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K Sigler

Publications and source records attributed to K Sigler.

At least 73 records · Page 4Linked to original sources
PubMed1993

Fluorescent staining with bromocresol purple: a rapid method for determining yeast cell dead count developed as an assay of killer toxin activity.

A method is described for detecting yeast cells with plasma membrane damage, based on cell staining with bromocresol purple (BCP) which has a convenient fluorescence after entering the cells at pH below 5.2. The method was used to determine the activity of Saccharomyces cerevisiae pore-forming killer toxin K1 in commonly used lethal units. The BCP test is rapid and as precise as the plating test.

Bromcresol Purple↗
PubMed1993

Factors affecting the susceptibility of sensitive yeast cells to killer toxin K1.

Optimum conditions for action of the killer toxin K1 on sensitive strain S. cerevisiae S6 were established. Maximum killing was reached in a very narrow pH range of 4.5-4.6. Maximum susceptibility to toxin was displayed by highly energized fresh cells from the early exponential phase in the presence of an external energy source (at least 200 mmol/L glucose). Further, maintenance of maximum membrane potential was necessary for killer action, as documented by decreasing toxin activity in the presence of increasing concentrations of KCl. The killing was strongly stimulated in the presence of millimolar concentrations of Ca2+ and Mg2+.

Calcium↗
PubMed1993

Effect of hydrogen peroxide on sugar transport in Schizosaccharomyces pombe. Absence of membrane lipid peroxidation.

Stationary unaerated cells of S. pombe containing endogenous substrates but not energized by any exogenous ones take up 2-deoxy-D-glucose, 6-deoxy-D-glucose, D-xylose and D-arabinose actively over diffusion equilibrium. The active uptake is inhibited by 20-100 mmol/L H2O2 which causes an increase in KT but has no effect on Jmax. This "competitive inhibition" indicates that H2O2 affects directly the sugar binding sites of the transporters. The ATP-binding site of the plasma membrane H(+)-ATPase is also affected by 100 mmol/L H2O2; the KT decreases 7-fold, Jmax about 2.5-fold. These effects are not likely to be mediated by membrane lipid peroxidation which appears to be lacking in S. pombe, and this lack may be one of the reasons for the high resistance of this yeast to H2O2. Because of this S. pombe represents a suitable system for studying direct effects of oxidants on membrane proteins.

Antimycin A↗
PubMed1993

Response of catalase activity and membrane fluidity of aerobically grown Schizosaccharomyces pombe and Saccharomyces cerevisiae to aeration and the presence of substrates.

Intracellular catalase (EC 1.11.1.6) activity of permeabilized aerobically grown cells of Schizosaccharomyces pombe was insensitive to cell aeration and inhibition of protein synthesis, and was only mildly enhanced by the presence of glucose and ethanol via de novo protein synthesis. By contrast, the intracellular catalase activity of Saccharomyces cerevisiae, which, in freshly harvested cells, was two to three times lower than that in Sch. pombe, increased on aeration without substrates or with ethanol and was inhibited on aeration with glucose following cell permeabilization. The enhanced intracellular activity was due to de novo protein synthesis while the inhibitory effect of glucose, absent in Sch. pombe, was caused by one of the major glucose metabolites, succinate. The intact-cell catalase activity of both yeasts increased greatly during aeration. In Sacch. cerevisiae, this increase was again prevented by glucose. In parallel, export of catalase to the cell surface increased in both yeasts. This was especially conspicuous in Sch. pombe aerated in the presence of ethanol, and may represent a protective mechanism against the damaging effects of ethanol. The cell-surface-bound catalase activity was confirmed in isolated plasma membranes of both yeasts. The fluidity of the plasma membrane increased during aeration. This effect was further stimulated by the presence of glucose and to a lesser extent by ethanol. Both yeasts exhibited increased extracellular catalase activity during aeration which could not be caused entirely by cell lysis. In Sch. pombe this activity was strongly enhanced by the presence of ethanol.

