stimulated net excretion of K⁺ and Cl^– in Nepenthesand of Na⁺ and Cl^– in Limonium but had no effects on K⁺in nectar. Thus, FC and ABA had opposing effects on ion excretionby the salt eliminating glands of Limonium and Nepenthes. Bothcompounds, however, had similar effects on sugar secretion ofnectary glands which was either inhibited or unaffected by FCand ABA. It is suggested that the effects of FC and ABA on ion excretionby gland cells could be reconciled with literature showing FC-stimulationand possible ABA-inhibition of proton pumps at the plasmalemmaof plant cells. Nepenthes hookeriana, Limonium vulgare, Limonium pectinatum, Acer platanoides, salt-glands, nectaries, excretion, fusicoccin, abscisic acid, proton pump     

Release of Guttation Fluid from Passive Hydathodes of Intact Barley Plants. II. The Effects of Abscisic Acid and Cytokinins

Guttation was used as a non-destructive way to study the flowof water and mineral ions from the roots and compared with parallelmeasurements of root exudation. Guttation of the leaves of barley seedlings depends on age andon the culture solution. Best rates of guttation were obtainedwith the primary leaves of 6- to 7-day-old seedlings grown onfull mineral nutrient solution. The growing leaf tissue becomessaturated with K⁺ below 1.5 mM K⁺ in the medium, whereas K⁺concentration in the guttated fluid still increases furtheras K⁺ concentration in the medium is raised. At 3 mM K⁺ averagevalues of guttation were 1.4–2.4 mm³ h^–1 per plantwith a K⁺ concentration of 10–20 mM; for exuding plantsthe flow was 4.2–7.6 mm³ h^–1 per plant and K⁺ concentration35–55 mM. Abscisic acid (ABA) at 10^–6 to 10^–4 M 0–2h after addition to the root medium increased volume flow ofguttation and exudation and the amount of K⁺ exported. Threeh after addition of ABA both volume and amount of K⁺ were reduced.There was an ABA-dependent increase in water permeability (Lp)of exuding roots shortly after ABA addition. Later Lp was decreasedby 35 per cent and salt export by 60 per cent suggesting aneffect of ABA on salt transport to the xylem apart from itseffect on Lp. Benzyladenine (5 x 10^–8 to 10^–5 M)and kinetin (5 x 10^–6 M) progressively reduced volumeflow and K⁺ export in guttation and exudation and reduced Lp. Guttation showed a qualitatively similar response to phytohormonesas found here and elsewhere using exuding roots. Hordeum vulgare L., barley, guttation, abscisic acid, cytokinins, benzyl adenine, kinetin     

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of Nostoc muscorum

ATPase activity in the cell membrane of a salt-stressed cyanobacterium,Nostoc muscorum M-14, was examined cytochemically by three differentstaining protocols. Application of Hulstaert's method resultedin distinct precipitation of the reaction products of ATPaseinside the cell membrane exclusively. No reaction products wereformed when ATP was replaced by GTP or when dicyclohexylcarbodiimideor N-ethylmaleimide was present in the reaction mixture. Bycontrast, low levels were detectable after the reaction in thepresence of ouabain. Bafilomycin did not affect the formationof products. Mayahara's method, which is considered to demonstratethe reaction of Na⁺,K⁺-ATPase activity, revealed the presenceof a ouabain-sensitive Na⁺,K⁺-ATPase in the cell membrane, whileWachstein-Meisel's method revealed the presence of an ATPaseactivity that was resistant to ouabain. It appears, therefore,that cell membranes of Nostoc muscorum contain both ouabain-sensitiveATPase and ouabain-insensitive ATPase. Comparison of the stainingprofiles of salt-stressed cells with those of control cellssuggested that a high-salt environment activates the ouabain-sensitiveNa⁺,K⁺-ATPase, which seems likely to be involved in the effluxof Na⁺ ions. (Received February 7, 1995; Accepted August 9, 1995)     

The Potassium Relations of Lemna minor L.: II. THE MECHANISM OF POTASSIUM UPTAKE

The experiments described in this paper concern the nature ofthe K⁺-influx mechanism in Lemna minor L. It is establishedthat K⁺ influx is ‘site restricted’ and that theaffinity of the system involved is in close agreement with thatof the well-documented System I mechanisms that are found inmany plants (Epstein, 1972). Experiments with ATP and CCCP suggestthat K⁺ influx is an active process, that the influx machineryresides at the plasmalemma and that this machinery containsan ATPase activity. Membrane vesicles isolated from the plantsabsorb K⁺(Rb⁺) with an affinity comparable to that of the systeminvolved in physiological K⁺ influx.     

