Equilibrium and Ion Exchange Characteristics of Potassium and Sodium Accumulation by Barley Roots

Potassium ion and Na⁺ influx and efflux rates into and from excised barley roots are compared with the maximum capacity of accumulation. Potassium ion and Na⁺ influx and efflux involve a cation exchange that is independent of simultaneous exchange of the accompanying anion. These exchange fluxes depend on the concentration and cation composition of the solutions from which they originate. Selective differences between K⁺ and Na⁺ fluxes are sufficient to account for a cationic distribution within the roots that differs markedly from that of the external solution and that persists for extended time periods. The accumulation maximum is a cation exchange equilibrium with the cation influx and efflux rates approaching equality. The equilibrium level is independent of the individual cation fluxes and the external solution concentration. It is a finite quantity which appears to be determined by the internal anion concentration including accumulated as well as endogenous anions.     

Cation and anion balance in the xylem exudate of tobacco roots

Summary The sum of Na, K, Ca, Mg in the exudate of tobacco generally exceeded the sum of mineral anions. Insufficient organic acids were present to account for the differences and bicarbonate appeared to be the other anion involved. Amino acids were present in very low concentrations relative to mineral cations. When nitrate salts only were in the external solutions, the anions were mostly, but not entirely, nitrate. When chloride salts only were in the external solutions, the cations far exceeded the level of mineral anions in the exudate. It is postulated that nitrate is actively transported when nitrate salts are in the external solution regardless of the cation, but when anions other than nitrate are in the external solution, the cations are actively transported with the anions passively following. Nitrate transport was via a symplasm, but that of the other anions seemed to be different. When bicarbonate is the only anion in the external solution and when present at relatively high concentrations (5 × 10⁻³ M or higher), the volume of exudate is decreased. It appears that the organic acids which were synthesized as a result of the bicarbonate absorption were not transferred to the xylem vessels.     

Active ion uptake and maintenance of cation-anion balance: A critical examination of their role in regulating rhizosphere pH

The processes responsible for maintenance of cation-anion balance in plants and their relation to active ion accumulation and changes in rhizosphere pH are outlined and discussed. The major processes involved are: (1) accumulation and degradation of organic acids which occur in the plant mainly as organic acid anions (and their transfer within the plant) and (2) extrusion of H⁺ or OH^– into the rhizosphere. The relative importance of the two processes is determined by the size of the excess anion or cation uptake. Indeed, plants typically absorb unequal quantities of nutritive cations (NH₄ ⁺+Ca²⁺+ Mg²⁺+K⁺+Na⁺) and anions (NO₃ ^–+Cl^–+SO₄ ^2–+H₂PO₄ ^–) and charge balance is maintained by excretion of an amount of H⁺ or OH^– which is stoichiometrically equal to the respective excess cation or anion uptake. The mechanisms and processes by which H⁺ and in particular OH^– ions are excreted in response to unequal cation-anion uptake are, however, poorly understood.The contemporary view is that primary active extrusion of H⁺, catalyzed by a membrane-located ATPase, is the major driving force for secondary transport of cations and anions across the plasma membrane. However, the fact that net OH^– extrusion often occurs (since excess anion absorption commonly takes place) implies there is a yet-to-be characterized OH^– ion efflux mechanism at the plasma membrane that is associated with anion uptake. There is, therefore, a need for future studies of the uptake mechanisms and stoichiometry of anion uptake; particularly that of NO₃ ^– which is often the predominant anion absorbed. Another related phenonenon which requires detailed study in terms of cation-anion balance is localized rhizosphere acidification which can occur in response to deficiencies of Fe and P.     

Relationship of Cell Sap pH to Organic Acid Change During Ion Uptake

Excised roots of barley (Hordeum vulgare, var. Campana) were incubated in KCl, K₂SO₄, CaCl₂, and NaCl solutions at concentrations of 10⁻⁵ to 10⁻² n. Changes in substrate solution pH, cell sap pH, and organic acid content of the roots were related to differences in cation and anion absorption. The pH of expressed sap of roots increased when cations were absorbed in excess of anions and decreased when anions were absorbed in excess of cations. The pH of the cell sap shifted in response to imbalances in cation and anion uptake in salt solutions as dilute as 10⁻⁵ n. Changes in cell sap pH were detectable within 15 minutes after the roots were placed in 10⁻³ n K₂SO₄. Organic acid changes in the roots were proportional to expressed sap pH changes induced by unbalanced ion uptake. Changes in organic acid content in response to differential cation and anion uptake appear to be associated with the low-salt component of ion uptake.     

