Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures II. Relationship between 515-nm absorbance change and rapid H+ uptake in chloroplasts after a short flash illumination

The absorbance change at 515 nm induced by a short (7.6 µsec)light flash in spinach chloroplasts was studied at sub-roomtemperatures in relation to rapid H⁺ uptake into chloroplasts. Lowering of temperature caused a marked decrease in the rateof recovery of 515-nm absorbance change after a flash illumination.Initial rate of rapid H⁺ uptake, measured with absorbance changeof bromcresol purple (BCP), was also reduced at lower temperatures,in a parallel fashion. Half-recovery time of the absorbancechange at 515 nm and rise-time of the pH-indicating absorbanceincrease of BCP coincided well at each temperature studied.Values of the calculated activation energy for these two processeswere almost the same. The parallelism between the 515-nm absorbance change and therapid H⁺ uptake after a single flash illumination was also observedwhen the electric field decay and/or H⁺ translocation were acceleratedby ionophorous antibiotics, carbonylcyanide m-chlorophenylhydrazoneor phenazine methosulfate. From these results, it is suggestedthat the rapid H⁺ uptake into chloroplast is chemically coupledto electron transfer and at the same time diffusion- (or transport-)controlled. Membrane potential, reflected in the 515-nm absorbancechange is dissipated with the rapid H⁺ influx. A model for theelectron-transfer-coupled H⁺ translocation involving a plastosemiquinoneloop is presented. Dissipation of the illumination-formed inside-positivemembrane potential by the influx of H⁺ is explained by the model. (Received September 17, 1976; )     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures I. Participation of structural changes of thylakoid membranes in light-induced 515-nm absorbance change

Light-induced absorbance change at 515 nm in spinach chloroplastswas studied in the temperature range from –2?C to 27?C.Lowering of temperature had no marked effect on the extentsof initial "light-on" spike and the steady-state change overthe temperature range examined, whereas the rate of recoveryof the 515-nm change was significantly reduced at lower temperatures.Above 15?C, recovery of the 515-nm change after continuous illuminationshowed a first-order kinetics. In contrast, the recovery wascomposed of a fast and a slow phases at lower temperatures. The fast phase of the recovery of the 515-nm change was acceleratedby carbonyl cyanide m-chlorophenylhydrazone, valinomycin plusK⁺ or sodium tetraphenylboron, while the slow phase was completelyeliminated in glutaraldehyde-fixed chloroplasts. Light-inducedchange in absorbance at 546 nm, an indicator of structural changesof membrane, showed almost the same dependency on temperatureas the slow phase of the recovery of the 515-nm change. Theseresults suggest that not only electric field formation acrossthe thylakoid membrane but also structural or conformationalchanges in the membrane participate in the 515-nm absorbancechange observed under steady illumination. (Received July 5, 1976; )     

Anion-dependent changes of energy transfer in chloroplasts II. Relationships of the coupling factor to light-induced proton transport and absorbance change at 515 nm

Roles of the coupling factor in light-induced proton transportand 515-nm absorption change were investigated in chloroplastswashed with high concentrations of Tris salts (pH 7.2). Washingthe chloroplasts with Tris-HCl and Tris-HNO₃ buffers diminishedboth the light-induced pH rise and absorbance change at 515-nm,while Tris-H₂SO₄ buffer was much less effective. Inhibited activitiescould be restored by replacement of the coupling factor afterextraction with EDTA. N,N'-dicyclohexylcarbodiimide also restoredboth activities. Effects of various anions on the proton pumpand 515-nm shift were also investigated. The order of effectivenesswas NO₃^–>Cl^–>SO₄^2–. The role of thecoupling factor and its mode of action; the action mechanismsof Tris and anionsn energy transducing processes in chloroplasts,photophosphorylation, proton transport and absorbance changeat 515 nm, are discussed. ¹Present address: Biology Department, College of Science andEngineering, Ryukyu University, Naha, Okinawa, Japan. (Received June 27, 1972; )     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles II. Energy conversion in chloroplasts and different types of subchloroplast particles

The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH₄Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH₄Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC₄Cl, but in subchloroplast particles the additionalinhibitory effect of NH₄Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH₄Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH₄Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH₄Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. ²Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; )     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles I. Ion transport, ionic environment and the light-induced 475-515 nm absorbance change

