The speed-controlling reactions in fermentation of quickly dried yeast

Several factors which influence the speed of fermentation of quickly dried yeast are investigated. If the yeast is washed and the bulk of coenzymes and phosphate is removed, addition of 0.5 μmole diphosphopyridine nucleotide (DPN) and 0.5 μmole adenosine triphosphate (ATP) per cc. is necessary for maximal speed. In the optimal pH range, which lies between 6.6 and 6.2, and with optimal amounts of cofactors, there is no influence of nicotinamide mononucleotide (NMN), but with suboptimal amounts of DPN, the speed is raised by synthesis to DPN.The dialyzate of boiled juice contains factors which raise the speed of washed yeast by 20–30% of the maximum obtained in the presence of the usual cofactors. A phosphate concentration of 0.01–0.02 M is likewise necessary even if the phosphate is only partly esterified. At pH 6.0 to 5.9 the speed is less than half that at pH 6.5.Fermentation is completely absent without either K⁺ or NH₄⁺ and without Mg. The optimal amount of the monovalent ions is 5 × 10^?2M. Sodium alone is unable to allow fermentation but is only slightly harmful if enough K⁺ or NH₄⁺ is present.Addition of small amounts of phosphoglycerate at optimal potassium phosphate concentration and pH increases the rate of sugar fermentation and gives rise to an extra CO₂ formation during the time of phosphoglycerate decomposition of about 3 to 5 times the amount added.     

Interaction of phosphate with monovalent cation uptake in yeast

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb⁺ uptake shows saturation kinetics and the phosphate concentration at which halfmaximal inhibition is observed is equal to the K_m of phosphate for the sodiumindependent phosphate uptake mechanism. The kinetic coefficients of Rb⁺ and Tl⁺ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased.In the case of Na⁺ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na⁺ uptake via a sodium-phosphate cotransport mechanism.Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimenthylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium eptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Der Einfluß von Natrium- und Kaliumionen auf die Phosphataufnahme bei Ankistrodesmus braunii

Summary The uptake of phosphate as influenced by sodium and potassium ions was investigated in the light and in the dark. It was found to be a function of the external phosphate concentration. At a low concentration (up to 10^–5 mol/l) in the presence of Na⁺ phosphate is quickly absorbed and hence phosphate is the limiting factor for further labelling. In the presence of K⁺ phosphate uptake is constant over a long period.The enhancement of phosphate uptake by Na⁺ is also found when the external concentration of P is raised up to 10^–4 mol/l. Then the gross uptake proceeds over six hours, with the greatest Na⁺-dependent increase occurring in the label of the TCA-insoluble phosphate fraction (P_u).The phosphate uptake is strongly dependent on the pH of the reaction mixture. In the presence of Na⁺ it is highest between pH 5.6 and 7. As the uptake in the presence of K⁺ parallels the dissociation curve of the dihydrogen form H₂PO ₄ ^– , the Na⁺-enhancement is optimal in the alkaline pH range (pH 8).On the basis of a comparison between the pH-dependence of phosphate uptake and the dependence of the uptake on the external phosphate concentration analysed by a method of enzyme kinetics, it is suggested that Ankistrodesmus metabolically transports H₂PO ₄ ^– but not HPO ₄ ⁼ . Moreover, it is concluded from the absence of light stimulation and the weak inhibition of the uptake by DCMU or CCCP in the presence of K⁺ that at low P-concentrations the diffusion is limiting the uptake. Only at higher concentrations is an active phosphate uptake measured.Furthermore it is concluded that the observed Na⁺-stimulation of the ³²P-labelling of the TCA-soluble and insoluble compounds inside the cell is indirect and depends only on the action of Na⁺ and K⁺ ions at the first transport site in the plasmalemma.     

Functional Characterization of a Wheat NHX Antiporter Gene TaNHX2 That Encodes a K+/H+ Exchanger

The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K⁺ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K⁺/H⁺ exchange activity but very little Na^+/H⁺ exchange compared with controls transformed with the empty vector; Na⁺/H⁺ exchange was not detected with concentrations of less than 37.5 mM Na⁺ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K⁺/H⁺ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K⁺ homeostasis.     

Die pH-Abhängigkeit der Aufnahme von H2PO 4 - , SO 4 = , Na+ und K+ und ihre gegenseitige Beeinflussung bei Ankistrodesmus braunii

