Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

Effects of Aging on the ATP- and Pyrophosphate-dependent Pumping of Protons across the Tonoplast Isolated from Pumpkin Cotyledons

Tonoplast vesicles were isolated from 7- to 26-day-old pumpkincotyledons by an improved floating method, and the activitiesof pyrophosphatase (PPase) and adenosine triphosphatase (ATPase)in tonoplast vesicles, as well as rates of PPase- and ATPase-dependentpumping of protons across tonoplast vesicles, were measured.PPase activity and the rate of pyrophosphate-dependentproton-pumpingdecreased more rapidly than loss of chlorophyll from cotyledons,and the pumping on day 14 was only 10% of that on day 7, whilePPase activity was still more than 30% of that on day 7. Bycontrast, ATPase activity and the rate of ATP-dependent proton-pumpingincreased until day 14. In this latter case, the changes inboth activity and pumping were not major and were parallel toone another until day 21. However, a rapid decrease was observedonly in the rate of pumping on day 26, at which time an apparentloss of fresh weight was observed in cotyledons. The relationshipbetween the aging of pumpkin cotyledons and functional changesin vacuoles is discussed in terms of ATP- and pyrophosphate-dependentproton-pumping across the tonoplast. The two proton pumps inthe tonoplast, H⁺-ATPase and H⁺-pyrophosphatase, appear to playdifferent roles during the growth and senescence of pumpkincotyledons. ¹Plant EcoPhysiology Laboratory, Tohoku National AgriculturalExperiment Station, Shimo-Kuriyagawa, Morioka, Iwate, 020-01Japan.     

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)     

Stimulation of the Extrusion of Protons and H+-ATPase Activities with the Decline in Pyrophosphatase Activity of the Tonoplast in Intact Mung Bean Roots under High-NaCl Stress and Its Relation to External Levels of Ca2+ Ions

Extrusion of protons as a response to high-NaCl stress in intactmung bean roots was investigated at different external concentrationsof Ca²⁺ ions ([Ca²⁺]_ex). The extrusion of protons was graduallyenhanced in the roots exposed to 100 mM NaCl, and high [Ca²⁺]_exdiminished this enhancement of the extrusion. Vesicles of plasmalemmaand tonoplast were prepared from the roots and the H⁺-translocatingATPase (H⁺-ATPase) activities associated with the two typesof membrane and the H⁺-pyrophosphatase (H⁺-PPase) activity ofthe tonoplast were assayed. The plasmalemma ATPase was stimulatedin parallel with dramatic increases in the intracellular concentrationof Na⁺([Na⁺]_in). High [Ca²⁺]ex prevented the increase in [Na⁺]inand diminished the stimulation of ATPase activity. The tonoplastATPase showed a rapid response to salt stress and was similarlystimulated even at high [Ca²⁺]M. The activities of both ATPaseswere, however, insensitive to concentrations of Na⁺ ions upto 100 HIM. By contrast, H⁺-PPase activity of the tonoplastwas severely inhibited with increasing [Na⁺]in under salt stressand recovered with high [Ca²⁺]ex. These findings suggest thathigh-NaCl stress increases the intracellular concentration ofNa⁺ ions in mung bean roots, which inhibits the tonoplast H⁺-PPase,and the activity of the plasmalemma H⁺-ATPase is thereby stimulatedand regulates the cytoplasmic pH. (Received March 26, 1991; Accepted December 13, 1991)     

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     

Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H⁺ pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H⁺ pumping activities. N,N′-dicyclohexylcarbodiimide inhibited the ATPase strongly (I₅₀ = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4′-Stilbenedisulfonic acid affected both enzyme activities and their associated H⁺ pumping activities. This is in contrast to the H⁺-PPase of higher plants which is 4,4′-stilbenedisulfonic acid insensitive.     

