Comparison of Isoprene Emission, Intercellular Isoprene Concentration and Photosynthetic Performance in Water-Limited Oak (Quercus pubescens Willd. and Quercus robur L.) Saplings

Abstract: The influence of prolonged water limitation on leaf gas exchange, isoprene emission, isoprene synthase activities and intercellular isoprene concentrations was investigated under standard conditions (30 °C leaf temperature and 1000 μmol photons m^-2 s^-1 PPFD) in greenhouse experiments with five-year-old pubescent oak ( Quercus pubescens Willd.) and four-year-old pedunculate oak ( Quercus robur L.) saplings. Net assimilation rates proved to be highly sensitive to moderate drought in both oak species, and were virtually zero at water potentials (Ψ_pd) below - 1.3 MPa in Q. robur and below - 2.5 MPa in Q. pubescens . The response of stomatal conductance to water stress was slightly less distinct. Isoprene emission was much more resistant to drought and declined significantly only at Ψ_pd below - 2 MPa in Q. robur and below - 3.5 MPa in Q. pubescens . Even during the most severe water stress, isoprene emission of drought-stressed saplings was still approximately one-third of the control in Q. robur and one-fifth in Q. pubescens . Isoprene synthase activities were virtually unaffected by drought stress. Re-watering led to partial recovery of leaf gas exchange and isoprene emission. Intercellular isoprene concentrations were remarkably enhanced in water-limited saplings of both oak species during the first half of the respective drought periods with maximum mean values up to ca. 16 μl l^-1 isoprene for Q. pubescens and ca. 11 μl l^-1 isoprene for pedunculate oak, supporting the hypothesis that isoprene serves as a short-term thermoprotective agent in isoprene-emitting plant species.     

High carbon dioxide and sun/shade effects on isoprene emission from oak and aspen tree leaves

Abstract. Isoprene (2-methyl 1, 3-butadiene) is emitted from many plants, especially trees. We tested the effect of growth at high CO₂ partial pressure and sun versus shade conditions on the capacity of Quercus rubra L. (red oak) and Populus tremuloides Michx. (quaking aspen) leaves to make isoprene. Oak leaves grown at high CO₂ partial pressure (65 Pa) had twice the rate of isoprene emission as leaves grown at 40Pa CO₂. However, aspen leaves behaved oppositely, with high CO₂-grown leaves having just 60-70% the rate of isoprene emission as leaves grown in 40 Pa CO₂. Similar responses were observed from 25 to 35 °C leaf temperature during assay. The stimulation of isoprene emission by growth at high CO₂ and the stimulation in high temperature resulted in isoprene emission consuming over 15% of the carbon fixed during photosynthesis in high-CO₂ grown oak leaves assayed at 35 °C. Leaves from the south (sunny) sides of trees growing in natural conditions had rates of isoprene emission double those of leaves growing in shaded locations on the same trees. This effect was similar in both aspen and oak. The leaves used for these experiments had significantly different chlorophyll a/b ratios indicating they were functionally sun (from the sunny locations) or shade leaves (from the protected locations). Because the metabolic pathway of isoprene synthesis is unknown, we are unable to speculate about how or why these effects occur. However, these effects are more consistent with metabolic control of isoprene release rather than a metabolic leak of isoprene from metabolism. The results are also important for large scale modelling of isoprene emission and for predicting the effect of future increases in atmospheric CO₂ level on isoprene emission from vegetation.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

The composition and function of thylakoid membranes from pea plants grown under white or green light with or without far-red light

Pea plants ( Pisum sativum L. ev. Greenfeast) were grown for 2 to 3 weeks in while (˜ 50 μmol photons m⁻² s⁻¹; 400–700 nm) or green (˜ 30 μmol photons m⁻² s ⁻¹ 400–700 nm) light (16 h day/8 h night), with or without far-red light. Supplementary far-red light decreased leaf area and increased internodal length in both white and green light, demonstrating that phytochrome influenced leaf size and plant growth. However, there was no effect of far-red light on chlorophyll a /chlorophyll b ratios, chlorophyll-protein composition, the stoichiometry of electron transport complexes or photosynthetic function of isolated thylakoids. These results suggest that phytochrome is ineffective in modulating the composition and function of thylakoids in pea plants grown at low irradiance. One possible explanation of the ineffectiveness of phytochrome on thylakoids is discussed in terms of the drastic attenuation of red relative to far-red light in green tissue.     

