Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots

Plasma membrane H⁺-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H⁺-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg²⁺. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H⁺-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H⁺-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986)     

A Comparison Between the ATPase and Proton Pumping Activities of Plasma Membranes Isolated from the Stele and Cortex of Zea mays Roots

Plasma membrane vesicles of high purity, determined by markerenzyme assays, were obtained by phase partitioning microsomalfractions from stelar and cortical tissues of Zea mays (cv.LG11) roots. ATP hydrolytic activities in both of the plasmamembrane fractions were inhibited by vanadate, SW26 and erythrosinB, but were insensitive to nitrate. Activity in both fractionsexhibited a marked pH optimum of 6·5 and displayed typicalMichaelis-Menten kinetics. A high substrate specificity wasapparent in both the stele and cortex plasma membrane fractions,while the lower fractions, after phase partitioning, showedlower specificity for nucleotide substrates. Specific activitiesof the stele (67·8 µmol Pi mg^–1 h^–1)and cortex (78·4 µmol Pi mg^–1 h–¹)plasma membrane H⁺ -ATPases were very similar. Proton pumping activities in microsomal membrane fractions fromstele and cortex were inhibited by nitrate and insensitive tovanadate. Homogenization of stele and cortex tissue in the presenceof 250 mol m^–3 KI resulted in microsomal fractions exhibitingvanadate-sensitive, nitrate-insensitive proton pumping activity,suggesting a plasma membrane origin for this activity. SW26was also an effective inhibitor of proton pumping activity,although results indicated an interaction between SW26 and thefluorescent probes quinacrine and acridine orange. The results are discussed in relation to models for the transportof ions into the stele and are consistent with a role for theH⁺ -ATPase activity in this process. Key words: ATPase, cortex, plasma membrane, stele, Zea mays     

Effects of Two Plasmalemma ATPase Inhibitors on H+ Extrusion and Intracellular pH in Elodea densa Leaves

Beffagna, N. and Romani, G. 1988. Effects of two plasmalemmaATPase inhibitors on H⁺ extrusion and intracellular pH in Elodeadensa leaves.—J. exp. Bot. 39: 1033–1043. Elodea leaves in the dark show very little exchange of H⁺ withthe medium in the external pH range between 5.0 and 6.0. Thepresence of fusicoccin and potassium in the medium markedlystimulates H⁺ extrusion. Fusicoccin- and K⁺ -induced H⁺ extrusionis inhibited by either erythrosin B (EB) or Na-orthovanadate,two inhibitors of H⁺ transporting plasma membrane ATPase. EBcompletely inhibits it from the first 30 min of treatment, whensupplied at pH 5.5 at a concentration of 30 mmol m^–3.Vanadate also inhibits H⁺ extrusion, this effect becoming evidentonly after 45 min of treatment. After this time inhibition iscomplete with 250 mmol m^–3 vanadate but only partial forlower concentrations. In the presence of either inhibitor the intracellular pH, measuredas cell sap pH, is significantly lowered. When the intracellularpH changes are determined on vacuole and, separately, on cytoplasmby the weak acid and base distribution method, acidificationof both compartments is found to accompany the blocking of H⁺extrusion by either of the inhibitors. Key words: Intracellular pH, vanadate, erythrosin B, H⁺pumping     

Inhibition of Malate Synthesis by Vanadate: Implications for the Cytosolic Alkalinization Induced by Vanadate Concentrations Partially Inhibiting the Plasmalemma H+ Pump

The effects of Na-orthovanadate, at concentrations only partiallyinhibiting net H⁺ extrusion, were determined on vacuolar andcytosolic pH by the weak base and weak acid distribution atequilibrium. Treatment with vanadate induces in Elodea densaleaves and in Arabidopsis thaliana seedlings a moderate acidificationof both cell sap and vacuole. Conversely, it induces an alkalinizationof cytosol, this effect being in apparent contrast with a conditionof reduced activity of the H⁺-transporting plasmalemma ATPase,which should be associated with a cytosolic acidification. InArabidopsis seedlings treated with vanadate, the increase inpH of both cytosol and external medium is associated with adecrease in cell sap buffer capacity, more evident for highervanadate concentrations, and particularly marked in the pH rangebetween 3·5 and 5·5. In these conditions, themalate content is strongly reduced, its decrease almost completelyaccounting for the decrease in cell sap buffer capacity. Anin vitro analysis of the vanadate effect on phosphoenolpyruvatecarboxylase indicates that the decrease in malate content seemssubstantially due to an inhibiting effect of vanadate on thisenzyme. These results stress that the in vivo use of vanadateas an inhibitor of the plasmalemma H⁺-ATPase must be taken withcaution; in particular, for studying the correlations betweenchanges in net H⁺ extrusion and changes in cytosolic pH andrelated processes. Key words: Vanadate, malate, cytosolic pH, Elodea densa, Arabidopsis thaliana     

Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall

Net fluxes of H⁺and Ca²⁺were measured in the mesophyll tissueof broad bean (Vicia faba L.) leaves and in protoplasts derivedfrom these cells. NaCl at 90 m M enhanced H⁺extrusion in bothprotoplasts and tissue, but in different ways. Proton extrusionwas inhibited by vanadate, suggesting the involvement of theplasma membrane H⁺-ATPase in cell responses to salinity. Therewas virtually no effect of NaCl on the net Ca²⁺flux in protoplasts,while in the tissue a large transient Ca²⁺efflux followed thesalt treatment. Salt-induced Ca²⁺efflux was essentially independentof external Ca²⁺concentrations in the range 0.1 to 10 m M. Also,Ca²⁺flux responses were ‘saturated’ above 50 m MNaCl. It is suggested that almost all the measured Ca²⁺fluxoriginates from Na⁺/Ca²⁺and H⁺/Ca²⁺ion exchange in the cellwall. This conclusion was supported by the results of modellingcation exchange in the cell wall. Copyright 2000 Annals of BotanyCompany Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba     

Nitrate-Sensitive ATPase Activity and Proton Pumping in Guard Cell Protoplasts of Commelina

ATPase activity was measured in crude homogenates of guard cellprotoplasts of Commelina communis L. using a linked enzyme assay.A low level of azide-sensitive ATPase activity was detectedwith a pH optimum of 6.8. This activity was stimulated by 0.01%(v/v) Triton X-100, and the pH optimum shifted to pH 7.4. Nitrate-sensitiveATPase activity was measured in the presence of azide and showeda pH optimum around pH 8.0. Proton pumping activity in a mixedpopulation of vesicles from GCP was monitored using fluorescencequenching of quinacrine. Mg-ATP dependent proton pumping wasobserved at pH 8.0, but not at pH 6.6. The activity at pH 8.0was inhibited by nitrate and DCCD but not vanadate. These dataindicate that activity of the tonoplast proton pump was beingmeasured. There was, however, no evidence for a tonoplast cation(K⁺)/proton antiporter under these assay conditions as potassiumdid not reduce the initial rate of pH gradient formation orincrease the rate of collapse of a pre-formed gradient afterinhibition of the pump. Key words: Tonoplast ATPase, proton pump, guard cell protoplasts, Commelina     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Effects of Intracellular Vanadate on Electrogenesis, Excitability and Cytoplasmic Streaming in Nitellopsis obtusa

Vanadate, which is known to inhibit the plasma membrane H^$-ATPaseof Neurospora, was applied intracellularly to internodal cellsof Nitellopsis by use of the intracellular perfusion technique.It inhibited electrogenesis and H^$-extrusion, evidence thatthe electrogenic pump of the Characeae plasmalemma is the H^$-extrudingone. The concentration of vanadate for the half-maximal inhibitionof the activity of the pump was 5 µM. The membrane potentialand H^$-extrusion occasionally recovered from vanadate inhibitionsfor reasons that are unknown. Membrane excitability, which isdependent on Mg?ATP, was not inhibited by vanadate, which suggeststhat the ATPase involved with membrane excitability differsfrom that of the H^$ pump. Cytoplasmic streaming took place evenwhen the cell was perfused with the medium containing 1 mM vanadate,which indicates that the vanadate-insensitive actomyosin systemis concerned with the motive force generation of the streaming. (Received August 27, 1981; Accepted April 13, 1982)     

In Vivo Treatment of Barley Roots with Vanadate Increases Vacuolar H+-Translocating ATPase Activity of the Tonoplast-Enriched Membrane Vesicles and the Level of Endogenous ABA

Evidence for an increase in the activity of the vacuolar H⁺-translocatingATPase of barley roots, accompanied by qualitative change, inresponse to exogenously applied abscisic acid (ABA) has beenreported previously [Kasai et al. (1993b) Plant Cell Physiol.34: 1107]. In the present study, such an increase in the H⁺-translocatingATPase activity was shown to occur upon treatment of barleyroots with vanadate. Furthermore, the level of endogenous ABAin the roots was shown to increase significantly as a resultof this treatment. The present observations provide furthersupport for the proposed physiological significance of cellularABA in the regulation of the vacuolar H⁺-translocating ATPaseactivity. (Received September 3, 1993; Accepted December 16, 1993)     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Vanadate Sensitive ATPase and Phosphatase Activity in Guard Cell Protoplasts of Commelina

Flicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPaseand phosphatase activity in guard cell protoplasts of Commelina.—J.exp. Bot. 38: 642–648. Phosphatase activity was measured in extracts of guard cellprotoplasts of Commelina communis L. using the artificial substratep-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.Ammonium molybdate (Ol mol m^–3) and sodium vanadate (1–0mol m^–3) gave almost complete inhibition of phosphataseactivity at pH 60. ATPase assays were, therefore, conductedin the presence of 0–2 mol m ^–3 molybdate and vanadatewas used as a specific inhibitor of plasmamembrane ATPase activity.Vanadate sensitive ATPase activity showed a pH optimum of 6.6and activity was stimulated by KC1. These properties are characteristicof plasmamembrane proton pumping ATPases in other systems andsuggest that proton extrusion in guard cells could be mediatedby a similar enzyme. The maximum ATPase activity is sufficientto account for all the proton flux observed during the stomatalopening response. Key words: ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate     

H+ Efflux and Trans-Root Potential Measured while Increasing the Temperature of Solutions Bathing Excised Roots of Zea mays

Kennedy, C. D. and Gonsalves, F. A. N. 1988. H⁺ efflux and trans-rootpotential measured while increasing the temperature of solutionsbathing excised roots of Zea mays.—J. exp. Bot. 39: 37–49. Novel temperature-ramp procedures have been used to measureH⁺ efflux and trans-root potential of excised roots of Zea mays(var. Fronica). Two types of experiment were performed: (1),increasing temperature from 17°C, and (2), pre-cooling theroots to 1°C before starting the temperature ramp. The ratesof increase of temperature for H⁺ efflux and trans-root potentialexperiments were 0·5 and 2·1°C min^–1respectively The H⁺ scans revealed strong sharp maxima at 30°C and 32°C,for non-pre-cooled and pre-cooled roots respectively, the latterbeing significantly smaller. The trans-root potential scansfor the pre-cooled roots showed a corresponding maximum at 30°C,which was inhibited by KCN (1-0 mmol dm^–3) with or withoutSHAM (10 mmol dm^–3), or Hg²⁺ (1, 10, 100 µmol dm^–3)in the bathing solutions. Some of the evidence suggests thatthese maxima are associated with electrogenic H⁺ pumping, mediatedby a plasma membrane-bound ATPase. However, no correspondingmaximum was observed in the trans-root potential scans for non-pre-cooledroots, the potential remaining at about — 75 m V from20°C to 35°C. As there is a 7-fold increase in H⁺ effluxbetween 20°C and 30°C, the relationship between netH+ efflux and electrogenic proton pumping in these roots isby no means clear. Some possibilities are considered here. Pre-cooled and non-pre-cooled roots show clear maxima in thetrans-root potential scans at about 46°C, at which temperaturethere is a slight net H⁺ influx. This, and other less prominentfeatures observed, are briefly discussed. Key words: H⁺ efflux, trans-root potential, temperature-ramp procedure, Zea mays, roots     

ATPase and Proton Pumping Activities in Cotyledons and other Phloem-Containing Tissues2Ricinus communis

ATPase activity was investigated in phloem-containing tissuesof Ricinus communis in relation to its proposed role in phloemloading. Cytochemical staining of cotyledons revealed an ATP-hydrolysingactivity on the plasma membrane of the sieve tube/companioncell complex. Microsomal fractions prepared from cotyledonsand main veins contained a Mg²⁺-dependent ATPase activity whichshowed low stimulation by KC1 particularly at pH 6.5. The pHoptimum was at pH 8.5 to 90, although the effect of azide indicatedthe presence of mitochondrial ATPase. At pH 6.5, the cited optimumfor plasma membrane ATPase, the activity showed strong inhibitionby PCMBS, vanadate and DCCD. A high pyrophosphatase activitywas observed at pH 8.5. Acidification of the medium by intactcotyledons was increased by fusicoccin and inhibited by PCMBS,NEM and vanadate. Proton pumping by microsomal vesicles as measuredby quinacrine fluorescence was also inhibited by PCMBS, NEMand vanadate. Sucrose uptake by cotyledon discs showed stronginhibition by PCMBS, NEM and CCCP but was little affected byvanadate. Sucrose uptake varied with the developmental stageof the cotyledons and this correlated with microsomal ATPaseactivity measured at pH 6.5, although the precise cellular originof this activity is not certain. The results are discussed inrelation to the role of ATPase activity and proton pumping inphloem loading. Key words: ATPase, phlocm loading, proton pumping, Ricinus communis, sucrose     