Characterization of ATPase Activity Associated with Plasma Membrane from Vigna Hypocotyls

A plasma membrane fraction was isolated from the hypocotylsof cowpea {Vigna unguiculata) by a combination of differentialcentrifugation and sucrose density gradient centrifugation.The ATPase activity of this fraction was dependent on divalentcations (Mn²⁺>Mg²⁺>Co²⁺>Ca²⁺>Fe²⁺>Zn²⁺>Ni²⁺)but was not further stimulated by monovalent cations (K⁺ and/orNa⁺). The pH optimum for the activation of ATPase by Mg²⁺ was7.0. This fraction hydrolyzed ATP or UTP as a substrate andthe ATPase activity obeyed a Michaelis-Menten type of kinetics.The K_m for MgATP ranged from 0.65 to 1.1 mM. The ATPase activitywas inhibited by inhibitors such as N, N'- dicyclohexylcarbodiimide,diethylstilbestrol and triphenyltin chloride, all of which arereported to block proton (H⁺) transport in plant cells, butwas insensitive to those of mitochondrial ATPase such as oligomycinand sodium azide. The ATPase activity was not stimulated bytreatment with ionophores (e.g., carbonyl cyanide p-trifluoromethoxyphenylhydrazone,3,5-di-ter-butyl-4-hydroxybenzilidenemalononitrile and valinomycin⁺KCl)which would be expected to dissipate the electrochemical potentialdifference of H⁺ or the membrane potential difference. The characteristics of the ATPase are compared with those ofplasma membrane ATPases of other plants and its possible rolein H⁺-transport is discussed. ¹ Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu 505-01, Japan or Laboratory for Plant EcologicalStudies, Faculty of Science, Kyoto University, Kyoto 606, Japan. (Received April 20, 1984; Accepted August 14, 1984)     

Effect of CO2 on Volume Change of Guard Cell Protoplast from Vicia faba L

Guard cell protoplasts (GCP) were isolated from epidermal stripsof Vicia faba L. by enzymatic digestion. The presence of non-osmoticvolume in the protoplast was suggested by the relationship betweenprotoplast volume and the mannitol concentration of the suspendingmedium. Light illumination caused swelling of GCP only whenKCl was present in the suspending medium. Dark treatment causedshrinking of GCP irrespective of the presence of 10 mM KCl.In the presence of 10 µM abscisic acid (ABA), GCP shrank.Light-induced swelling was suppressed at concentrations of ambientCO₂ higher than that in normal air. Promotion of swelling wasnot always observed at lower CO₂ concentration. These volumechange responses to light, ABA and CO₂ suggest that GCP retainsits physiological activity as a guard cell. The osmotic contributionof K⁺ to volume increase was lower than expected. Ambient CO₂seems to have some effect on the contribution of K⁺ to osmoregulationof GCP. (Received January 30, 1982; Accepted June 25, 1982)     

Localization of the NaCl-Sensitive Membrane Fraction in Cucumber Roots by Centrifugation on Sucrose Density Gradients

Cucumber plants (Cucumis sativus L. cv. Shogoin) were treatedwith 200 HIM NaCl for one day and the root microsomal membraneswere fractionated by centrifugation on a continuous densitygradient of 10% to 45% sucrose, and the activity of K⁺-Mg²⁺-ATPasewas studied. The most significant difference in the ATPase activitybetween control and NaCl-stressed roots was detected in themembranes sedimented at sucrose concentrations of 40% to 45%.The ATPase activity associated with those fractions was vanadate-sensitive,and the membranes contained much lower levels of calcium andphospholipids after the treatment with NaCl. The putative plasmamembrane fraction collected after centrifugation on a discontinuousgradient of 34% and 45% sucrose appeared to originate from theplasma membrane, as judged by sensitivity to inhibitors andpH optimum of the ATPase activity. NaCl-stress caused a significantreduction in K⁺-Mg²⁺- ATPase activity despite the similarityin polypeptide components detected by SDS-polyacrylamide gelelectrophoresis on disc gels. The effects of NaCl-stress onthe levels of calcium and phospholipids in membranes are discussed,with reference to the disintegration of the membranes. (Received April 7, 1989; Accepted September 19, 1989)     