Leaching of solutes by the action of acidic rain: a comparison of efflux from twigs and single needles of Picea abies (L. Karst.)

Abstract. Leaching of inorganic cations (K⁺, Mg²⁺) and in some cases of inorganic anions and sugars from detached twigs and single needles of spruce Picea abies L. Karst.) in the presence of acid rain (H₂SO₄, 1 mol m^?3) or salt solutions (Na₂ SO₄, 1 mol m^?3) was examined under laboratory conditions. Cation leaching (as percentage of the total water soluble ion content of the tissue per hour) was: K⁺: 0.01-0.02%; Mg²⁺: 0.005-0.01%; Ca²⁺: 0.1-0.2%. Leaching rates of anions were even lower than that and concentrations in the leachate were often below the detection limit of anion chromatography. Spraying with H₂SO₄ (pH 2.95, 1 mol m^?3) increased leaching only transiently. Similar effects were found when Na₂SO₄ was used instead of H₂SO₄. The transiently enhanced leaching was apparently due to H⁺/cation or cation/cation exchange at the twig or leaf surfaces. Feeding of K⁺ or Al³⁺ through the stems increased leaching of all cations within a few hours, again demonstrating rapid ion exchange in the apoplast. Leaching of potassium and magnesium from single needles occurred at similar relative rates as from twigs. Loss of Ca²⁺ ions, however, was even smaller from needles than from twigs. Apparently, a large part of the Ca²⁺ lost from twigs originated from the bark and not from the needles. Efflux of ions from longitudinal needle sections was about 1000 times taster than the rates obtained with intact needles, indicating that the cuticle was the main barrier Preventing solute loss. In relation to the total amount of mineral nutrients in trees, leaching is considered to be too small to be the primary cause of damage to trees stressed by acid rain, as has been suggested in the literature.     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Effects of pH Changes on Ion and 2,4-D Uptake of Wheat Roots

The quantitative relationships between pH-dependent ion and 2,4-D uptake in winter wheat seedlings (Triticum aestivum L. cv. Yubileynaya 50) have been investigated. The movement of various ions (potassium, phosphate, nitrate and ammonium) and 2,4-D across the root membranes was monitored with radioactive and stable isotope tracer methods. It was found that the H₊ ion concentration of the absorption solution strongly influences the 2,4-D uptake of the roots. Simultaneously, the 2,4-D uptake stimulates secretion of H⁺ into the absorption solution, that is, a H⁺ efflux can accompany the uptake of 2,4-D. This finding is consistent with the acid secretion theory of auxin and fusicoccin action. At pH 4 the 2,4-D uptake was much higher than at pH 6, thereby inhibiting the ion uptake and increasing the phytotoxicity in the plant. The results indicate that 2,4-D enters the root cells rapidly at the lower pH, mostly as undissociated molecules. With reference to the 2,4-D concentration in the roots at pH 4, a possible transport mechanism of the auxin herbicide is briefly discussed.     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

Correlation of proton release and ultraviolet difference spectra associated with metal binding by transferrin

