Discrimination between Alkali Metal Cations by Yeast : II. Cation interactions in transport

K⁺ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb⁺ is a competitive inhibitor of K⁺ uptake, but Li⁺, Na⁺, and Cs⁺ act like H⁺. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K⁺ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K⁺ transport competitively. Ca⁺⁺ and Mg⁺⁺ in relatively low concentrations partially inhibit K⁺ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K⁺ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.     

Studies on the mechanism of K+ transport in yeast.

The effect of uncouplers and diffusible acids on K⁺ transport was studied in yeast.Although the K⁺ transport system seems to depend on ATP to function, the effects of uncouplers are not due primarily to its action on the energy conserving systems of the cell.Other uncouplers with different structures to that of DNP showed also an inhibitory effect on K⁺ transport, which agrees with their reported ability to conduct protons through membranes.Uncouplers, besides inhibiting K⁺ uptake, produce an efflux of this cation; however, the rate of efflux produced is quantitatively important only when the cells have previously taken up the cation; there seems to exist a mechanism which prevents the loss of cations by yeast.In the absence of substrate, at pH 8.5, with 0.5 m KCl, TCS produces the efflux of H⁺, and when ⁸⁶Rb⁺ was used as a substitute for K⁺, an increase of the entrance of the cation could be detected in the presence of the uncoupler. It seems that the effect of the uncoupler depends on the direction of the combined H⁺ and K⁺ gradients, or the electrochemical potential of the cell.As reported by other authors, weak diffusible acids increase the uptake of K⁺ by yeast, and this effect is not due to changes in the metabolism, but to the magnitude of the entrance of the molecules to the yeast cell.It was found that the efflux of the acids (H₂CO₃), on the other hand, can produce an efflux of K⁺, which means that anions are important not only for the entrance of the cations, but for its permanence within the cell as well.The data seem to be in agreement with the hypothesis of the existence of a proton pump, responsible for the creation of an electrochemical potential, involved in K⁺ transport. At low pH, this pump seems to be activated by the transport of K⁺ into the cell.     

Specific inhibition by macrocyclic polyethers of mitochondrial electron transport at site I

The macrocyclic polyethers dibenzo-18-crown-6 (XXVIII) and dicyclohexyl-18-crown-6 (XXXI) inhibit the valinomycin-mediated K⁺ accumulation energized by glutamate, -ketoglutarate, malate plus pyruvate or isocitrate but not that promoted by succinate, ascorbate plus TMPD or ATP. The polyethers inhibit the oxidation of the former group of substrates without preventing either the oxidation of succinate or ascorbate plus TMPD or the hydrolysis of ATP.The substrate oxidation inhibited by the macrocyclic polyethers is relieved in intact mitochondria by increasing the concentration of K⁺ in the medium. It is also completely reverted by supplementing the medium with valinomycin, Cs⁺ and phosphate, or else by the addition of vitamin K₃.In submitochondrial sonic particles the macrocyclic polyethers inhibit the oxidation of NADH as well as the ATP-driven reversal of electron flow at the site I of the electron transport chain. They also block the oxidation of NADH in non-phosphorylating Keilin-Hartree particles as well as in Hatefi's NADH-coenzyme Q reductase. The polyethers do not inhibit electron transport in mitochondria from the yeast which lack the first coupling site.The inhibition of electron transport by the polyethers do not require of the addition of alkali metal cations such as K⁺ in intact mitochondria or other membrane preparations.It is established that the macrocyclic polyethers XXVIII and XXXI, already characterized as mobile carrier molecules for K⁺ in model lipid membranes, inhibit electron transport at site I of the electron transport chain from mitochondrial membranes.It is suggested that the ability of the polyethers to coordinate alkali metal cations in aqueous versus lipid environments, but not K⁺ transportper se, is related to their rotenone-like induced inhibition of electron flow in mitochondrial membranes.Supported in part by a Grant from the Research Corporation.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Alkali Metal Cation Transport and Homeostasis in Yeasts