Summary Ion uptake was studied using ³²P, ³⁵S, ²²Na and ⁴²K as tracers in synchronized cells of Ankistrodesmus, which were slightly starved with respect to the ions to be investigated. In the light and in the dark, phosphate uptake is maximal between pH 5.5 and 6.5. Whereas Na⁺ in comparison to K⁺ enhances phosphate uptake in the light (8 to 9-fold) and in the dark, Ca⁺⁺ exerts only a slightly stimulatory effect. The stimulation of phosphate binding by Na⁺ occurs rapidly, even after less than 5 sec of incubation, and also in the presence of an equimolar concentration of K⁺.The pH-dependence of Na⁺-uptake in the light and in the dark is comparable to a dissociation curve: Na⁺-uptake increases with decreasing extracellular H⁺-concentration and is inversely proportional to phosphate uptake in the absence of Na⁺. The light:dark ratio of Na⁺-uptake at pH 8 amounts to 7:1. Mere adsorption of Na⁺ is similarly dependent on the pH. K⁺ strongly competes with Na⁺-uptake, even at pH 8. K⁺-uptake proceeds in a quite different manner from Na⁺-uptake and has an optimum at pH 7.Sulfate is taken up linearly in a biphasic process as a function of time; the pH-optimum lies between pH 7.5 and 8. K⁺ but not Na⁺ slightly enhances sulfate uptake.The Na⁺-enhancement of phosphate uptake can be related neither to a sodium-potassium exchange pump nor to a photosynthesis-dependent ion-exchange reaction.The results suggest that the uptake of phosphate, Na⁺ and K⁺, and the influence of alkali cations on phosphate uptake, but not sulfate uptake, are strongly dependent on fixed charges of the plasmalemma or even of the cell wall. These fixed charges may even prevent an active ion uptake.     

A new alkalitolerant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast strain is a promising model for dissecting properties and regulation of Na<Superscript>+</Superscript>-dependent phosphate transport systems

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na⁺-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na⁺-dependent systems specifically activated by Na⁺. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na⁺/P_i-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH 9.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1607–1615.Original Russian Text Copyright © 2004 by Zvyagilskaya, Persson.     

Apparent Three-Site Kinetics of Cs+-Uptake by Yeast

The concentration dependence of the uptake rate of Cs⁺ in yeast at low pH is described by a cubic rate equation rather than by a quadratic rale equation. This points to the involvement of three sties in the translocation of Cs⁺ across the yeast cell membrane. The possibility that one of the three sites is only apparent due to interaction of Cs⁺ with the electrical double layer of the yeast cell membrane is also considered.     

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.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K⁺] above 12 mM and peaked at about 40 mM KCl. In normal calcium buffers, phosphate labeling of PI but not PA declined sharply with [KCl] above 40 mM. In low calcium buffers, the phosphate labeling response was greatly attenuated for both lipids, but PI labeling did not decline at higher [K⁺].The phosphate labeling response was confined to PA and PI, and was specific for the increase in [K⁺]₀. The same response was seen in constant (105 mM) sodium buffers, and atropine had no effect. The specific radioactivity of ATP was increased by elevated potassium, but not enough to account for the increased labeling of PA. Further, this appeared to be a result of the loss of stored ATP rather than an increase in turnover.Increasing [K⁺]₀ produced a decline in [³H]inositol incorporation into PI in parallel with the increase in its labeling by ³³PO₄. This was the same in constant sodium and in low calcium buffers. It could be attributed to an inhibition of synaptosomal uptake of labelled inositol from the medium. Synaptosomal inositol content was unaffected.Elevated potassium had a greater effect on PA labeling than on PI, and it was more effective in increasing phosphate labeling of PA than was acetylcholine (ACh). When ACh and elevated potassium were combined at their maximally effective concentration, they acted synergistically to stimulate phosphate incorporation into PA but elevated potassium blocked the increase in [³H]inositol incorporation into PI normally produced by ACh. These results indicate that elevated potassium and ACh act upon the same population of synaptosomes, but affect different biochemical steps. Elevated potassium probably effects phospholipid labeling by a calcium dependent increase in diglyceride production from lipids other than PA or PI.     

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.     

Uptake by Yeast: Interaction of Rb+, Na+ and Phosphate

It has been shown that addition of phosphate to phosphate deficient yeast gives rise to an immediate increase in the rate of Na⁺ uptake and an immediate decrease in the rate of Rb⁺ uptake. In addition, phosphate uptake is enhanced specifically by Na ions presumably by a process with a very high affinity for phosphate with a K_m of about 2 × 10⁻⁶M at pH 7.2, whereas the K_m for phosphate uptake of the Na⁺ independent process amounts to 1.3 × 10⁻⁴M.     

The role of intracellular pH in the regulation of cation exchanges in yeast

1. When yeast oxidizes propan-2-ol in the presence of KCl no uptake of K⁺ occurs. 2. When propionate is added to suspensions containing propan-2-ol, or if the suspensions are bubbled with CO₂, a considerable uptake of K⁺ occurs. 3. Maximum K⁺ uptake occurs at a propionate concentration of 2mm. 4. The addition of 20mm-propionate to the suspension lowers the intracellular pH of the yeast from a resting value in the region of 6.2 to approx. 5.6. 5. When K⁺ uptake is measured in the presence of 20mm-propionate, progressive changes in the rate of K⁺ uptake and intracellular pH occur. The optimum rate of K⁺ uptake occurs at an intracellular pH of 5.70. 6. The effect of both intra- and extra-cellular pH on K⁺–K⁺ exchange was studied and an optimum rate was found at an extracellular pH of 5.35, the corresponding intracellular pH being 6.44. 7. When a Na⁺-loaded yeast oxidizes propan-2-ol in the presence of KCl, a steady efflux of Na⁺ and influx of K⁺ occurs. The addition of 10mm-propionate to the suspension markedly inhibited the Na⁺ efflux but only slightly decreased the K⁺ influx. 8. The effect of both extra- and intra-cellular pH on Na⁺ efflux was studied with propan-2-ol and with glucose. The results can be best interpreted in terms of intracellular pH changes, and an optimum was obtained at approx. pH6.40.     

Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K+ reduction and acidification

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K _o ⁺ ] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K⁺ concentration. As well as changes in serum [K _o ⁺ ], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K _o ⁺ -dependent, and the suggestion has been made that pH and K _o ⁺ may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K _o ⁺ . It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K⁺ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K⁺ channels in the open vs inactivated states of the channels by physiological H⁺ and K⁺ concentrations represents an important pathway to control Kv channel function in health and disease.     

Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae

Maintenance of ion homeostatic mechanisms is essential for living cells, including the budding yeast Saccharomyces cerevisiae. Whereas the impact of changes in phosphate metabolism on metal ion homeostasis has been recently examined, the inverse effect is still largely unexplored. We show here that depletion of potassium from the medium or alteration of diverse regulatory pathways controlling potassium uptake, such as the Trk potassium transporters or the Pma1 H⁺‐ATPase, triggers a response that mimics that of phosphate (Pi) deprivation, exemplified by accumulation of the high‐affinity Pi transporter Pho84. This response is mediated by and requires the integrity of the PHO signaling pathway. Removal of potassium from the medium does not alter the amount of total or free intracellular Pi, but is accompanied by decreased ATP and ADP levels and rapid depletion of cellular polyphosphates. Therefore, our data do not support the notion of Pi being the major signaling molecule triggering phosphate‐starvation responses. We also observe that cells with compromised potassium uptake cannot grow under limiting Pi conditions. The link between potassium and phosphate homeostasis reported here could explain the invasive phenotype, characteristic of nutrient deprivation, observed in potassium‐deficient yeast cells.     

On the Concept of Resting Potential—Pumping Ratio of the Na+/K+ Pump and Concentration Ratios of Potassium Ions Outside and Inside the Cell to Sodium Ions Inside and Outside the Cell

In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na⁺/K⁺ pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na⁺/K⁺ pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na⁺/K⁺ pump to prove that the three Na⁺ to two K⁺ pumping ratio of the Na⁺/K⁺ pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na⁺/K⁺ pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is ?30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na⁺/K⁺ pump and the ion concentration ratios play a role in the evolution of animal cells.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

THE ACCUMULATION OF ELECTROLYTES : VI. THE EFFECT OF EXTERNAL pH

It would be natural to suppose that potassium enters Valonia as KCl since it appears in this form in the sap. We find, however, that on this basis we cannot predict the behavior of potassium in any respect. But we can readily do so if we assume that it penetrates chiefly as KOH. We may then say that under normal conditions potassium enters the cell because the ionic activity product (K) (OH) is greater outside than inside. This hypothesis.leads to the following predictions: 1. When the product (K) (OH) becomes greater inside (because the inside concentration of OH^- rises, or the outside concentration of K⁺ or of OH^- falls) potassium should leave the cell, though sodium continues to enter. Previous experiments, and those in this paper, indicate that this is the case. 2. Increasing the pH value of the sea water should increase the rate of entrance of potassium, and vice versa. This appears to be shown by the results described in the present paper. It appears that photosynthesis increases the rate of entrance of potassium by increasing the pH value just outside the protoplasm. In darkness there is little or no growth or absorption of electrolytes. The entrance of potassium by ionic exchange (K⁺ exchanged for H⁺ produced in the cell), the ions passing as such through the protoplasmic surface, does not seem to be important.     

Purification and properties of trout gill AMP deaminase

Summary The AMP deaminase has been purified 450–500 fold from 20,000 g supernatants from trout gill. The procedure comprised cellulose phosphate and DEAE-cellulose chromatography. The gill appeared to contain different isoenzymes as indicated by different chromatographic behaviour on cellulose phosphate and different heat stabilities. The two major isoenzymes were compared with respect to their pH optima and the effect of temperature, ATP and inorganic phosphate. The pH optimum is about pH 6.7 at low substrate concentration. A second optimum is found in phosphate buffer. The substrate saturation curve is hyperbolic, even in the absence of KCl or ATP. ATP is an activator of the enzyme in the absence of KCl, but is without effect in the presence of monovalent cations. Among the monovalent cations tested, Na⁺ is the most potent activator followed by K⁺ and NH ₄ ⁺ . Inorganic phosphate is an inhibitor of gill AMP deaminase increasing the affinity for its substrate but having no effect on the maximal velocity or the Hill coefficient. The inhibition by phosphate is partially reversed by ATP. ADP and GTP are competitive inhibitors of the enzyme. In addition, the enzyme showed negative cooperativity in the presence of ATP or GTP.