Effects of cordycepin on IAA- and fusicoccin-stimulated cell enlargement, proton extrusion and potassium uptake in maize coleoptiles

In maize (Zea mays F, XL 640 A, DEKALB) coleoptiles, cordycepin (3′-deoxyadenosine) is very active in preventing the cell elongation. H⁺ extrusion and K⁺ uptake induced by IAA and, to a much lesser degree, the same phenomena induced by fusicoccin (FC). Cordycepin, while depressing uridine incorporation into RNA, does not decrease the ATP level or significantly influence the pyruvate level and leucine incorporation into proteins in this material. These results support the hypothesis that one or more proteins. whose synthesis is dependent upon short half-life mRNAs, are essential for a full response to IAA. while this requirement is only partial in FC-stimulation of growth. They also confirm the view that auxin- or FC-induced activation of H⁺/K⁺ exchange plays an important role in mediating the effects of these compounds on cell enlargement.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.     

Effects of hyper-osmotic stress on K+ fluxes, H+ extrusion, transmembrane electric potential difference and comparison with the effects of fusicoccin

The stimulation of H⁺ extrusion by hyper-osmotic stress (0.2–0.3 M mannitol) in cultured cells of Arabidopsis thaliana (L.) Heynh. was shown to be associated with an inhibition of Cl^? efflux, whereas hypo-osmotic stress, inhibiting H⁺ extrusion, early and strongly stimulated Cl^? efflux. In this paper, we investigate the contribution of other factors [K⁺ transport and transmembrane electric potential difference (E_m)] to the hyper-osmotic-induced activation of the plasma membrane (PM) H⁺-ATPase. The effects of mannitol (MA) on K⁺ transport and on E_m were compared with those of fusicoccin (FC) since the modes of action of osmotica and of the toxin in stimulating H⁺-ATPase activity seem to differ at least in some steps. The changes in H⁺ extrusion induced by hyper- or hypo-osmotic stress were opposite and could be reversed by the application of the respective opposite stress. The effect of MA on H⁺ extrusion was dependent on the presence of K⁺ (or Rb⁺) similarly to that of FC, while Na⁺ and Li⁺, which also stimulated the FC effect, were ineffective on that of MA. The MA effect was independent of the anions (Cl^?, SO₄^2?, NO₃^?) accompanying K⁺. K⁺ net uptake and K⁺ influx were stimulated by both MA and FC. Tetraethylammonium (TEA⁺) and Cs⁺ inhibited both MA- and FC-induced H⁺ extrusion, suggesting the involvement of K⁺ channels. MA (0.2 M) induced a strong hyperpolarization of E_m both in the absence and in the presence of K⁺. The hyperpolarizing effect of MA was also found when the cells were already hyperpolarized by FC, and was rapidly reversed by removing the osmoticum from the medium. In the presence of the lipophilic cation tributylbenzylammonium (TBBA⁺), MA was no longer able to stimulate H⁺ extrusion, while FC still stimulated it. In cells pretreated with TBBA⁺, which strongly depolarized E_m, the subsequent addition of FC repolarized it, while the hyperpolarizing effect of MA was lacking. On the contrary, in cells pretreated with Erythrosine B (EB), E_m was strongly depolarized and the following addition of FC did not hyperpolarize it, while the hyperpolarizing effect of MA was still observed. These results suggest that the mechanism of MA in activating H⁺ extrusion and K⁺ uptake is different from that of FC. The rise in net K⁺ uptake seems to be driven by the activation of some hyperpolarizing system that does not seem to depend on a direct activation of PM H⁺-ATPase, but rather on the inhibition of Cl^? efflux induced by hyper-osmotic stress.     

Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K-Induced Cytosol Alkalinization

In maize root segments fusicoccin induced a consistent increase in cell sap pH (taken as representative of vacuolar pH). This effect was markedly enhanced by the presence of K⁺ in the medium, whereas in the absence of fusicoccin K⁺ did not significantly influence cell sap pH. Treatment with a weak acid at 2 mm concentration inhibited the uptake of a different (¹⁴C-labeled) weak acid fed at a lower concentration, thus suggesting that acidification of the cytoplasm inhibits weak acid uptake. Fusicoccin and K⁺ increased the rate of uptake of 5,5-dimethyloxazolidine-2,4-dione, butyric acid, or isobutyric acid slightly when fed separately, strongly when fed in combination. The synergism between fusicoccin and K⁺ in stimulating weak acid uptake was parallel to that observed for the stimulation of H⁺ extrusion. Application of the weak acid distribution method to a condition of `quasi-equilibrium' indicated that fusicoccin induces a cytosolic pH increase of about 0.14 unit. These results are interpreted as providing circumstantial evidence that fusicoccin- and K⁺- induced stimulation of H⁺ extrusion led to an alkalinization of the cytosol, and that other early metabolic responses, such as an increase in malate level, are a consequence of the increase in cytosolic pH.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Role of the plasma membrane H<Superscript>+</Superscript>-ATPase in auxin-induced elongation growth: historical and new aspects

This article will cover historical and recent aspects of reactions and mechanisms involved in the auxin-induced signalling cascade that terminates in the dramatic elongation growth of cells and plant organs. Massive evidence has accumulated that the final target of auxin action is the plasma membrane H⁺-ATPase, which excretes H⁺ ions into the cell wall compartment and, in an antiport, takes up K⁺ ions through an inwardly rectifying K⁺ channel. The auxin-enhanced H⁺ pumping lowers the cell wall pH, activates pH-sensitive enzymes and proteins within the wall, and initiates cell-wall loosening and extension growth. These processes, induced by auxin or by the "super-auxin" fusicoccin, can be blocked instantly and specifically by a voltage inhibition of the H⁺-ATPase due to removal of K⁺ ions or the addition of K⁺-channel blockers. Vice versa, H⁺ pumping and growth are immediately switched on by addition of K⁺ ions. Furthermore, the treatment of segments either with auxin or with fusicoccin (which activates the H⁺-ATPase irreversibly) or with acid buffers (from outside) causes an identical transformation and degradation pattern of cell wall constituents during cell-wall loosening and growth. These and other results described below are in agreement with the acid-growth theory of elongation growth. However, objections to this theory are also discussed.     

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

H extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments treated with permeant weak acids

The rapid uptake of weak acids permeant in the uncharged form is accompanied in maize and wheat root segments by a hyperpolarization of the transmembrane electrical potential and an increase in K⁺ uptake, suggesting a stimulation of the plasmalemma H⁺ pump. The evaluation of weak acid-induced H⁺ extrusion must take into account the alkalinization of the medium due to the rapid uptake of the uncharged form of the acid, partially masking the proton pump-mediated extrusion of H⁺. The data corrected for this interference show that the lipophilic butyric acid and trimethyl acetic acid induce in maize and in wheat root segments a significant increase in `real' H⁺ extrusion, roughly matching the increase in net K⁺ uptake. The presence of K⁺ significantly increases the rate of uptake of the weak acid, possibly as a consequence of an alkalinization of the cytosol associated with K⁺ absorption. In maize root segments, the effects of fusicoccin and those of butyric acid on both K⁺ uptake and H⁺ extrusion are clearly synergistic, thus suggesting distinct modes of action. These results support the view that the activity of the plasmalemma H⁺ pump is regulated by the value of cytosolic pH.     

Relationship between ATP Level and Activity of Fusicoccin-stimulated H/K-Exchange System in Plant Tissues

The treatment with fusicoccin causes a slight but significant decrease (about 15%) in the ATP level in pea-internode and maize-coleoptile segments. This decrease is detectable within 15 minutes and is accompanied by a parallel increase in O₂ uptake. Sodium azide inhibits O₂ uptake and completely blocks the stimulation of O₂ uptake by fusicoccin in both pea and coleoptile segments. Benzohydroxamic acid does not affect either basal or fusicoccin-induced O₂ uptake in maize-coleoptile sections. The drop of ATP level induced by various treatments (sodium arsenate, 2-deoxyglucose, limiting O₂, and anaerobiosis) is accompanied by a parallel inhibition of K⁺ uptake in maize coleoptiles treated with or without fusicoccin. These results are consistent with the hypothesis that ATP is the energy source for the fusicoccin-activated H⁺/K⁺-exchange system.     