Chloroplast structure in normal and pigment-deficient soybeans grown in continuous red or far-red light

Clark L1, a normal green soybean [ Glycine max (L.) Merrill] and Clark y₉y₉, a backross-developed isoline exhibiting pigment deficiency, were grown under continuous red (11 W m⁻² and far-red (9 W m⁻²) light. Chloroplast thylakoids from the unifoliolate leaf (9–10 days old) were isolated and analyzed for pigments, pigment-protein, membrane polypeptides, electron transport and ultrastructural differences. Chloroplasts of soybean plants grown under far-red light have decreased chlorophyll a to chlorophyll b ratio, increased light-harvesting complexes, and grana structure with few stroma-type thylakoids. Photosystem II/photosystem I ratios (PSII/PSI) are higher in far-red due to decreased synthesis of PSI reaction center and/or less antenna associated with PSI.     

Isoprene emissions and photosynthesis in three ferns – The influence of light and temperature

A study was designed to determine the rates of isoprene emission and photosynthesis in three fern species [ Dicksonia antarctica Labill., Thelypteris decursive-pinnata (Van Hall) Ching and Thelypteris kunthii (Desv.) Morton] and the independent influence of light and temperature on these processes. The plants were conditioned in a growth chamber and then transferred to a controlled environment gas-exchange chamber. Samples of the chamber atmosphere were collected; isoprene was concentrated cryo-genically and measured by gas chromatography. Only small amounts of isoprene were detected around the ferns in the dark. Isoprene emissions increased with increasing levels of photosynthetic photon flux density (PPFD) in all three species; 50% of the maximum emission occurred at PPFD levels of 130 to 500 μmol m−² s−¹. Maximum isoprene emissions occurred between 35 and 39°C which is a lower temperature maximum than reported for angiosperms and gymnosperms. The increased emissions with temperature were primarily associated with increased biosynthetic rates for isoprene. Carbon lost through isoprene accounted for 0.02 to 2.6% of the carbon fixed during photosynthesis, depending on the PPFD level, temperature and fern species.     

Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper

We studied the sequence of several metabolic reactions, representative for oxidative damage and protection, in primary leaves of Phaseolus vulgaris (cv. Limburgse vroege) as a function of root assimilation of a toxic sublethal Cu concentration (630 μ M ). A transient increase of products of membrane peroxidation was observed in the primary leaves during the period of Cu uptake. This rise was mainly due to the oxidizing properties of copper itself and not to a stimulation of the lipoxygenase (EC 1.13.11.12) activity. In our experimental conditions, membrane lipid peroxidation and K⁺-leakage were not directly related; during at least three days after Cu application to the roots, when products of lipid peroxidation were already detected in the leaf, permeability of the cytoplasmic membrane for K⁺ was improved. However, Cu stimulated the capacity of catalase (EC 1.11.1.6) and ascorbate peroxidase (EC 1.11.1.11). These enzymes protect the tissue against oxidative stress since at least the hydrogen peroxide content was significantly reduced. Superoxide dismutase (EC 1.15.1.1) was not involved in this defense mechanism.     

Zinc-dependent changes in ESR signals, NADPH oxidase and plasma membrane permeability in cotton roots

The effect of Zn²⁺ on the plasma membrane permeability and superoxide radical (O₂^-) formation in roots was studied with cotton ( Gossypium hirsutum L. cv. Delta-pine 15/21) plants grown in nutrient solution with different Zn²⁺ supply. Compared to Zn-sufficient plants, the plasma membrane permeability of Zn-deficient plants was increased as indicated by a 3-, 5- and 2.5-fold increase in root cell leakage of K⁺, NO₃^- and organic carbon compounds, respectively. Resupply of Zn²⁺ to Zn-deficient plants for 12 h substantially decreased this leakage. The effects of Zn²⁺ on membrane permeability were closely correlated with the levels of O₂^- measured by electron spin resonance (ESR) spectroscopy in the microsomal membrane fraction and in the cytosol fraction of root cells. The amplitudes of the O₂^- -derived Tiron ESR signal also coincided with a O₂^- -generating oxidase activity which was strongly dependent on the presence of NADPH and FAD. The results suggest that Zn²⁺ directly affects the integrity of the plasma membrane, at least in part, by interfering with O₂^- generation by a membrane-bound NADPH oxidase.     