H+/OH- Excretion and Nutrient Uptake in Upper and Lower Parts of Lupin (Lupinus angustifolius L.) Root Systems

The cultivation of narrow-leafed lupins (Lupinus angustifoliusL.) increase rates of subsoil acidification, and this is thoughtto be partly related to their pattern of nutrient uptake andH⁺/OH^- excretion. The main hypothesis of this study was thatH⁺ and OH^- excretion is not distributed evenly over the entirelength of the root system but is limited to zones where excesscation or anion uptake occur. Seedlings of nodulated lupinswere grown in solution culture using vertically split pots thatallowed the upper and lower zones of the root system to be suppliedwith varying concentrations of K⁺ and NO^-₃. Net H⁺/OH^- excretionwas equated to the addition of NaOH/HCl required to maintaina constant pH in the nutrient solution during a 4-d treatmentperiod and nutrient uptake was measured by depletion from solutionin each zone of the split pots. The excess of cation over anion uptake was positively correlatedwith H⁺ excretion in each rooting zone. In zones where K⁺ wassupplied at 1200 µM, cation uptake was dominated by K⁺and up to twice as much H⁺ was excreted than in zones whereK⁺ was absent. In zones where NO^-₃ was supplied at 750 µM,the anion/cation uptake was balanced, however H⁺ excretion continuedto occur in the zone. When NO^-₃ was supplied at 5000 µM,anion uptake exceeded cation uptake but there was no OH^- excretion.Organic acid anions may be excreted by lupins to maintain theirinternal electroneutrality when anion uptake exceeds cationuptake. Rhizosphere pH would not increase unless the pK_a ofthe excreted organic anions was greater than the external pH.Copyright1993, 1999 Academic Press Lupinus angustifolius L., H⁺/OH^- excretion, nutrient uptake, cation-anion balance, vertical split root     

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)     

Differential stimulation by Cl- of H+ transport and ATPase activity in purified plasma membrane vesicles from maize (Zea mays L.) roots

Maize (Zea mays L.) root plasma membranes purified by the aqueouspolymer two-phase technique have previously been shown to bevery low in tonoplast H⁺ -ATPase and H⁺ -PPase activities. Westernblots of a similar preparation showed that, compared to a microsomalfraction, there was practically no reaction with antibodiesto the tonoplast enzymes, but a strong reaction with an antibodyto the plasma membrane H⁺ -ATPase. Freeze/thaw treatment ofthe plasma membrane vesicles increased the proportion with aninsideout orientation to about 40%. This preparation was usedto demonstrate that substitution of KCl for K₂S0₄ resulted ina 14-fold stimulation of H⁺ transport, but an increase in ATPaseactivity of less than 10%. In contrast to its effect on tonoplastvesicles, Cl^– had only a small effect on the membranepotential of plasma membrane vesicles, assayed by oxonol V fluorescencequench recovery. To account for the apparent variability inthe H⁺/ATP coupling ratio, it may be necessary to devise a modelthat takes into consideration the possibility of non-linearbehaviour with respect to the membrane potential of the protonleak and/or of slip in the ATPase. Key words: ATPase, plasma membrane, anion stimulation, proton transport     

The Influence of NO-3 and NH+4 Nutrition on the Gas Exchange Characteristics of the Roots of Wheat (Triticum aestivum) and Maize (Zea mays) Plants

Respiratory oxygen consumption by roots was 1·4- and1·6-fold larger in NH⁺₄-fed than in NO^-₃-fed wheat (Triticumaestivum L.) and maize (Zea mays L.) plants respectively. Higherroot oxygen consumption in NH⁺₄-fed plants than in NO^-₃-fedplants was associated with higher total nitrogen contents inNH⁺4-fed plants. Root oxygen consumption was, however, not correlatedwith growth rates or shoot:root ratios. Carbon dioxide releasewas 1·4- and 1·2-fold larger in NO⁺3-fed thanin NH⁺₄-fed wheat and maize plants respectively. Differencesin oxygen and carbon dioxide gas exchange rates resulted inthe gas exchange quotients of NH^-₄-fed plants (wheat, 0·5;maize, 0·6) being greatly reduced compared with thoseof NO^-₃-fed plants (wheat, 1·0; maize, 1·1). Measuredrates of HCO^-₃ assimilation by PEPc in roots were considerablylarger in 4 mM NH⁺₄-fed than in 4 NO^-₃ plants (wheat, 2·6-fold;maize, 8·3-fold). These differences were, however, insufficientto account for the observed differences in root carbon dioxideflux and it is probable that HCO^-₃ uptake is also importantin determining carbon dioxide fluxes. Thus reduced root extension in NH⁺₄-fed compared with NO^-₃-fedwheat plants could not be ascribed to differences in carbondioxide losses from roots.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize assimilation, ammonium assimilation, root respiration     