Summary: The maintenance of appropriate intracellular concentrations of alkali metal cations, principally K⁺ and Na⁺, is of utmost importance for living cells, since they determine cell volume, intracellular pH, and potential across the plasma membrane, among other important cellular parameters. Yeasts have developed a number of strategies to adapt to large variations in the concentrations of these cations in the environment, basically by controlling transport processes. Plasma membrane high-affinity K⁺ transporters allow intracellular accumulation of this cation even when it is scarce in the environment. Exposure to high concentrations of Na⁺ can be tolerated due to the existence of an Na⁺, K⁺-ATPase and an Na⁺, K⁺/H⁺-antiporter, which contribute to the potassium balance as well. Cations can also be sequestered through various antiporters into intracellular organelles, such as the vacuole. Although some uncertainties still persist, the nature of the major structural components responsible for alkali metal cation fluxes across yeast membranes has been defined within the last 20 years. In contrast, the regulatory components and their interactions are, in many cases, still unclear. Conserved signaling pathways (e.g., calcineurin and HOG) are known to participate in the regulation of influx and efflux processes at the plasma membrane level, even though the molecular details are obscure. Similarly, very little is known about the regulation of organellar transport and homeostasis of alkali metal cations. The aim of this review is to provide a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants.     

Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1

The wheat root high-affinity K⁺ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter. At toxic millimolar levels of sodium (Na⁺), HKT1 mediates low-affinity Na⁺ uptake while potassium (K⁺) uptake is blocked. In roots, low-affinity Na⁺ uptake and inhibition of K⁺ uptake contribute to Na⁺ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K⁺ and Na⁺ over Rb⁺, Cs⁺ and Li⁺. In addition, Rb⁺ and Cs⁺, and an excess of extracellular K⁺ over Na⁺, are shown to partially reduce or block HKT1-mediated K⁺-Na⁺ uptake. Furthermore, K⁺, Rb⁺ and Cs⁺ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high-affinity Rb⁺ uptake at approximately 15-fold lower rates than for K⁺. Rb⁺ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35-fold lower HKT1 conductance for Rb⁺. A model for HKT1 activity is presented involving a high-affinity K⁺ binding site and a high-affinity Na⁺ binding site, and competitive interactions of K⁺, Na⁺ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K⁺ and Na⁺ uptake in plants are discussed.     

Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

Effects of monovalent cations on derepression of phosphate transport in yeast

The effect of monovalent cations on derepression of phosphate transport was studied. It was found that ammonium, K⁺ and Rb⁺ accelerate the derepression of phosphate transport produced by glucose in yeast (Saccharomyces cerevisiae). Na⁺ and Li⁺ were ineffective in accelerating derepression; Cs⁺ produced only a minor stimulation. The concentration range of both K⁺ and NH₄⁺ that accelerated derepression was similar to that required for transport to occur. In the case of ammonium, the effects seem to depend exclusively on the so-called low-affinity transport system. The effect was strongly dependent on pH, with an optimum around 6; however, the increase in the pH of the medium did not produce in itself a high increase of the depression. Derepression was dependent on the presence of glucose, and it was very low with ethanol as substrate. The mechanism seems to depend on the ability that both K⁺ and NH₄⁺ have to decrease the membrane potential of the cell while transported, and not on the capacity to produce the alkalinization of the cell interior. In addition, the phenomenon depends on the presence of glucose as substrate, which indicates the involvement of some product of glucose metabolism in the mechanism, and possibly some relation to catabolic repression.     