A variety of metal ions can bind to the iron-transport protein, transferrin, at two specific sites. For each metal ion, a carboxylate anion is concomitantly bound. Six metal ions which were examined fall into two classes based on proton release and ultraviolet spectral changes which accompany binding to the protein. Class II ions, which include Cu²⁺ and Zn²⁺, release approximately 2 H⁺/metal bond. Class III ions, which include Fe³⁺, Ga³⁺, Al³⁺, and VO²⁺, release approximately 3 H⁺/metal bound. The increase in absorbance near 242 nm, characteristic of tyrosine ionization, has the ratio 0.55–0.75 for class II:class III ions. Both Fe³⁺ and Cu²⁺ form metal-transferrin-oxalate complexes in the presence of excess C₂O₄^2?. Fe³⁺ releases close to 3 H⁺/metal whether forming oxalate or bicarbonate complexes with transferrin. Binding of Cu²⁺ to transferrin releases 2 H⁺/metal in the presence of C₂O^2?₄ or HCO₃^?. Since equal numbers of H⁺/metal are released for both anions, it is likely that the bicarbonate ion does not lose its proton, and remains as HCO₃^? in transferrin. These results are interpreted in terms of possible combinations of ligands at the metal binding sites.     

The Rates of Interexchange of Ions Across Fixed Charge Membranes in Bi-Ionic Systems

This paper examines the applicability of the Nernst-Planck approach in treating the relationship between the initial rates at which critical ions interexchange across permselective membranes in bi-ionic systems and the rates of self-exchange of these ions across the same membranes. Data are presented for five species of univalent cations with two types of cation permeable membranes, a polystyrene sulfonic acid-collodion matrix membrane, and an oxidized collodion membrane; five species of univalent anions were studied with a protamine-collodion matrix anion permeable membrane. Except with systems involving H⁺ ion, the experimentally found relationships between the rates of interexchange and the rates of self-exchange were in agreement, in most cases within ±5%, with the values calculated from an expression in which interaction between the critical ions in the membrane is not taken into account. In systems with H⁺ ion, the experimental rates of interexchange were from 27% to 40% less than calculated values.     

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     

Separation of tonoplast and plasma membrane H+-ATPase from Zucchini hypocotyls by consecutive sucrose and glycerol gradient centrifugation

Summary In order to isolate tonoplast and plasma membrane vesicles involved in ATP-dependent proton transport we devised a preparative procedure with two consecutive centrifugations. Three fractions were obtained on a sucrose step gradient: light microsomes, heavy microsomes, and a mitochondria-rich fraction. The light and heavy microsomal fractions were each recentrifuged on an isopycnic glycerol density gradient. Recentrifugation of light microsomes resulted in two fractions with H⁺-ATPase activity, one equilibrating at a density less than 1.11 g/cm³ and one equilibrating at a density of about 1.17g/cm³. Comparison with marker enzyme activities suggests that the upper fraction was enriched in tonoplast, and the dense fraction with plasma membrane. In addition to marker enzyme content, H⁺ transport in the H⁺-ATPase-containing fractions was further characterized with respect to pH dependence, cation and anion dependence, and uncouplers and inhibitors. H⁺ transport in all fractions was strongly dependent on the presence of halides but no specific stimulation by potassium or any other monovalent cation was found. Of the anions tested, malate and fumarate preferentially stimulated H⁺ transport in the tonoplast-enriched fraction. It is suggested that a Ca²⁺/H⁺ antiporter is present in all fractions. Only H⁺-ATPase in the plasma membrane-enriched fractions was sensitive to nystatin, an uncoupler, and to orthovanadate, an inhibitor. The tonoplast fraction was more sensitive to nitrate than the plasma membrane-enriched fraction, and all fractions showed some sensitivity to high concentrations of oligomycin. Oligomycin sensitivity was not due to the presence of mitochondria.     

Cation fluxes in excised and intact roots in relation to specific and varietal differences