Kinetics of net H+, Ca2+, K+, Na+, NH+4, and Cl- fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues

Although temperature-induced changes in membrane structure and activity seem to be central to chilling stress perception, the specific details of temperature effects on plant nutrient acquisition remain obscure. In this study, we have undertaken a comparative study of low temperature effects on the activity of plasma membrane transporters of different ions in corn ( Zea mays L.) leaf and root tissues by non-invasive measurements of net ion fluxes using ion-selective microelectrode (the MIFE) technique. Kinetics of net H⁺, Ca²⁺, K⁺, Na⁺,     and Cl^– fluxes were measured as plant tissues recovered after short-term (3 h) chilling stress. The major findings can be summarized as follows: (1) The critical temperatures, under which the recovery of the activity of plasma membrane transporters took place, were found to be the same for all ions measured and are likely to be associated with the phase transition of membrane lipids. (2) The most pronounced was the reduction in net uptake of K⁺ and     (3) Chilling treatment caused a significant net influx of Cl^– and Na⁺ in the leaf tissue. (4) For the same species, the critical temperatures for membrane-transport processes in roots were 2–2.5°C lower than in leaves. Possible physiological significance of these findings is discussed.     

The effects of high temperature on isoprene synthesis in oak leaves

Isoprene emission from plants is highly temperature sensitive and is common in forest canopy species that experience rapid leaf temperature fluctuations. Isoprene emission declines with temperature above 35 °C but the temperature at which the decline begins varies between 35 and 44 °C. This variability is caused by the rate at which leaf temperature is increased during measurement with lower temperatures associated with longer measurement cycles. To investigate this we exposed leaves of red oak (Quercus rubra L.) to temperature regimes of 35–45 °C for periods of 20–60 min. Isoprene emission increased during the first 10 min of high temperature exposure and then decreased over the next 10 min until it reached steady state. This phenomenon was common at temperatures above 35 °C but was not noticeable at temperatures below that. The response was reversible within 30 min by lowering leaf temperature to 30 °C. Because there is no storage of isoprene inside the leaf, this behaviour indicates regulation of isoprene synthesis in the leaf. We demonstrated that the variability in isoprene decline results from regulation and explains the variability in the temperature response. This is consistent with our theory that isoprene protects leaves from damage caused by rapid temperature fluctuations.     

Ca(2+) and Mg(2+)/ATP independently trigger homotypic membrane fusion in gastric secretory membranes

Exocytic activation of gastric parietal cells represents a massive transformation. We studied a step in this process, homotypic fusion of H,K-ATPase-containing tubulovesicles, using R18 dequenching. Ca²⁺ and Mg²⁺/ATP each caused dramatic dequenching, reflecting a change in R18 distribution from 5% to 65–90% of the assay's membranes in 2.5 min. These stimuli also triggered fusion between tubulovesicles and liposomes. Independent confirmation that dequenching represented membrane fusion was established by separating tubulovesicle–liposome fusion products on density gradients. Only agents that trigger fusion allowed the transmembrane H,K-ATPase to move to low-density fractions along with R18. EC₅₀ for Ca²⁺-triggered fusion was 150 n m and for Mg²⁺/ATP-triggered fusion 1 m m , the latter having a Hill coefficient of 2.5. ATP-triggered fusion was specific for Mg²⁺/ATP, required ATP hydrolysis, and was insensitive to inhibition of NSF and/or H,K-ATPase. Fusion initiated by either trigger caused tubulovesicles to become resistant to subsequent challenge by either trigger. Ca²⁺-and Mg²⁺/ATP-triggered fusion required protein component(s) in tubulovesicles, though this was required in only one of the fusing membranes since tubulovesicles fused well with liposomes containing no proteins. Our data suggest that exocytosis in parietal cells is triggered by separate but interacting pathways and is regulated by self-inhibition.     

A Conserved, Lipid-Mediated Sorting Mechanism of Yeast Ist2 and Mammalian STIM Proteins to the Peripheral ER

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS^Ist2) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS^Ist2 targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P₂ at the PM created ER–PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca²⁺ entry (SOCE). The CSS^Ist2 and the C-terminus of the ER-located Ca²⁺ sensor STIM2 were sufficient to bind PI(4,5)P₂ and PI(3,4,5)P₃ at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P₂, suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.     

Enhancement of the light-triggered electrical response in plant cells following their de-energization with uncouplers

Light-triggered membrane potential changes in cells of a liverwort Anthoceros are greatly enhanced by the ionophorous uncouplers nigericin and monesin. Stimulation of the light-triggered electrical response (LTER) by nigericin occurred concomitantly with inhibition of a slow decline in the chlorophyll fluorescence, which suggests that the transmembrane pH gradient in thylakoids is not essential for generation of LTER at the plasma membrane. The extent of monensin-stimulated LTER remained high under a diminished driving force for the ionophore-induced proton-cation exchange across the plasma membrane (elevation of the external Na⁺ concentration from 1 to 50 m M ), which indicates that energy uncoupling in chloroplasts is more related to the electric response enhancement than the induction of the H⁺/K⁺(Na⁺) exchange at the plasma membrane. Enhancement of LTER by ionophores occurs in parallel with stimulation of light-triggered pH changes (alkalinization) in the vicinity of the cell surface, which suggests an association of trans-membrane H⁺ fluxes with LTER. The results are consistent with the hypothesis that illumination produces a temporary inhibition of the plasma membrane H⁺ pump with a subsequent activation of gated channels and transient rapid depolarization of the cell.     