Stimulation of potassium cycling in mitochondria by long-chain fatty acids

Nonesterified long-chain fatty acids (myristic, palmitic, oleic and arachidonic), added at low amounts (around 20 nmol/mg protein) to rat liver mitochondria, energized by respiratory substrates and suspended in isotonic solutions of KCl, NaCl, RbCl or CsCl, adjusted to pH 8.0, induce a large-scale swelling followed by a spontaneous contraction. Such swelling does not occur in alkaline solutions of choline chloride or potassium gluconate or sucrose. These changes in the matrix volume reflect a net uptake, followed by net extrusion, of KCl (or another alkali metal chloride) and are characterized by the following features: (1) Lowering of medium pH from 8.0 to 7.2 results in a disappearance of the swelling-contraction reaction. (2) The contraction phase disappears when the respiration is blocked by antimycin A. (3) Quinine, an inhibitor of the K⁺/H⁺ antiporter, does not affect swelling but suppresses the contraction phase. (4) The swelling phase is accompanied by a decrease of the transmembrane potential and an increase of respiration, whereas the contraction is followed by an increase of the membrane potential and a decrease of oxygen uptake. (5) Nigericin, a catalyst of the K⁺/H⁺ exchange, prevents or partly reverses the swelling and partly restores the depressed membrane potential. These results indicate that long-chain fatty acids activate in liver mitochondria suspended in alkaline saline media the uniporter of monovalent alkali metal cations, the K⁺/H⁺ antiporter and the inner membrane anion channel. These effects are presumably related to depletion of mitochondrial Mg²⁺, as reported previously [Arch. Biochem. Biophys. 403 (2002) 16], and are responsible for the energy-dissipating K⁺ cycling. The uniporter and the K⁺/H⁺ antiporter are in different ways activated by membrane stretching and/or unfolding, resulting in swelling followed by contraction.     

Four Pathogenic Candida Species Differ in Salt Tolerance

The virulence of Candida species depends on many environmental conditions, including extracellular pH and concentration of alkali metal cations. Tests of the tolerance/sensitivity of four pathogenic Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. parapsilosis) to alkali metal cations under various growth conditions revealed significant differences among these species. Though all of them can be classified as rather osmotolerant yeast species, they exhibit different levels of tolerance to different salts. C. parapsilosis and C. albicans are the most salt-tolerant in general; C. dubliniensis is the least tolerant on rich YPD media and C. glabrata on acidic (pH 3.5) minimal YNB medium. C. dubliniensis is relatively salt-sensitive in spite of its ability to maintain as high intracellular K⁺/Na⁺ ratio as its highly salt-tolerant relative C. albicans. On the other hand, C. parapsilosis can grow in the presence of very high external NaCl concentrations in spite of its high intracellular Na⁺ concentrations (and thus lower K⁺/Na⁺ ratio) and thus resembles salt-tolerant (halophilic) Debaryomyces hansenii.     

Barium modifies the concentration dependence of active potassium transport by insect midgut

Summary The rate of active K⁺ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K⁺] over the range 20 to 70mm K⁺ in the absence of any divalent cation. Addition of Ba⁺⁺ to the hemolymph (K⁺ uptake) side introduces a linear component to the concentration dependence, such that active K^– transport is decreased at [K⁺] of 55mm or less, but increased transiently at higher [K⁺]. As [Ba⁺⁺] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba⁺⁺ the concentration dependence is dominated by the linear component. The effect of Ba⁺⁺ cannot easily be accounted for by simple competition with K⁺ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K⁺ transport possesses a substantial linear component in solutions containing 5mm Ca⁺⁺ and 5mm Mg⁺⁺ (the alkali earth metal concentrations of standard lepidopteran saline).     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Direct NMR detection of the "invisible" alkali metal cations tightly bound to G-quadruplex structures

We report the first direct solution NMR detection of the alkali metal cations (²³Na⁺, ³⁹K⁺, and ⁸⁷Rb⁺) residing inside G-quadruplex channel structures formed by guanosine 5′-monophosphate and a DNA oligomer, d(TG₄T). In solution, these channel alkali metal cations are tightly bound to the G-quadruplex structure and have been considered to be “invisible” to NMR spectroscopy for many years. Our finding that it is possible to directly observe these alkali metal cations by NMR spectroscopy provides a new tool for studying cation binding affinity and dynamics in G-quadruplex DNA.     

The activation of intestinal brush border sucrase by alkali metal ions: an allosteric mechanism similar to that for the Na+-activation of nonelectrolyte transport systems in intestine.