Summary In this short survey differences between species and varieties in the four major mechanisms that affect selective uptake of potassium and sodium to the plant within the root are considered. These include influx selectivity, K⁺/Na⁺ exchange at the plasmalemma, and selectivity at the tonoplast as well as at the symplasm-xylem boundary. The affinity of various plants for potassium influx in system 1 is rather uniform although varietal differences in barley have been observed. Differences are much more pronounced for sodium influx, for which Helianthus showed rather high and Fagopyrum rather low affinity. There is substantial variation between species in the efficiency of K⁺/Na⁺ exchange at the plasmalemma of cortical root cells; the three cereals Hordeum, Triticum, and Secale were highly efficient while K⁺/Na⁺ exchange in Atriplex, Helianthus and Allium was poor, even if the cytoplasmic sodium content was accounted for. Apparently there was no direct relation between salt tolerance and K⁺/Na⁺ exchange. The observed differences in the efficiency of K⁺-dependent sodium extrusion or K⁺/Na⁺ exchange were not due to the use of excised roots, they were observed also when roots of whole seedlings were investigated. At the tonoplast a 11 exchange of vacuolar potassium for sodium has been observed in roots of Hordeum. By this exchange sodium ions are removed from the symplasm and potassium ions are recovered from vacuoles and thus made available for transport to the shoot. Indications for specific differences in this exchange have been observed; the exchange appears to be more efficient in Helianthus than in Hordeum roots. More comparative studies are needed here. At the boundary between symplasm and xylem vessels selectivity can be set up during xylem release of cations and there are reports that suggest a preference for sodium (Lycopersicum cheesemanii, Solanum pennellii, and Suaeda) and for varietal differences amongst tomatoes. Selectivity at this boundary, the plasmalemma of the xylem parenchyma cells was described in this paper by the selectivity ratio of transport that relates the rates of xylem transport to the cytoplasmic sodium and potassium concentrations. Based on this ratioAtriplex hortensis was shown to discriminate for sodium during xylem release while there was little selectivity in Hordeum and possibly some discrimination in favour of K⁺ in Allium roots. The data are shortly discussed in relation to salt tolerance and to the breeding of salt-tolerant crop varieties.     

Sodium and potassium binding by rat liver cell microsomes

The effects of ion concentration, pH, and presence of competing ions on the sodium and potassium binding properties of rat liver cell microsomes were studied. Typical adsorption isotherms were obtained in the concentration dependence studies, with saturation being reached when 1.2 to 1.4 m.eq. cations were retained per gm. of microsome Kjeldahl nitrogen. The retention was shown to be due to a binding to specific sites rather than to a trapping of the cations. The binding showed a sharp pH dependence in the range 6.0 to 7.5. The presence of one cation depressed the binding of the other, indicating that Na⁺ and K⁺ as well as H⁺ ions compete for the same sites. Potassium was bound slightly more strongly than sodium, while hydrogen was bound about 10⁵ times more strongly than either. Calculations show that the binding follows the simple mass law. Similarities between adsorption by microsomes and adsorption by synthetic cation exchange resins are discussed and compared to some of the characteristics of electrolyte behavior in living systems. A possible ion exchange elution, active cation transport mechanism is suggested, involving the preferential elution of Na⁺ out of the cell by H⁺ ions produced by metabolism.     

Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site

The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na⁺ and K⁺ conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3–5 and TM8 that should function as an anion exchanger and a cation leak.     

Potassium Translocation into the Root Xylem

Abstract: Potassium is the most abundant cation in cells of higher plants and plays vital roles in plant growth and develop ment. Since the soil is the only source of potassium, plant roots are well adapted to exploit the soil for potassium and supply it to the leaves. Transport across the root can be divided into three stages: uptake into the root symplast, transport across the symplast and release into the xylem. Uptake kinetics of potassium have been studied extensively in the past and sug gested the presence of high and low affinity systems. Molecular and electrophysiological techniques have now confirmed the existence of discrete transporters encoded by a number of genes. Surprisingly, detailed characterisation of the transpor ters using reverse genetics and heterologous expression shows that a number of the transporters (AKT and AtKUP family) func tion both in the low (μM) and high (mM) K⁺ range. Electrophy siological studies indicate that K⁺ uptake by roots is coupled to H⁺, to drive uptake from micromolar K⁺. However, thus far only Na⁺ coupled K⁺ transport has been demonstrated (HKT1). Ion channels play a major role in the exchange of potassium be tween the symplast and the xylem. An outward rectifying chan nel (KORC) mediates potassium release. Cloning of the gene en coding this channel (SKOR) shows that it belongs to the Shaker super-family. Both electrophysiological and genetic studies demonstrate that K⁺ release through this channel is controlled by the stress hormone abscisic acid. Interestingly, xylem par enchyma cells of young barley roots also contain a number of in ward rectifying K⁺ channels that are controlled by G-proteins. The involvement of G-proteins emphasises once more that po tassium transport at the symplast/xylem boundary is under hor monal control. The role of the electrical potential difference across the symplastxylem boundary in controlling potassium release is discussed.     