The light-induced absorbance change at 515 nm and the light-inducedhydrogen ion uptake in chloroplasts are sensitive to physicaltreatment and to changes in ionic environment. High concentrationsof salts (chlorides) were inhibitory to the 515-nm absorbancechange. This inhibition was stronger in chloroplasts than insubchloroplast particles. In subchloroplast particles, NH₄Clwas slightly stimulatory for the 515-nm change at low concentrations({small tilde}0.5 mM), as was the case with photophosphorylation. Tetraphenylboron (TPB), as a permeant anion, inhibited the 515-nmchange and the rate of hydrogen ion uptake. Tetraphenylarsonium(TPA) and tetraphenylphosphonium (TPP), both permeant cations,diminished the 515-nm change but did not affect the hydrogenion uptake. These results are analyzed in connection with adiscussion of the significance of the membrane potential andhydrogen ion gradient in the energy conversion of chloroplastsand subchloroplast particles. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. (Received February 5, 1974; )     

Electron transfer and energy dependent reactions in glutaraldehyde-fixed chloroplasts

The effects of GA fixation on electron transfers in photosystemsI and II in photosynthesis and energy dependent reactions ofchloroplasts, such as changes in light scattering, H⁺ uptakeand 515-nm absorbance, were investigated. Fixation of chloroplastswith GA resulted in a lowering of the DCIP and MV photoreductions.DCIP photoreduction activity in fixed chloroplasts was not restoredin the presence of DPC, an electron donor to photosystem II,but was significantly stimulated by DPC when chloroplasts werefixed after aging. The results suggest that the inhibitory effectof GA fixation on photosystem II differs in its mechanism fromthose of treatments such as heating, Tris-washing and aging.The oxidation-reduction reaction of P700 was depressed by GAfixation. Energy dependent reactions in fixed chloroplasts were more markedlydepressed than were electron transfers. Fixed chloroplasts showeda slight conformational response in the presence of PMS. Analysis of the emission spectrum and the induction of chlorophylla fluorescence in fixed chloroplasts suggested that the twopigment systems were partially disordered and that the correspondingprimary photochemical processes were inhibited. (Received November 21, 1972; )     

Acceleration of the Decay of Membrane Potential after Energization of Chloroplasts in Intact Zea mays Leaves

The relationship between dissipation of the flash-induced membranepotential across the thylakoid membrane and the high energystate was studied in Zea mays leaves. The dark decay of theflash-induced 515-nm absorbance change was accelerated by shortpreillumination of the leaf. No acceleration of the decay bypreillumination was observed when leaves were incubated in argonor CO² gas or treated with DCMU. These effects of preilluminationand incubation were reversible. The delayed fluorescence from chlorophyll a was reversibly decreasedby incubating leaves in argon or CO² gas, though the modes ofdepression were somewhat different from each other. In leavesincubated in argon or CO² gas, the phase of slow decrease ofthe intensity of prompt fluorescence during illumination reversiblydisappeared. The results suggested that the dissipation of membrane potentialgenerated by a flash was accelerated after the energizationof chloroplasts in leaves, probably by increased H^– permeabilityof the thylakoid membrane. O2 was important in maintaining (indarkness) and forming (under illumination) the high energy statein chloroplasts in intact leaves. (Received October 1, 1980; Accepted December 15, 1980)     

Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Induction of the H+ Release from Thylakoid Membranes by Illumination in the Presence of Protonophores at High Concentrations