Non-specificity of Salt Effects on Mg2+dependent ATPase from Grass Roots

The effect of tris, choline, and ethanolamine chlorides on theactivity of Mg^2–dependent ATPase in membrane fractions(cell walls, mitochondria, and microsomes) of Zea mays L. (cv.Neve Yaar 22), Avena saliva L. (cv. Mulga), and Hordeum vulgareL. (cv. Omer) was compared with the effect of KC1 and NaCl.Considerable salt effects on apparent Mg²⁺ATPase activity werefound only at relatively high pH values (8.2) at which Mg²⁺.ATPaseactivity was low in the absence of monovalent cation salts.The Mg²⁺-dependent ATP hydrolysis by ATPases from all the membranefractions increased in the presence of at least one of the organiccations to the same extent as in the presence of KCI or NaCl.The monovalent organic cations are only very slowly absorbedby corn roots in comparison with K⁺ and Na⁺. It is concluded that monovalent salt effects on ATPase fromthese plant roots are not cation specific and not related tothe capability of root cells to absorb cations. Present evidencefor the existence of a cation-transport ATPase in plant tissueis critically reviewed.     

Proton Pumping Pyrophosphatase in a High Density Fraction of Radish Microsomes

Radish (Raphanus sativus L.) microsomal vesicles show a vanadate-?nd nitrate-insensitive, and imidodiphosphate-sensitive electrogenictransport of protons dependent upon addition of inorganic pyrophosphate(PP) or ADP. The activity is detectable in preparations from24 h-old seedlings and increases about 3 fold in vesicles from72 h-old seedlings. The ADP-dependent proton uptake, being preventedby inorganic pyrophosphatase, used as a PP scavenging system,can be ascribed to enzymes utilizing ADP and producing PP whichappears the only substrate for the proton pumping PPase. TheH⁺-PPase has a K_m of ca. 10 µM for the translocating functionand 20 µM for the hydrolytic activity. It has a pH optimumnear to 7.0 and is stimulated by certain monovalent cations(K⁺, Rb⁺ and Cs⁺). The majority of this activity is associatedwith a high density (35–45% sucrose interface) fractionwhich is enriched for vanadate-sensitive, nitrate-insensitiveATPase activity. (Received September 11, 1989; Accepted December 22, 1989)     

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves ofCitrus sinensis L. Osbeck is affected by the presence of thefruitlet on the shoot. Previous work gave evidence that gibberellinsmay be involved in this 'fruit effect'. In the present workwe have studied the effect of gibberellic acid (GA₃) on ¹⁴C-sucroseuptake by leaf discs and whether its action could be due toa modulation of the plasma membrane ATPase, which maintainsthe H⁺ gradient that drives H⁺/sucrose co-transport. The effect of GA₃ on ¹⁴C-sucrose uptake depended on the osmolarityof the assay medium. At 300 mOsm a reduction in the uptake ratewas observed. The inhibitory effect of the hormone disappearedafter preincubating the leaf discs with para-chloromercuri-phenylsulphonicacid (PCMPS), a sulphydril binding inhibitor. ATPase activityof isolated plasma membrane vesicles was inhibited by IAA treatments,while GA₃ or ABA did not affect this enzyme, even after a 3h preincubation period. However, in the absence of a surfactantin the assay medium, GA₃, together with turgor pressure, modulatedplasma membrane ATPase activity, possibly through modificationsof membrane permeability. The hormone effect on ¹⁴ C-sucroseuptake may involve action on the sucrose carrier.Copyright 1994,1999 Academic Press Abscisic acid, Citrus sinensis, gibberellic acid, indoleacetic acid, orange, osmotic pressure, plasma membrane ATPase, ¹⁴C-sucrose uptake     