Possible Linkage between NADH-Oxidation and Proton Secretion in Zea mays L. Roots

A possible involvement of two different systems in proton translocationand the correlation of this factor to growth rates were measuredsimultaneously by means of a pH stat and an optical system.Ferricyanide, which can accept electrons at the plasmalemma,led to an immediate increase of net H⁺ -efflux but also decreasedroot growth rate. The reduced form, ferrocyanide, inhibitednet H⁺ -effluxwithout changing the growth rate. Thus, corn rootgrowth was not determined by proton secretion exclusively. Vanadatestrongly inhibited net H⁺ -efflux by the roots but did not preventthe stimulating effect of fcrricyanide. Moreover, the extentof enhancement of net H⁺ -effluxby ferricyanide was exactlythe same in vanadate pretreated as in untreated roots. Alcoholswere used to try to increase the intracellular NADH level throughthe action of the cytoplasmic alcohol dehydrogenase presentin the roots and coleoptiles. Alcohols, known to be substratesfor alcohol dehydrogenase such as propan- 1-ol, ethanol andbutan-l -ol increased net H⁺ -effluximmediately but methanoland secondary alcohols which are not substrates had no effecton proton secretion. The K_m values of alcohol dehydrogenasefor the alcohols correspond only partly to their effects onproton secretion. However, the specificlty observed suggeststhat increased H⁺ -efflux arose from reduction of endogenousNAD by ADH and consequent increased membrane NADH-oxidasc activitytrans locating protons and electrons out of the cells. Decreased oxygen concentrations slowed proton secretion at valuesfar higher than are necessary to saturate cytochrome c oxidase.The results of these experiments suggest two distinct systemscontributing to proton efflux. Key words: ADH, proton transport, redox chain     

Electrophysiological Evidence for an Electrogenic Proton Pump and the Proton Symport of Glucose in the Marine Fungus Dendryphiella salina

The marine hyphomycete Dendryphiella salina (Suth.) Nicot &Pugh has a resting membrane potential of –250 mV (insidenegative). The respiratory inhibitors sodium azide and FCCPinduced a rapid but reversible depolarization of the membraneof at least 180 mV; sodium azide also caused alkalinizationof the medium. Vanadate brought about significant depolarizationbut this was not always reversible. EDTA induced depolarizationthough to a lesser extent. DIDS and SITS caused a depolarizationof around 30–70 mV which was readily reversible, N-ethylmaleimideirreversibly depolarized the membrane by 180–200 mV. Ouabainhad no effect. When external concentrations of H⁺ , K⁺ , Na⁺or Cl^– were changed singly, only changes in H⁺ affectedmembrane potential, with shifts decreasing with increasing pH.Glucose and 3-O-methyl glucose depolarized the membrane in aconcentration-dependent manner which was enhanced by starvationof the hyphae. Recovery occurred in the presence of the hexose.Glucose caused an alkalinization of the medium, with time characteristicssimilar to the membrane potential changes. It is concluded thatthere is an electrogenic proton pump and a proton—glucosesymporter in D. salina. The retention of proton-based transportsystems suggests a terrestrial origin for the fungus. Key words: Marine fungi, Dendryphiella salina, membrane potential, electrogenic proton pump, proton symport, hexose     

Increase in Proton-Transport Activity of Tonoplast Vesicles as an Adaptive Response of Barley Roots to NaCl Stress

H⁺-Transport activity of the vesicles prepared from barley rootswas studied at the early phase after application of NaCl stress.The activity reached maximal level at 3 days after the treatmentwith 200 mM NaCl which moderately reduced the growth. This activityincrease could be suppressed in the presence of cycloheximideand actinomycin D. The properties of the membrane vesicles associated with H⁺-transportactivity prepared from both control and NaCl-stressed rootssuggested that it was of tonoplast origin based on the followingfindings: optimal pH at 7.5, strong inhibition by nitrate butnot by vanadate, and stimulation by chloride. The density gradient centrifugation of vesicles with DextranT70 did not show any detectable difference in the distributionpatterns of H⁺-transport activities between control and NaClstressedroots. Furthermore, K_m values for ATP of the H⁺-transport activityof vesicles prepared from control and NaCl-stressed roots werethe same. Therefore, H⁺-transport activity with properties similarto those of the control roots was increased by NaCl stress.The results are discussed in terms of an adaptive mechanismof barley against salt stress. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received April 18, 1988; Accepted July 20, 1988)     