Tributylbenzylammonium (TBBA+)-dependent fusicoccin (FC)-induced H+ extrusion in maize roots: relationship between the stimulating effects of TBBA+ on H+ extrusion and on Cl- efflux

The mechanism of the stimulating effect of lipophilic cations on H⁺ extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [³H]tributylbenzylammonium ([³H]TBBA⁺), the most active lipophilic cation on H⁺ extrusion, indicated that although a relevant fraction of TBBA⁺ taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA⁺ uptake and that of H⁺ extrusion. On the other hand, the lipophilic cations active on H⁺ extrusion (TBBA⁺ and dibenzyldimethylammonium (DDA⁺)), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl^-, while tetramethylammonium (TMA⁺), inactive on H⁺ extrusion, did not. The stimulation of Cl^- efflux by TBBA⁺ was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA⁺-induced H⁺ extrusion. These results suggest that the stimulation of H⁺ extrusion by TBBA⁺ depends on its effect on Cl^- efflux rather than on its penetration into the cells.Abbreviations DDA⁺ dibenzyldimethylammonium - FC fusicoccin - 3-O-MG 3-O-methyl glucose - PD transmembrane electric potential difference - TBBA⁺ tributylbenzylammonium - TCF tissue concentration factor - TMA⁺ tetramethylammonium - TPB^- tetraphenylboron     

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     

Relation between permeability to potassium and sodium ions and fusicoccin-stimulated hydrogen-ion efflux in barley roots

Summary Measurements are described of fusicoccin (FC)-stimulated H⁺ efflux in barley (Hordeum vulgare L.) roots when K⁺ and Na⁺ concentrations were varied. In low-salt roots H⁺ efflux was stimulated in both 5 mM KCl and NaCl. In salt-saturated roots H⁺ efflux was stimulated more effectively in KCl than in NaCl solution. The stimulation of H⁺ efflux thus is parallel with the selectivity of these different root preparations for K⁺ and Na⁺ and with estimates of permeability ratios (P _Na/P _K) determined from electrical measurements. It is suggested that the results support electrogenic coupling between FC-stimulated H⁺ efflux and cation uptake.     

Evidence that the partial resistance of the Arabidopsis 5-2 mutant to fusicoccin depends on a decreased capacity of the H+ pump to respond to activating factors

Measurements of H⁺ extrusion activity K⁺ influx, and Es bm in 3-d-old seedlings of the 5-2 mutant of Arabidopsis thaliana (which is partially insensitive to fusicoccin) showed the following, (i) The reduced response of 5-2 to fusicoccin (FC) does not depend on the penetration of FC to its site of action, or on decreased affinity of the FC receptor, (ii) The reduced response of H⁺ and K⁺ transport to FC does not depend on an impairment of the K⁺ absorption system, (iii) The mutation can influence the H⁺ extrusion system independently of the presence of FC. In the presence of factors other than FC known to activate the plasma membrane H⁺-ATPase (e.g. a cytosol-acidifying treatment), the response in 5-2 is about 50% lower than in wt. (iv) When both genotypes grow in optimal conditions, the rate of fresh weight increase and stem elongation is higher in wt than 5-2. These data indicate that the 5-2 mutation affects some intrinsic component of the H⁺-extrusion machinery, the limiting effect of which becomes considerable when either the physiological or the experimental conditions induce a high level of proton pump activity. An alteration either of the ATPase itself or of a factor controlling its activity is compatible with our observations.     