Activation of intestinal brush border sucrase by alkali-metal ions is described by an allosteric, noncompulsory mechanism involving two distinct sites: one for sucrose and another for the metal activator. Both Na⁺ and K⁺ activate guinea pig sucrase but K⁺ has ten times more affinity for the metal site. Li⁺ is inert. Values for the dissociation constants for the interaction of sucrase with either sucrose, Na⁺ or K⁺ have been calculated for the guinea pig, rat, and hamster. Qualitatively, the activation of sucrase by alkali metal ions is similar to that described for the Na⁺-activation of amino acid and sugar transport in intestine. However, the Na-binding site in the two systems is apparently quite different. In the guinea pig, the Na-dissociation constant is in the order of 10^?3m for sucrase, about two orders of magnitude smaller than that for transport. Also, K ⁺ is a strong activator of guinea pig sucrase, but an inhibitor of intestinal sugar transport.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

The respiration of brain mitochondria and its regulation by monovalent cation transport

The Na⁺ and K⁺ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose.Respiration was uncoupled by Na⁺ and less so by K⁺. Uncoupling was maximal in the presence of EDTA plus P_i and was decreased by Mg²⁺. Maximal uncoupler-stimulated respiration rates were inhibited by Na⁺ but largely unaffected by K⁺. The inhibition by Na⁺ was relatively insensitive to Mg²⁺. Membrane Na⁺ and K⁺ conductances as well as neutral exchanges (Na⁺/H⁺ and K⁺/H⁺ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations.Cation conductance, i.e. electrophoretic Na⁺ or K⁺ permeation, was increased by EDTA (Na⁺ > K⁺) and decreased by Mg²⁺. Magnesium preferentially suppressed Na⁺ conductance so as to reverse the cation selectivity (K⁺ > Na⁺). Neutral cation/H⁺ exchange rates (Na⁺ > K⁺) were not influenced by chelator or Mg²⁺.The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K⁺ conductor valinomycin. The metabolic influences of Na⁺ and K⁺ can be explained in terms of coupled flow of these ions with protons and their effect upon the H⁺ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae

The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H⁺). The buffering action of passively excreted metabolites (CO₂, organic acids) and cell surface polyelectrolytes contributed only 15–40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 mmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence $\text{K}{}^{\mathrm{+}}\text{>}\text{Rb}{}^{\mathrm{+}}\text{>>}\text{Li}{}^{\mathrm{+}}\text{～-}\text{Cs}{}^{\mathrm{+}}\text{～-}\text{Na}{}^{\mathrm{+}}$. The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO₂ and organic acids, net extrusion of H⁺ and K⁺ (in K⁺-free media; in K⁺-containing media this is preceded by an initial rapid K⁺ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Multiple Effects of Lysophosphatidylcholine on the Activity of the Plasma Membrane H+-ATPase of Radish Seedlings*

We analyzed the effect of lysophosphatidylcholine (lysoPC) on the activity of the plasma membrane (PM) H⁺-AT-Pase measured at pH 6.3 or 7.5 in inside-out PM vesicles isolated from germinating radish seeds. LysoPC stimulated PM H⁺-ATPase at both pHs, but the dependence of the effect on lysoPC concentration was different: at pH 6.3 maximal stimulation was observed with 40 to 200 μg ml^?1 lysoPC, while at pH 7.5 a sharp peak of activation was observed at about 50 μg ml^?1 lysoPC, higher concentrations becoming dramatically inhibitory; this inhibitory effect was considerably reduced in the presence of 10% (v/v) glycerol. In trypsin-treared PM lysoPC stimulated the H⁺-ATPase activity assayed at pH 6.3, but only marginally that assayed at pH 7.5. LysoPC increased both V_max (from 190 to 280nmol min^?1 mg^?1 prot) and apparent K_M (from 0.15 to 0.3 mM) of the H⁺-ATPase at pH 6.3, while it increased V_max (from 120 to 230 nmol min^?1 mg^?1 prot) and decreased apparent K_m (from 0.8 to 0.4 mM) at pH 7.5. Low concentrations of Nacetylimidazole (10 to 50 mM), which modifies tyrosine residues, abolished the stimulation by lysoPC of the PM H⁺-ATPase activity at pH 7.5, but not that observed at pH 6.3. These results indicate that lysoPC influences the PM H⁺-ATPase through different mechanisms, and that its effect can only partly be ascribed to its ability to hamper the inhibitory interaction of the regulatory C-terminal domain with the catalytic site. N-acety-limidazole did not affect the stimulation of PM H⁺-ATPase by controlled trypsin treatment or by fusicoccin, indicating that the requirement for the tyrosine residue(s) modified by low Nacetylimidazole concentrations is specific for lysoPC-induced displacement of the C-terminal domain.