The Effect of Salinity Changes Upon the Physiology of Eulittoral Green Macroalgae from Antarctica and Southern Chile: II INTRACELLULAR INORGANIC IONS AND ORGANIC COMPOUNDS

The effects of hypo- and hypersaline treatments ranging from7–68% on the intracellular inorganic ion and organic soluteconcentrations were determined in the eulittoral green macroalgaeUlothrix implexa, Ulothrix subflaccida, Enteromorpha bulbosa,Acrosiphonia arcta, and Ulva rigida from Antarctica and SouthernChile. The main inorganic cations were K⁺, Na⁺, and Mg²⁺ inall species. The major osmolyte in E. bulbosa, A. arcta, andU. rigida was K⁺ at increasing salinities. In both Ulothrixspecies, however, K⁺ levels declined during hypersaline stressand Na⁺ concentrations rose significantly. The main inorganicanions were Cl^-, SO²_4-, and PO³_4- in all algae, while E. bulbosaand U. rigida also contained NO⁺₃. A. arcta showed an extremelyhigh SO^2-₄ content. The organic solutes proline, sucrose, andß-dimethylsulphoniopropionate (DMSP) played an importantrole in osmotic acclimation. The occurrence of three organicosmolytes suggests an additional function of these solutes ascryoprotectants in the cold-water macroalgae investigated.     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

Na+/H+ Antiporter in Tonoplast Vesicles from Rice Roots

The Na⁺/H ⁺ antiporter in vacuolar membranes transports Na⁺from the cytoplasm to vacuoles using a pH gradient generatedby proton pumps; it is considered to be related to salinitytolerance. Rice (Oryza sativa L.) is a salt-sensitive crop whosevacuolar antiporter is unknown. The vacuolar pH of rice roots,determined by ³¹P-nuclear magnetic resonance (NMR), increasedfrom 5.34 to 5.58 in response to 0.1 M NaCl treatment. Transportof protons into the tonoplast vesicles from rice roots was fluorometricallymeasured. Efflux of protons was accelerated by the additionof Na⁺. Furthermore, the influx of ²²Na⁺ into the tonoplastvesicles was accelerated by a pH gradient generated by proton-translocatingadenosine 5'-triphosphatase (H⁺-ATPase) and proton-translocatinginorganic pyro-phosphatase (H⁺-PPase). We concluded that thisNa⁺/H⁺antiporter functioned as a Na⁺ transporter in the vacuolarmembranes. The antiporter had a K_m of 10 mM for Na⁺ and wascompetitively inhibited by amiloride and its analogues. TheKⁱ values for 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyI)-amiloride(EIPA), and 5-(N, N-hexamethylene)-amiloride (HMA) were 2.2,5.9, and 2.9 µ M, respectively. Unlike barley, a salt-tolerantcrop, NaCl treatment did not activate the antiporter in riceroots. The amount of antiporter in the vacuolar membranes maybe one of the most important factors determining salt tolerance. ¹This work was supported by a grant from Bio-Media Project ofthe Japanese Ministry of Agriculture, Forestry and Fisheries(BMP96-III-1).     

V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration

We investigated for the presence of avacuolar-type H⁺-ATPase (V-ATPase) in the human eccrinesweat duct (SD). With the use of immunocytochemistry, ananti-V- ATPase antibody showed a strong staining at the apicalmembrane and a weaker one in the cytoplasm. Cold preservation followedby rewarming did not alter this staining pattern. With the use of thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein onisolated and perfused straight SD under HCO-free conditions and in the absence of Na⁺, proton extrusion wasdetermined from the recovery rate of intracellular pH(dpH_i/dt) following an acid load. Oligomycin (25 µM), an inhibitor of F-type ATPases, decreaseddpH_i/dt by 88 ± 6%, suggesting a role foran ATP-dependent process involved in pH_i recovery.Moreover, dpH_i/dt was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor ofV-ATPases, whereas 10 µM bafilomycin A₁, another specificinhibitor of V-ATPases, was required to decrease dpH_i/dt by 73%. These results strongly suggestthat a V-ATPase is involved in proton secretion in the human eccrine SD.

