Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

Regulation of the Cytoplasmic pH of Chara corallina in the Absence of External Ca2+: Its Significance in Relation to the Activity and Control of the H+ Pump

Effects of removal of external Ca²⁺ on the cytoplasmic pH (pH_c)of Chara corallina have been measured with the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) as a function of external pH (pH₀) and of the externalconcentration of K⁺. Removal of Ca²⁺ always decreased pH_c whenpH₀ was below about 6.0; the decrease was about 0.2–0.4units at pH₀ 5.0, increasing to about 0.5 units at pH₀ 4.3.When pH₀ was 6.0 or higher the removal of Ca²⁺ had little orno effect on pH_c. This situation was not altered by changingthe concentration of K⁺, though in some experiments at pH₀ 5.0–5.2there was a slight decrease in pH₀ (about 0.2 units) when K⁺was increased from 0.2 to 2.0 mol m^–3, an effect apparentlyreversed when K⁺ was higher (5.0 or 10.0 mol m^–3). Theresults suggest that H⁺ transport continues in the absence ofexternal Ca²⁺, despite previous suggestions to the contrary,and that the H⁺ pump does not necessarily run near thermodynamicequilibrium with its chemical driving reaction. They indicate,rather, that the H⁺ pump is under kinetic control and providefurther evidence for the inadequacy of present models for theoperation of the H⁺ pump in charophyte cells, especially inrelation to its proposed role in regulating pH_c. Key words: Chara corallina, Cytoplasmic pH, Calcium     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata     

Effects of Cations on the Cytoplasmic pH of Chara corallina

Smith, F. A. and Gibson, J.–L. 1985. Effects of cationson the cytoplasmic pH of Chara corallina.—J.exp. Bot.36: 1331–1340 Removal of external Ca²⁺ from cells of Chara corallina lowersthe cytoplasmic pH, as determined by the intracellular distributionof the weak acid 5,5–dimethyloxazolidine2–,4–dione(DM0), when the external pH is below about 60. This effect isreversed, at least partially, by addition of the following cationsto Ca²⁺-free solutions: tetraethylammonium (TEA⁺) and Na⁺ at5 or 10 mol m^-3, Li⁺ and Cs⁺ (10 mol m^-3), or Mg²⁺, Mn²⁺ andLa³⁺ (02 or 05 mol m^-3). Under the same conditions, increasesin pH sometimes, but not always, occur in the presence of 10mol m^-3 K⁺ or Rb⁺ The results are discussed in relation to the major transportprocesses that determine pH and the electric potential differenceacross the plasma membrane, namely fluxes of H⁺ and of K⁺. Thesimplest explanation of the effects of the various cations testedin this study is that they primarily affect pHi_c via changesin influx of H⁺ but direct effects on the H⁺ pump or on K⁺ fluxesmay also be involved Key words: Chara corallina, cytoplasmic pH, cations, H⁺transport     

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)     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

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     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes

Insulin enhancesNa⁺-K⁺ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na⁺-K⁺ pump function in rabbit ventricularmyocytes. Pump current (I_p) was measured using thewhole-cell patch-clamp technique at test potentials(V_ms) from 100 to +60 mV. When theNa⁺ concentration in the patch pipette([Na]_pip) was 10 mM, insulin caused aV_m-dependent increase in I_p.The increase was ~70% when V_m was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK⁺ and K⁺-congeners were excluded from thepatch pipettes. When [Na]_pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inI_p involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved.

     

Demonstration of Voltage Dependency of Light-induced Potential Change in Chara

The electromotive force (E_m) of the plasma membrane of the tonoplast-freecell of Chara australis decreased when the electrogenic pumpwas stopped by removing ATP or Mg²⁺ from the cell. Such a cellshowed a rapid light-induced potential change (rLPC). Threefactors were considered to be responsible for the generationof rLPC; removal of Mg-ATP, stoppage of the electrogenic pumpand membrane depolarization per se. Tonoplast-free cells having enough Mg-ATP occasionally showedsmall E_m (–87— –116 mV) due to stoppage ofthe electrogenic pump. Since the rLPC was induced in such cells,removal of Mg-ATP cannot be the factor. Cells having large E_mdue to active pump activity also showed rLPC when the potentialdifference across the plasma membrane (V_m) was depolarized byan outward electric current; evidence that not the stoppageof the pump but membrane depolarization is a necessary conditionfor the generation of rLPC. In the rLPC Vm always changed in the negative direction. However,calculation of E_m revealed the existence of a reversal potential[ (E_m)rev] toward which E_m converged from either more negativeor more positive values. The (E_m)rev approximately coincidedwith the equilibrium potential for K⁺ across the plasma membrane.Intracellular anions occupying lower positions in the lyotropicseries inhibited rLPC. (Received February 9, 1981; Accepted May 16, 1981)     

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     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

Salinity-induced Malate Accumulation in Chara

Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m^–3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na⁺ or Li⁺salts. Duringion absorption from various salts of K⁺ and Rb⁺ vacuolar cationconcentration exceeded Cl^– concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m₃^–,with accumulation occurring at rates of up to 0.45 mol m^–3h^–1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO₃^– concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H⁺efflux at a rate of about40 nmol m^–2 s^–1from the cell. The results presentedargue against cytoplasmic Cl^–, HCO₃^– or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity     

Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

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)     

Characteristics of the Wave of Depolarization Induced by Wounding in Bidens pilosa L.

Heating locally the hypocotyl of Bidens pilosa L. elicits awave of depolarization. The mechanism of the wave has been investigatedby means of microelectrophysiological techniques. The amplitudeof the transmembrane potential variation induced by an extracellularion concentration change (K⁺, Na⁺, Ca²⁺, Cl^–) was thesame in the resting conditions as during the slow wave. At pH4.0, the amplitude of the slow wave was reduced by 56% comparedwith the control performed at pH 7.0. In the presence of theuncoupler CCCP, the slow wave was not observed. The Ca²⁺ -chelatorEGTA and the Ca2²⁺ -channel blocker La³⁺ reduced, respectively,the amplitude of the slow wave by 78% and 68%. These resultsindicate the involvement of Ca²⁺ in triggering the slow wave.A transient modification of the electrogenic H⁺ pump activity(inactivation-activation) and of the transmembrane H⁺ flux inthe slow wave are discussed. Key words: Slow wave (of depolarization), wounding, electrogenic pump, calcium, Bidens pilosa L     

L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake

The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H⁺ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The K_m values of thethree affinity sites were 1.4 x 10^–3 M, 1.3 x 10^–4M, 4.3 x 10^–5 M; the maximum velocity values were 3.3x 10^–8, 4.5 x 10^–9, 1.8 x 10_–9 mol/10 min/4x 10⁶ cells. (Received April 18, 1981; Accepted August 25, 1981)