Transmembrane potential-mediated coupling between H+ pump operation and K+ fluxes in Elodea densa leaves hyperpolarized by fusicoccin, light or acid load

In isolated Elodea densa leaves, the relationships between H⁺ extrusion (-ΔH⁺), K⁺ fluxes and membrane potential (E_m) were investigated for two different conditions of activation of the ATP-dependent H⁺ pump. The ‘basal condition’ (darkness, no pump activator present) was characterized by low values of-ΔH⁺ and K⁺ uptake (ΔK⁺), wide variability of the ?ΔH⁺/ΔK⁺ ratio, relatively low membrane polarization and E_m values more positive than EK for external K⁺ concentrations (|K⁺]_o of up to 2mol m^?3. A net K⁺ uptake was seen already at [K⁺]_o below 1 mol m^?3, suggesting that K⁺ influx in this condition was a thermodynamically uphill process involving an active mechanism. When the H⁺ pump was stimulated by fusicoccin (FC), by cytosol acidification, or by light (the ‘high polarization condition’), K⁺ influx largely dominated K⁺ and C^? efflux, and the ?ΔH⁺/ΔK⁺ ratio approached unity. In the range 50 mmol m^?3?5 mol m^?3 [K⁺]₀, E_m was consistently more negative than E_K. The curve of K⁺ influx at [K⁺]₀ ranging from 50 to 5000mmol m^?3 fitted a monophasic, hyperbolic curve, with an apparent half saturation value = 0–2 mol m^?3. Increasing |K⁺]₀ progressively depolarized E_m, counteracting the strong hyperpolarizing effect of FC. The effects of K⁺ in depolarizing E_m were well correlated with the effects on both K⁺ influx and ?ΔH⁺, suggesting a cause-effect chain: K⁺₀ influx → depolarization → activation of H⁺ extrusion. Cs⁺ competitively inhibited K⁺ influx much more strongly in the ‘high polarization’ than in the ‘basal’ condition (50% inhibition at [Cs⁺]/[K⁺]₀ ratios of 1:14 and 1:2, respectively) thus confirming the involvement of different K⁺ uptake systems in the two conditions. These results suggest that in E. densa leaves two distinct modes of interactions rule the relationships between H⁺ pump, membrane polarization and K⁺ transport. At low membrane polarization, corresponding to a low state of activation of the PM H⁺-ATP^ase and to E_m values more positive than E_K, K⁺ influx would mainly     

Effects of Cr on proton extrusion, potassium uptake and transmembrane electric potential in maize root segments

Abstract Proton extrusion of maize root Zea mays segments, was inhibited by the presence of Cr (o.n. + 6; present in solution as CrO₄^2-, Cr₂O₇^2-) in the incubation medium: the minimum inhibiting concentration was 2 × 10^?3 mol m^?3 and the inhibition progressively increased with Cr concentration. Cr inhibited proton extrusion. Also, when this activity was stimulated by the presence of K⁺ or fusicoccin (FC) in the incubation medium, the K⁺ and FC stimulating effect was still present when proton extrusion was inhibited by Cr. In addition, Cr inhibited K⁺ uptake. This inhibition was higher (50%) at K⁺ concentrations up to 1 mol m^?3 lower (15%) at higher K⁺ concentrations. This result indicates that the system responsible for K⁺ uptake operating at low K⁺ concentrations is more sensitive to Cr inhibition. Cr had no effect on transmembrane electric potential (PD). The depolarizing and hyper-polarizing effect of K+ and FC, respectively, were not affected by Cr; but Cr enhances the depolarizing effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCP). These results indicate that Cr inhibited the proton translocating mechanism coupled with K⁺ uptake, but did not change the net transport of charges through the plasmalemma. The Cr effect is discussed, taking into account the possibility of a direct effect of Cr at the membrane level or, alternatively, of an effect on some metabolic processes controlling membrane function.     