     

Nitrate Stimulation of Mobilization of Seed Reserves in Temperate Cereals: Importance of Water Uptake

Relationships between nitrate (NO^-₃) supply, uptake and assimilation,water uptake and the rate of mobilization of seed reserves wereexamined for the five main temperate cereals prior to emergencefrom the substrate. For all species, 21 d after sowing (DAS),residual seed dry weight (d.wt) decreased while shoot plus rootd.wt increased (15–30%) with increased applied NO^-₃concentrationfrom 0 to 5–20 mM . Nitrogen (N) uptake and assimilationwere as great with addition of 5 mM ammonium (NH⁺₄) or 5 mMNO^-₃but NH⁺₄did not affect the rate of mobilization of seedreserves. Chloride (Cl^-) was similar to NO^-₃in its effect onmobilization of seed reserves of barley (Hordeum vulgare L.).Increased rate of mobilization of seed reserves with additionalNO^-₃or Cl^-was associated with increases in shoot, root and residualseed anion content, total seedling water and residual seed watercontent (% water) 21 DAS. Addition of NH⁺₄did not affect totalseedling water or residual seed water content. For barley suppliedwith different concentrations of NO^-₃or mannitol, the rate ofmobilization of seed reserves was positively correlated (r >0.95)with total seedling water and residual seed water content. Therate of mobilization of seed reserves of barley was greaterfor high N content seed than for low N content seed. Seed watercontent was greater for high N seed than for low N seed, 2 DAS.Additional NO^-₃did not affect total seedling water or residualseed water content until 10–14 DAS. The effects of seedN and NO^-₃on mobilization of seed reserves were detected 10and 14 DAS, respectively. It is proposed that the increasedrate of mobilization of seed reserves of temperate cereals withadditional NO^-₃is due to increased water uptake by the seedlingwhile the seed N effect is due to increased water uptake bythe seed directly. Avena sativa L.; oat; Hordeum vulgare L.; barley; Secale cereale L.; rye; xTriticosecale Wittm.; triticale; Triticum aestivum L.; wheat; nitrate; seed; germination; seed reserve mobilization     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Examination of analytical methods for ions and saccharides in the extract of Phaseolus pulvini

With slight modifications, conventional assay procedures forK⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, NO₃^–, H₂PO₄^–, fructoseand fructose-yielding saccharides, and glucose were applicableto the extract of Phaseolus pulvini. About 10 ml of a hot-waterextract from about 30 mg fresh weight of the pulvini was sufficientfor separate measurement of the ions and saccharides named above. (Received August 7, 1979; )     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

ATP-Promoted Sorbitol Transport into Vacuoles Isolated from Apple Fruit

Sorbitol was transported actively into vacuoles isolated fromapple (Malus pumilla Mill, var domestica Schneid.) fruit flesh.The uptake was stimulated up to twofold by the addition of ATP,and the ATP dependent uptake showed a saturation curve as tothe substrate concentration. The optimum uptake of sorbitolwas pursued in the acidic range of pH 5 to 6. The K_m value forthe ATP dependent sorbitol uptake was about 5 mM. Sorbitol uptake was clearly inhibited by PCMB and uncouplers(CCCP and DCCD), and to a lesser extent by orthovanadate, butonly slightly by oligomycin. K⁺ stimulated sorbitol uptake.Sorbitol was converted to other sugars (glucose) only very slowlywhen transported across the tonoplast. This suggests that sorbitolis transported into vacuoles by a carrier mediated transportsystem coupled with H⁺- ATPase, localized on the tonoplast.Sucrose uptake into the vacuoles was also enhanced by ATP. (Received May 31, 1986; Accepted March 2, 1987)