Carbon Incorporation and Anion Dynamics as Synergistic Drivers for Ultrafast Diffusion in Superionic LiCB11H12 and NaCB11H12

The disordered phases of LiCB₁₁H₁₂ and NaCB₁₁H₁₂ possess superb superionic conductivities that make them suitable as solid electrolytes. In these materials, cation diffusion correlates with high orientational mobilities of the CB₁₁H₁₂^? anions; however, the precise relationship has yet to be demonstrated. In this work, ab initio molecular dynamics and quasielastic neutron scattering are combined to probe anion reorientations and their mechanistic connection to cation mobility over a range of timescales and temperatures. It is found that anions do not rotate freely, but rather transition rapidly between orientations defined by the cation sublattice symmetry. The symmetry‐breaking carbon atom in CB₁₁H₁₂^? also plays a critical role by perturbing the energy landscape along the instantaneous orientation of the anion dipole, which couples fluctuations in the cation probability density directly to the anion motion. Anion reorientation rates exceed 3 × 10¹⁰ s^?1, suggesting the underlying energy landscape fluctuates dynamically on diffusion‐relevant timescales. Furthermore, carbon is found to modify the orientational preferences of the anions and aid rotational mobility, creating additional symmetry incompatibilities that inhibit ordering. The results suggest that synergy between the anion reorientational dynamics and the carbon‐modified cation–anion interaction accounts for the higher ionic conductivity in CB₁₁H₁₂^? salts compared with B₁₂H₁₂^2?.     

Long-term effects of SO2 on plants,SO2 metabolism and regulation of intracellular pH

The impact of SO₂ on the ionic balance of plants and its implications for intracellular pH regulation was studied to find explanations for long-term effects of SO₂. When sulphur, taken up as SO₂ by the shoots of plants, is not assimilated in organic compounds, but stored as sulphate, an equivalent amount of H⁺ is produced. These H⁺ ions are not buffered chemically, but removed by metabolic processes.On the basis of knowledge on metabolic buffering mechanisms a conceptual model is proposed for the removal of shoot-generated H⁺ by (i) OH^- ions, produced in the leaves when sulphate and nitrate are assimilated in organic compounds and/or by (ii) OH^- ions produced by decarboxylation of organic anions (a biochemical pH stat mechanism). The form in which nitrogen is supplied largely determines the potential of the plant to neutralize H⁺ in the leaves during SO₂ uptake by the proposed mechanisms.In field experiments with N₂ fixing Vicia faba L. crops, the increase of sulphate in the shoots of SO₂-exposed plants was equivalent in charge to the decrease of organic anion content, calculated as the difference between inorganic cation content (C) and inorganic anion content (A), indicating that H⁺ ions produced in the leaves following SO₂ uptake were partly removed by OH^- from sulphate reduction and partly by decarboxylation of organic anions.The appearance of chronic SO₂ injury (leaf damage) in the field experiment at the end of the growing period is discussed in relation to the impact of SO₂ on the processes involved in regulation of intracellular pH. It is proposed that the metabolic buffering capacity of leaf cells is related to the rates of sulphate and nitrate reduction and the import rate of organic anions, rather than to the organic anion content in the vacuoles of the leaf cells.     

Activation of wheat germ acetyl CoA carboxylase by potassium and rubidium.

Wheat germ acetyl CoA carboxylase requires certain alkali cations to exhibit maximal activity. Maximal activation results when 60 mM K⁺ or Rb⁺ are included in the assay mixture, whereas only marginal activation occurs in the presence of similar concentrations of Li⁺⁺ and Na⁺⁺. Cs⁺⁺ activates, but less effectively than K⁺ or Rb⁺. Since it is also possible to activate the enzyme maximally using 20 mM potassium isocitrate, but not 20 mM sodium isocitrate, activation of the wheat germ enzyme is due to a cation effect and not to citrate anion.