Antonio Peña

Studies on the mechanism of K+ transport in yeast

Abstract 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 86 Rb + 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 (H 2 CO 3 ), 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.

Effect of the pH of the incubation medium on glycolysis and respiration in Saccharomyces cerevisiae

Abstract An investigation was undertaken in order to explain the effect of pH (4.0 and 7.5) of the incubation medium on the anaerobic glycolysis and respiration in yeast, in which a higher ethanol production and glucose consumption, as well as a higher respiratory rate at the high pH are observed. The measurement of the changes in the concentrations of the metabolites of the glycolytic sequence, NADH, ADP, ATP, and Pi that appeared when the pH was suddenly increased, permitted us to detect an increase of ADP and Pi and a decrease of the ATP levels, which in conjunction with the changes in the concentrations of the glycolytic intermediates indicate that at the high pH there was an increase in the activity of an ATPase system which is responsible for the acceleration of glycolysis. This same ATPase activity could be shown under similar conditions with ethanol as substrate in respiring yeast, and the increased levels of ADP seem to be responsible also for the increased respiratory rate at the high pH. When the pH was increased from 4.0 to 7.5, this ATPase activity was induced if potassium ions were absent from the incubation medium, but no important changes in the levels of the adenosine phosphates were observed upon the pH increase when these ions were previously included. The data could be explained on the basis of the existence of an energy-requiring proton pump, which might be of the kind described by Mitchell; it would have the role of transporting H+ to the outside of the cell. Such an anisotropic system ought to be inhibited by increasing concentrations of H+ on the outside, and stimulated at the high pH. At the low pH, K+ would allow the function of this system, by exchanging for H+. At the high pH, this ATP-utilizing system could work without K+, and anions would leave the cell accompanying protons; in the presence of K+, the cation could be taken up by yeast in exchange for the same protons, lowering the amount of anions expelled, without a further increase of the ATP utilization. The results presented in this communication, furthermore do not support the idea that the pH of the yeast cell remains constant with variations of the pH of the incubation medium. This inconstancy has important implications for the regulation of both glycolysis and respiration in yeast as a consequence of the changes of the external pH.

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl

Debaryomyces hansenii showed an increased growth in the presence of either 1m KCl or 1m NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86 Rb+ and 22 Na+ . 86 Rb+ transport was saturable, with Km and Vmax values higher for cells grown in 1m NaCl. 22 Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1m KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+ /H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth.

Effects of Chitosan on Candida albicans: Conditions for Its Antifungal Activity

The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 μg·mL−1 produced (a) an efflux of K+ and stimulation of extracellular acidification, (b) an inhibition of Rb+ uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca2+. It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K+, it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca2+, (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL−1 are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells.

Sodium and potassium transport in the halophilic yeast Debaryomyces hansenii.

Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.6 M of either NaCl or KCl, accumulating high concentrations of Na+ or K+. After growth in NaCl or KCl, a rapid efflux of either accumulated cation was observed if the cells were incubated in the presence of KCl or NaCl, respectively, accompanied by a slower accumulation of the cation present in the incubation medium. However, a similar, rapid efflux was observed if cells were incubated in buffer, in the absence of external cations. This yeast shows a cation uptake activity of both 86Rb+ and 22Na+ with saturation kinetics, and much higher affinity for 86Rb+ than for 22Na+. The pH dependence of the kinetics constants was similar for both cations, and although Km values were higher at pH 8.0, there was also an increase in the Vmax values. The accumulation of 22Na+ was found to be increased in cells grown in the presence of 0.6 M NaCl. 86Rb+ was also accumulated more in these cells, but to a slightly greater extent. The inhibition kinetics of the uptake of 22Na+ by K+, and that of 86Rb+ by Na+ was found to be non‐competitive. It can be concluded that Na+ in D. hansenii is not excluded but instead, its metabolic systems must be resistant to high salt concentrations. Copyright

Studies on the mechanism of the stimulation of glycolysis and respiration by K+ in Saccharomyces cerevisiae

Abstract 1. The effect of K + on the respiration and glycolysis of yeast was investigated. Measurement of the levels of ADP, ATP and P i revealed that K + stimulates an enzymatic activity that diminishes ATP and increases ADP. This increase in the ADP level seems to be the responsible for the stimulation of both glycolysis and respiration. 2. When glucose was used as substrate, the increase in the levels of P i upon the addition of K + was found to be greater than either the ATP decrease or the ADP increase. This might mean that the ion induces the breakdown of ATP by an indirect mechanism, with another phosphorylated compound being hydrolyzed directly. The amount of ATP would decrease at the rate at which it was used to replace this hypothetical compound. 3. The studies on the mechanism by which K + induces the stimulation of glycolysis and respiration suggest that it is the presence of the ion inside the cell or some consequence of this that induces the changes necessary to produce the stimulation of “ATPase” activity responsible for the activation of glycolysis and respiration. The possibility that the presence of the ion outside the cell or the energy required for its transport causes the changes appears to have been eliminated.