Effect of Abscisic Acid on the K+ Efflux and Membrane Potential of Nicotiana tabacum L. Leaf Cells

K⁺ efflux from tobacco (Nicotiana tabacum L, cv. Samsun NN)leaf discs into the external medium was increased and the membranepotential (Em) changed in the positive direction with a changein pH from 8.0 to 4.0. E_m was affected by the external concentrationof KCl, greatly decreasing with a change in concentration from1 mM to 100 mM. The equilibrium potential of the membrane forK⁺ (E_k) was decreased in a Nernst fashion with increasing externalconcentrations of KCl. E_k is more positive than E_m above ca.50 µM KCl. Most of the experiments were carried out underconditions in which the difference between the electrochemicalpotential for K⁺ on the inside to the outside of the cell (µ_kis positive. Thus, K⁺ may passively flow to the outside of thecells accompanied by the depolarization of the membrane. Abscisic acid (ABA) increased the K⁺ efflux under conditionsof passive transport. K⁺ efflux was accelerated with an increasingconcentration of ABA, being maximal at 10^–4 M–10^–3M. This acceleration was due to the enhancement of the potassiummotive force (µ_k/F) which is the force causing the netpassive transport of K⁺. The membrane potential was decreasedfrom –205 mV to –170 mV by 2 x 10^–4 M ABAwithin 10 min. The depolarization was not transient, being lostfor at least 3 hr. These results show that ABA accelerated passive K⁺ efflux, whichaccompanied depolarization of the membrane. (Received June 22, 1981; Accepted August 24, 1981)     

Portasomes as Coupling Factors in Active Ion Transport and Oxidative Phosphorylation

SYNOPSIS. We propose that particles, 7–15 nm in diameter,observed on the apical plasma membranes of cation transportingcells of insect midgut, salivary glands, and Malpighian tubulesare modified F₁-F₀ coupling complexes such as those found onphosphorylating membranes of mitochondria, chloroplasts, andbacteria. We suggest the generic term, portasome, to describeall of these particles and point out that they are located onthe side of the membrane which is electronegative and has thelow cation concentration, i.e., on the input side in each case.Biophysical evidence identifies the portasome bearing membraneas the ion transporting membrane in several insect epithelia,some of which exhibit ion modulated ATPase activity. The activityof a K⁺-modulated ATPase from Manduca sexta midgut is increasedin portasome enriched plasma membrane fractions. We proposethat portasomes orient the scalar hydrolysis of negatively chargedMgATP^2– to less negatively charged MgADP thereby eliminatingthe attraction of MgATP^2– to K⁺ with the result that theK⁺ ions are ejected to the opposite side of the portasome bearingmembrane. This mechanism explains the coupling of the scalarhydrolysis of ATP to the vectorial active transport of K⁺ whichleads to the establishment of a K⁺ electrochemical gradient.The reverse process, but with an H⁺ ionophore replacing a K⁺ionophore in the portasome, would provide a mechanism for couplingthe vectorial flow of H⁺, driven by a proton electrochemicalgradient, to scalar ATP synthesis and thereby provide a mechanismfor oxidative phosphorylation. Electrogenic active potassiumion transport would appear to have evolved from oxidative phosphorylation.     

Circadian Rhythms of Potassium and Sodium Contents in the Growing Front of Neurospora crassa

The temporal changes of potassium (K⁺) and sodium (Na⁺) contentsin the growing front of Neurospora crassa (al-2, bd strain)grown on solid medium showed circadian rhythms which persistedfor at least 45 h in the dark. The K⁺ content reached a maximumat about 10 and 30 h after the transfer from light to darkness,while the Na⁺ content was at a minimum at these times. Boththe rhythms were set off by the light to dark transition andwere not observed in constant light. The phase of the circadianrhythm of conidiation of this strain was delayed by 5 h by exposureto 50 min of white light (photon fluence rate 20.7 W/m²) 7 hafter the light to dark transition. The same exposure significantlychanged the ratio of K⁺ to Na⁺ content in the growing frontmeasured 8 h after the exposure. ³ Present address: Pesticides Research Laboratory, TakarazukaResearch Center, Sumitomo Chemical Co., Ltd., 2-1, 4-chome,Takatsukasa Takarazuka, Hyogo 665, Japan. (Received June 26, 1984; Accepted January 11, 1985)     