The generally observed light-induced uptake of protons intothe thylakoid lumen is diminished by adding protonophores. Insteadof the H⁺ uptake, the release of protons was observed duringillumination in the presence of various protonophores at highconcentrations, namely, 1 µM nigericin, 10 µM carbonylcyanidem-chlorophenylhydrazone or 30 µM gramicidin. An uncoupler,NH₄C1 (4 mM), and a detergent, Triton X-100 (0.02%), also inducedthe H⁺ release but a K⁺ ionophore, valinomycin, did not. Theamount of H⁺ released reached about 100 nmol H⁺ (mg Chl)^–1at pH 7.5 under continuous illumination. The rate of the H⁺release was similar to that of the conventional H⁺ uptake butits dark relaxation was much slower than that of the H⁺ uptake.We compared the H⁺ release in protonophore-added thylakoidswith the previously reported H⁺ release in coupling factor 1(CF₁-depleted thylakoids. The H⁺ release in thylakoids withnigericin showed similar characteristics to that in CF₁-depletedthylakoids in terms of their responses to pH, phenazine methosulfateand light intensity. Both types of H⁺ release were relativelyinsensitive to DCMU and were stimulated somewhat by DCMU atlow concentrations (around 200 nM). Nigericin did not inhibitthe superoxide dismutase activity of the membranes. These resultsindicate that the H⁺ release in protonophore-added thylakoidsand that in CF₁ depleted thylakoids involve the same mechanismand that water-derived protons from PS II that result from animpairment of the activity of superoxide dismutase, as previouslyproposed, are not involved. Judging from the rate of electronflow and the lumenal acidification under the illumination, weconclude that the H⁺ release is a light-dependent scalar processwhich can be observed in thylakoid membranes with high H⁺ permeability.The H⁺ release of this type was not observed in mitochondriafrom rat liver or in chromatophores from Rhodobacter sphaeroides. (Received November 29, 1990; Accepted June 27, 1991)     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Discrimination and characterization of photosystem I-and photosystem II-induced 515-nm absorbance changes in chloroplasts II. Separate local fields in thylakoids indicated by differential responses of the 515-nm absorbance change

Flash-induced 515-nm and 475-nm absorbance changes in spinachchloroplasts were investigated in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU). DCMU reduced the magnitude of the 515-nmabsorbance change by half and almost completely diminished theabsorbance change at 475-nm. The reduction of the 475-nm absorbancechange paralleled the inhibition of the photosystem II (PS II)light reaction. When chloroplasts were illuminated with red or far-red light,the ratio of A₅₁₅/A₄₇₅ changed depending on the photosystemactivated. Wide variations in the A₅₁₅/A₄₇₅ ratio observed insubchloroplast particle preparations were probably due to theenrichment and activation of one of the photosystems. We suggest that the photosynthetic pigments in the thylakoidmembrane are heterogeneously distributed, and chlorophyll bmolecules that may be responsible for the 475- nm absorbancechange are affected by the local field formed by the PS II lightreaction. On the other hand, an electric field due to the PSI reaction probably induced the absorbance change at 515-nm (Received February 24, 1978; )     

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     

The Role of Phosphoenolpyruvate in Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of Maize

Phosphoenolpyruvate (PEP) was transported together with H⁺ inC₄ mesophyll chloroplasts. Medium alkalization and stromal acidificationdue to pyruvate uptake into maize mesophyll chloroplasts inthe light were partially inhibited by adding PEP. Thus, theH⁺ taken up by H⁺/pyruvate cotransport into mesophyll chloroplastsis released together with PEP in vivo. (Received August 5, 1994; Accepted October 3, 1994)     

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     

Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells

H⁺-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na⁺ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H⁺ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H⁺ activity and increased Na⁺ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H⁺-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na⁺ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na⁺ uptake and acid-base regulation mechanisms in zebrafish HR cells. ionocytes; Na⁺/H⁺ exchanger; skin; gill; embryo     

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)     

The Divalent Cation Pump and its Role in the Regulation of Malic Enzyme Activity in Purified Mitochondria from Potato Tuber

The control of the activity of the matrix-located malic enzyme(EC 1.1.1.39 [EC] ) by Mn²⁺ was investigated in Percoll-purified mitochondriafrom potato (Solarium tuberosum) tuber. Malic enzyme activitywas tightly controlled by the amount of Mn²⁺ available in thematrix space and could be stimulated by the addition of exogenousMn²⁺. A net uptake of Mn²⁺ into the matrix space of energizedmitochondria was measured. The uptake of Mn²⁺ was mediated bythe active cation pump present in the mitochondria. The activityof this cation pump was shown to be dependent on the membranepotential sustained by the activity of the respiratory chain.The uptake of Mn²⁺ was totally abolished in the presence ofan uncoupler and strongly depressed in the presence of rutheniumred, a specific inhibitor of the Ca²⁺-pump which is presentin animal mitochondria. Thus, the effect of Mn²⁺ on matrix-locatedMn²⁺-dependent malic enzyme was strongly influenced by the presenceof an uncoupler or of ruthenium red. In addition, this effectwas reduced in the presence of Ca²⁺. The possible physiologicalsignificance of the presence of this cation pump is discussedin relation to the presence of a matrix-located, NAD⁺-dependentmalic enzyme in plant mitochondria. (Received November 21, 1988; Accepted March 6, 1989)