Some characteristics of Ca2+ uptake by yeast cells

SummaryExperiments were performed to obtain information on: (i) the specific properties of Ca2+ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca2+ in yeast. These were obtained mainly by considering actual concentrations of Ca2+ in the medium after substracting the amounts bound to the cell. Akm of 1.9 μm and aVmax of 1.2 nmol (100 mg·3 min)−1 were calculated.The effects of some inhibitors and other cations on Ca2+ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca2+ uptake in yeast.The effects of Mg2+ on the uptake of Ca2+ agree with the existence of a single transport system for both divalent cations.The actions of Na+ and K+ on the transport of Ca2+ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.

Effect of ethidium bromide on Ca2+ uptake by yeast

SummaryEthidium bromide and other cationic dyes have been found to inhibit movalent cation uptake. This dye also produces in a K+-free medium an efflux of K+ which could be of the electrogenic type.The study of the effects of the same cationic dyes on Ca2+ uptake showed a large stimulation of the uptake rate of the divalent cation of more than tenfold.The analysis of the effects of one of the cationic dyes on Ca2+ uptake indicated that the efflux of K+ is of the electrogenic type and can drive the uptake of the divalent cation.Kinetic data on Ca2+ uptake indicate that, both under “normal” or under stimulated conditions, the divalent cation is taken up by the same transport system.The addition of ethidium bromide, besides, can stimulate the uptake of Mn2+ and14C-glycine and could be a good weapon to magnify and study some of the characteristics of ion transport systems in yeast.

Interaction of ethidium bromide with the transport system for monovalent cations in yeast

SummaryEthidium was found to be taken up by yeast cells in a process that, at certain concentrations has the main following characteristics: a) a substrate is required; b) it presents cooperative kinetics, withn, according to the Hill equation ≈3; c) ethidium can be concentrated more than 100-fold; d) the uptake is inhibited by Ca2+; e) the uptake of the dye is inhibited by monovalent cations with a selectivity pattern similar to that observed in their transport by yeast; f) ethidium inhibits the uptake of K+, and, at concentrations up to about 250 μm produces a competitive inhibition on the uptake of Rb+; and g) ethidium produces the same effects as K+ on respiration and the extrusion of H+. It is concluded that ethidium is taken up by yeast cells in a selective way by the same transport system normally employed for monovalent cation uptake.

Early metabolic effects and mechanism of ammonium transport in yeast

Studies were performed to define the effects and mechanism of NH+4 transport in yeast. The following results were obtained. Glucose was a better facilitator than ethanol-H2O2 for ammonium transport; low concentrations of uncouplers or respiratory inhibitors could inhibit the transport with ethanol as the substrate. With glucose, respiratory inhibitors showed only small inhibitory effects, and only high concentrations of azide or trifluoromethoxy carbonylcyanide phenylhydrazone could inhibit ammonium transport. Ammonium in the free state could be concentrated approximately 200-fold by the cells. Also, the addition of ammonium produced stimulation of both respiration and fermentation; an increased rate of H+ extrusion and an alkalinization of the interior of the cell; a decrease of the membrane potential, as monitored by fluorescent cyanine; an immediate decrease of the levels of ATP and an increase of ADP, which may account for the stimulation of both fermentation and respiration; and an increase of the levels of inorganic phosphate. Ammonium was found to inhibit 86Rb+ transport much less than K+. Also, while K+ produced a competitive type of inhibition, that produced by NH4+ was of the noncompetitive type. From the distribution ratio of ammonium and the pH gradient, an electrochemical potential gradient of around -180 mV was calculated. The results indicate that ammonium is transported in yeast by a mechanism similar to that of monovalent alkaline cations, driven by a membrane potential. The immediate metabolic effects of this cation seem to be due to an increased [H+]ATPase, to which its transport is coupled. However, the carriers seem to be different. The transport system studied in this work was that of low affinity.