Hg对油菜叶细胞膜的损伤及细胞的自身保护作用

砂基培养50d的油菜幼苗,用不同浓度的HgCl₂溶液灌溉后,研究叶细胞膜结构与功能及细胞保护系统的变化。结果表明,0.5mg·L^-1Hg污灌叶细胞膜结构与功能未见明显变化,细胞保护系统亦无变化;浓度大于1mg·L^-1时,叶组织细胞膜脂质过氧化水平升高,膜结构损伤,膜透性增大。1～10mg·L^-1Hg污灌,组织蛋白质含量升高,SOD、POD、CAT活性表现出不同程度的升高,细胞呈现出积极性自身保护作用;50mg·L^-1Hg污灌,组织蛋白质含量下降,SOD、POD、CAT活性持续下降,细胞自身积极性保护作用消失,说明细胞自身保护系统只能在一定范围内起积极保护作用。     

The Mechanism of Iron (III) Stimulation of Lipid Peroxidation

A study conducted on Fe²⁺ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe³⁺ addition but the stimulation did not present a maximum at a Fe²⁺/Fe³⁺ ratio approaching 1:1. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation was able to oxidize deoxyribose; the increased Fe²⁺ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triph-enylphosphine. Neither Fe²⁺ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe³⁺ effect on Fe²⁺ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe²⁺/Fe³⁺ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe³⁺ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe³⁺ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.     

Membrane potential generation by two reconstituted mitochondrial systems: Liposomes inlayed with cytochrome oxidase or with ATPase

Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB⁻), a penetrating anion probe, was used.

The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB⁻ uptake by liposomes. The PCB⁻ response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.

Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F₁and F_c, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB⁻ uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.

The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).     

Protective cytokinin action switches to damaging during senescence of detached wheat leaves in continuous light

The effect of exogenous application of the cytokinin meta -topolin [mT; N⁶-( meta -hydroxybenzyl)adenine] on artificial senescence of detached wheat leaves ( Triticum aestivum L. cv. Hereward) was studied and compared in leaves senescing under continuous light (100 µmol photons m⁻² s⁻¹) and darkness. Senescence-induced deterioration in structure and function of the photosynthetic apparatus was characterized by reduction in chlorophyll content, maximal efficiency of photosystem (PS) II photochemistry ( F _v/ F _m) and the rate of CO₂ assimilation, by increase in the excitation pressure on PSII (1 −  q _P) and a level of lipid peroxidation and by modifications in chloroplast ultrastructure. While in darkened leaf segments mT effectively slowed senescence-induced changes in all measured parameters, in light-senescing segments the effect of mT changed into opposite a few days after detachment. We observed an overexcitation of photosynthetic apparatus, as indicated by pronounced increases in the excitation pressure on PSII and in a deepoxidation state of xanthophyll cycle pigments, marked starch grain accumulation in chloroplasts and stimulation of lipid peroxidation in light-senescing leaf segments in mT. Possible mechanisms of acceleration of senescence-accompanying decrease in photosynthetic function and increase in lipid peroxidation during mT influence are discussed. We propose that protective mT action in darkness becomes damaging during artificial senescence in continuous light due to overexcitation of photosynthetic apparatus resulting in oxidative damage.     

Mechanism of calcium-dependent salt tolerance in cells of Nitellopsis obtusa: role of intracellular adenine nucleotides

Abstract. The tonoplasts of internodal cells of Nitellopsis were removed by perfusing the vacuoles with media containing a Ca² chelator, EGTA. Treatment of tonoplast-free cells with 100 mol m³ NaCl induces a large membrane depolarization, a drastic decrease in the membrane resistance and an increase in Na⁺ influx. These events are identical to those that occur in intact cells subjected to high NaCl. These responses to NaCl are prevented if 10 mol m³ Ca²⁺ is supplied together with 100 mol m³ NaCl. The protective effect of Ca²⁺ is evident only when the intracellular ATP concentration exceeds 0.1 mol m³ and does not occur full when the intracellular ATP is removed. AMP at concentrations greater than 0.5 mol m³ or 0.25 mol m³ AMPPNP can replace ATP. It is concluded that ATP does not act as an energy source nor as a substrate for protein phosphorylation. ATP seems to exert its effects as a coeffector with Ca²⁺ in regulating the Na⁺ permeability of the plasma membrane.