Potassium Transport in Non-Growing Corn Root Tissue as Affected by IAA and GA3

Experiments were done to determine if the spontaneous recoveryof non-growing segments of corn root (Zea mays L.) from excisioninjury is dependent on auxin. Washing the segments with 5 runindoleacetic acid (IAA) for 2 to 4 hours gave a small but significantincrease in K⁺ (⁸⁶Rb) influx, used here as a parameter reflectingrecovery of electrogenie H⁺-efflux pumping. This promotive effectwas obtained only after an hour of washing, and was sustainedby 100 nm gibberellic acid (GA₃). Any early responses to auxinwere obscured by an adverse reaction of the root cells to externalIAA which resulted in a transitory inhibition of H⁺ pumpingand K⁺ influx. Pretreatment of excised root tips with 10 µM IAA in thegrinding medium protected a plasmalemma-enriched fraction ofthe microsomes during isolation, giving increased uncoupler-sensitiveATPase activity. Non-growing root tissue thus shows three responses to auxin:an adverse reaction at the outer surface of the plasmalemmawhich blocks H⁺ pumping; a protective or restorative effecton the H⁺-ATPase; an increased capacity for K⁺ influx duringthe developmental phase of washing, which is augmented by thepresence of GA₃. (Received March 31, 1986; Accepted September 8, 1986)     

Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Light-induced Activation of Electrogenic H+ Extrusion and K+ Uptake in Elodea densa Depends on Photosynthesis and is Mediated by the Plasma membrane H+ ATPase

In Elodea densa leaves light strongly stimulates electrogenic,K ⁺-dependent, vanadate- and erythrosin B-sensitive H⁺ extrusionand hyperpolarizes the transmembrane electrical potential. Theseeffects of light are suppressed by treatment with DCMU, an inhibitorof photosynthesis, which has no effect on H⁺ extrusion in thedark. Light-induced H⁺ extrusion requires the presence of K⁺in the medium and is associated with increased K⁺ uptake andalkalinization of the cell sap. Light-induced H⁺ extrusion increaseswith increased CO₂ concentration. At constant CO₂ concentration(10⁴ parts 10^–6) the rate of H⁺ extrusion is stronglyenhanced by an increased light intensity up to 30 W m^–2.Different wavelengths, between 400 and 730 nm, induce a significantstimulation of both proton secretion and transmembrane potentialhyperpolarization. The stimulating effects of light on H⁺ extrusion, K⁺ uptakeand cell sap pH are very similar to those induced in the darkby fusicoccin, a toxin known to stimulate strongly ATP-driven,vanadate- and erythrosin B-sensitive H⁺ transport. In the light,the effects of fusicoccin are only partially additive to thoseof light, thus suggesting that the two factors influence thesame system. The identification of this system with the plasmamembrane H⁺-ATPase is indicated by the observed inhibition ofthe effects of either light or fusicoccin by the H⁺-ATPase inhibitorsvanadate and erythrosin B. These data indicate that the activation of electrogenic H⁺ extrusionand of K⁺ uptake by light is mediated by some products of photosynthesis.The mechanism and the possible physiological implications ofthis phenomenon are discussed. Key words: Photosynthesis, H⁺ pump, K⁺ uptake, Elodea densa     

The Regulation of Proton/Amino Acid Symport in Riccia fluitans L. by Cytosolic pH and Proton Pump Activity

In the aquatic liverwort Riccia fluitans the regulation of theplasma membrane H⁺/amino acid symport has been investigated.Cytosolic pH (pH_c), membrane potential (E_m) and membrane conductancehave been measured and related to transport data, (i) The releaseof [¹⁴C]amino acids is strongly stimulated by cytosolic acidification,induced by the external addition of acetic acid, a decreasein external K⁺, and in the change from light to dark. On average,a decrease in pHc of 0.5 to 0.6 units corresponded with a 4-foldstimulation in amino acid efflux. (ii) External pH changes havefar less effect on substrate transport than the cytosolic pHshifts of the same order. (iii) The inwardly directed positivecurrent, induced by amino acids, is severely inhibited by cytosolicacidification. (iv) Fusicoccin (FC) stimulates amino acid uptakewithout considerable change in proton motive force. (v) Whenthe proton motive force is kept constant, the uptake of aminoacids into Riccia thalli is much lower than when the pump isdeactivated. It is suggested that both the proton pump activityand cytosolic pH are the dominant factors in the regulationof the H⁺/amino acid symport across the plasma membrane of Ricciafluitans, and it is concluded that the proton motive force isnot a reliable quantity to predict and interpret transport kinetics. Key words: Amino acid, cytosolic pH, pH-sensitive electrode, proton motive force, regulation, Riccia fluitans