Bafilomycin A1 is a non-competitive inhibitor of the tonoplast H+-ATPase of maize coleoptiles

When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 10–45% sucrose gradient, bafilomycin A1-inhibitedATPase activity co-localized with the activities of the tonoplastmarker-enzymes, nitrate-Inhibited ATPase and K⁺-dependent pyrophosphatase.Thus, bafilomycin A1 is a specific inhibitor of the vacuolarH⁺-ATPase of maize coleoptiles. Inhibition of the vacuolar H⁺-ATPaseby bafilomycin A1 was strictly dependent upon the concentrationof the enzyme present in the assay medium, suggesting a stoichiometricassociation between bafilomycin A1 and the vacuolar H⁺-ATPase.In tonoplast-enriched preparations, half-maximal inhibitionwas obtained at 43 pmol bafilomycin A1 mg^–1 protein. BafilomycinA1 inhibited the vacuolar H⁺-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theV_max, of the H+ -ATPase, but had no effect on its K_m towardsATP. Key words: Bafilomycin A1, coleoptile, H⁺-ATPase (vacuolar), maize, Zea mays L     

Effects of Abscisic Acid and Cytoplasmic pH on Potassium and Chloride Efflux in Arabidopsis thaliana Seedlings

The effects of ABA, isobutyric acid (IBA) and nicotine on K⁺and Cl⁺ efflux were studied in Arabidopsis thaliana seedlings,and the role of pH_cyt, and E_m in the regulation of the effluxof these ions was discussed. The data show that treatments withIBA and nicotine influenced in opposite directions the effluxof either K⁺ or Cl^–: K⁺ efflux was increased by nicotineand reduced in the presence of IBA, whereas Cl^– effluxwas stimulated by IBA and decreased by nicotine treatment. Underall the conditions tested ABA induced cytoplasmic acidificationand inhibition of K⁺ and Cl^– net efflux. Experiments aimedto estimate the individual contribution of pH_cyt and E_m in modulatingK^– efflux indicated that, within the range of acidic pH_cytvalues, a regulation of K⁺ efflux was imposed by pH_cyt on thecontrol exerted by E_m, the efflux being inhibited by lower pH_cytvalues. Conversely, in the alkaline side of pH_cyt K⁺ effluxseemed linked only to the E_m values. These results are consistentwith the hypothesis that the decrease in K⁺ efflux observedin non-stomatal tissues in the presence of ABA may be mediatedby the cytoplasmic acidification induced by the hormone. (Received August 6, 1996; Accepted January 19, 1997)     

Characterization of membrane-bound Mg++-activated ATPase isolated from the lower epidermis of tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase was separated from thelower epidermis of tobacco leaves (Nicotiand tabacum L. SamsunNN) on stepwise sucrose density gradient centrifugation. Membrane-bound epidermal ATPase was localized in the interfaceof densities in sucrose of 1.12 to 1.16 in the sedimentary fractionbetween 1,500?g to 10,000?g from the homogenate of the lowerepidermis. The epidermal ATPase activity was activated by divalentcations (Mg⁺⁺>Mn⁺⁺Co⁺⁺>Fe⁺⁺>Zn⁺⁺>Ca⁺⁺) and furtherstimulated by KCl by ca. 20%. The pH optimum for Mg⁺⁺-activationof the epidermal ATPase was ca. 6.0. The enzyme hydrolyzed ATPmore rapidly than other nucleoside triphosphates. The optimumtemperature for activation of the epidermal ATPase activitywas ca. 40?C. 50% of the epidermal ATPase activity was lostin 18 min at 55?C and in 2.5 days at 2.5?C. The apparent Kmvalue of the epidermal ATPase was 4.7?10^–4 M and Vmaxwas 65.4 nmoles Pi/mg protein/min. The epidermal ATPase wasstrongly inhibited by N, N'-dicyclohexylcarbodiimide (DCCD)in vitro whereas oligomycin, carbonyl cyanide m-chlorophenylhydrazone(CCGP), indoleacetic acid (IAA) and abscisic acid (ABA) wereinsensitive to the epidermal ATPase activity. (Received May 23, 1978; )