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Ammonium and Methylammonium Transport in Egeria densa Leaves in Conditions of Different H+ Pump Activity

Previous data in Egeria densa leaves demonstrated a strong inhibitory effect of Cs⁺ on passive K⁺ influx and on K⁺-induced, ATP-dependent electrogenic proton extrusion. In this paper we analyzed, using the same material, the effects of Cs⁺ on ammonium (NH₄⁺) and methylammonium (CH₃NH₃⁺) transport in order to elucidate whether a common transport system for K⁺ and NH₄⁺ could be demonstrated. The effects of Cs⁺ on NH₄⁺- and CH₃NH₃⁺-induced titratable H⁺ extrusion (–ΔH⁺) and on transmembrane electrical potential difference (E_m) in E. densa leaves were analyzed in parallel. All experiments were run either in the absence or presence of fusicoccin, corresponding to low or high H⁺-ATPase activity and membrane hyperpolarization and leading, in this material, to respectively active or passive transport of K⁺. The results suggest the presence in E. densa leaves of two distinct pathways for NH₄⁺ uptake: one in common with NH₄⁺ and (with lower affinity) CH₃NH₃⁺, insensitive to Cs⁺, and a second system, operating at higher H⁺-ATPase activity and E_m hyperpolarization, strongly inhibited by Cs⁺ and impermeable to CH₃NH₃⁺. In agreement with this hypothesis, Xenopus laevis oocytes injected with the KAT1 RNA of Arabidopsis thaliana were permeable to K⁺ and NH₄⁺, but not to CH₃NH₃⁺.     

Plasmalemma Redox Chain and H Extrusion: II. Respiratory and Metabolic Changes Associated with Fusicoccin-Induced and with Ferricyanide-Induced H Extrusion

Ferricyanide reduction by Elodea densa leaves is associated with a release of protons in the cytoplasm, a fraction of the increase in protons being then extruded by the ATP-driven proton pump (20). The data presented here show that ferricyanide induces a marked increase in O₂ uptake, additive to that induced by fusicoccin plus K⁺, and here interpreted as depending on the utilization of ATP by the H⁺ pump. Glucose 6-phosphate and malate levels are markedly increased by fusicoccin plus K⁺. The simultaneous presence of ferricyanide reduces by about 50% the increase of malate, while it completely suppresses that of glucose 6-phosphate. The ferricyanide-induced decrease of malate is interpreted as due to the acidification of the cytosol associated with ferricyanide reduction, while the more marked decrease of glucose 6-phosphate might depend in part on the pH change and in part on a faster oxidation of this substrate. In fact, ferricyanide reduction is accompanied by a marked decrease of the incorporation into RNA ribose of C-1 as compared with C-2 of [¹⁴C]glucose. This suggests a stimulation of the release of C-1 as CO₂ at the level of the glucose 6-phosphate oxidation pathway, as expected if NADPH was the electron donor for ferricyanide reduction. These results are interpreted as confirming that the H⁺ efflux associated with ferricyanide reduction depends on the activation of the ATP-driven plasmalemma H⁺ pump. They also suggest that NADPH is used as an electron donor to some initial component of the plasmalemma redox system.     

Proton permeability and the regulation of potassium permeability in mitochondria by uncoupling agents

Summary The addition of agents that uncouple electron transfer from energy conservation (uncouplers) to state 4 mitochondria causes the following ion movements: K⁺ is extruded from the mitochondria in association with phosphate and possibly other anions, but not H⁺. Endogenous Ca⁺⁺ is extruded from the mitochondria, and H⁺ moves in to counter-balance the Ca⁺⁺ movement; some phosphate movement may be associated with Ca⁺⁺ extrusion. The rate and extent of K⁺ extrusion induced by uncoupler is dependent on the concentrations of external phosphate and divalent ions. Phosphate induces K⁺ extrusion, while Mg⁺⁺ and Mn⁺⁺ inhibit it. TheV _max of K⁺ transport is 300 moles K⁺/g protein per min. The K _m for FCCP-induced potassium extrusion is 0.25 M at pH 7.4. The inhibitory effect of Mg⁺⁺ is noncompetitive with respect to uncoupler concentration but competitive with respect to phosphate concentration. The experimental evidence does not support the existence of high H⁺ permeability in the presence of uncoupler. A correlation is observed between the rate of K⁺ extrusion and the energy reserves supplied from the high energy intermediate. The action of uncoupler in inducing K⁺ permeability is considered to arise through its action in depleting the energy reserves of mitochondria rather than through a specific activating effect of permeability by the uncoupler itself. The relationship of membrane potential to regulation of K⁺ permeability is discussed.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.