Aerobiosis↗
PubMed1991

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.

Benzothiazoles↗
PubMed1991

Activation of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae by addition of hydrogen peroxide.

Addition of hydrogen peroxide (greater than 10 mM) to aerated derepressed cells of S. cerevisiae in the absence of substrate caused a boost of endogenous respiration and both intra- and extracellular acidification, without any significant change in cellular ATP level. Furthermore, a hyperpolarization of the plasma membrane was indicated by an enhanced accumulation of tetraphenylphosphonium in the cells. The extracellular pH attained was as low as 3.5. The acidification could be suspended by the H(+)-ATPase inhibitors diethylstilbestrol and dicyclohexylcarbodiimide and was, in general, associated with an opposite flux of K+. K+ also stimulated the H(+)-ATPase activity in the purified plasma membrane fraction. These results are consistent with the plasma membrane H(+)-ATPase being involved in the H+ extrusion induced by H2O2 in the absence of substrate. Extended exposure of cells to H2O2 led eventually to an arrest of both respiration and ion fluxes that could be again lifted by depolarizing the plasma membrane. Along with differences in the cellular NADH/NAD+ ratio and in the participation of organic acids, this makes the H2O2-induced acidification distinct from that induced by glucose.

Buffers↗
PubMed1988

Acrylonitrile depletes glutathione without changing calcium sequestration in hepatic microsomes and mitochondria.

Acrylonitrile administered either in vivo or in vitro reduced the level of non-protein thiols, GSH and GSSG in rat liver (in vivo) and liver microsomes (in vitro). It neither influenced protein thiols nor calcium sequestration in the microsomes and mitochondria. The fact that the GSSG level was not increased indicates that a mere unoxidative depletion of GSH does not lead to impaired hepatocyte Ca homeostasis, which has been associated with decreased GSH:GSSG ratio. An opposite effect was caused by CCl4 which did not considerably change the protein and non-protein SH, but strongly decreased microsomal calcium sequestration.

Acrylonitrile↗
PubMed1985

Rubidium transport in X-irradiated human erythrocytes.

Outflow of 86Rb, a radioactive analogue of potassium, from human erythrocytes X-irradiated in vitro was studied with the following results. (1) The 86Rb level in the supernatants of irradiated and control cell suspensions reflected mainly 86Rb outflow and much less its active re-uptake. (2) The effect of irradiation on 86Rb outflow was more pronounced at a low temperature (4 degrees C) than at 37 degrees C; the lowest dose of X-radiation exhibiting a significant effect on 86Rb outflow at 4 degrees C was 2.5 Gy. (3) K/Rb exchange did not seem to play an appreciable role in radiation-induced 86Rb outflow. (4) Calcium and its accumulation in irradiated cells was not found to be the cause of the effect of radiation on 86Rb outflow. (5) The effect of radiation on 86Rb outflow was higher in low Na medium but it was not inhibited by bumetanide. Rb/Na counter- or co-transport do not therefore seem to be involved in radiation-induced Rb+ outflow.

Biological Transport↗
PubMed1982

Acidification power: indicator of metabolic activity and autolytic changes in Saccharomyces cerevisiae.

Acidification power, defined as the sum of the spontaneous pH change determined after suspending yeast cells in water and the substrate-induced pH change after addition of glucose to the resulting suspension, reflects the level of cellular energy sources. Its use as an indicator of metabolic state of the cells was tested during a 120-h aerobic starvation. Its changes coincided with changes in cell viability, initial rate of endogenous oxygen consumption rate, cell ATP, extra- and intracellular buffering capacity, and the ability of cell-free extract to produce acidity by glucose fermentation. It was used as a sensitive marker of metabolic changes occurring during starvation, on treatment with glycolytic and respiratory inhibitors, and at elevated temperature.

Adenosine Triphosphate↗
PubMed1982

ATPases: common and unique features within a group of enzymes.

An attempt is made to survey ATPases with respect to features common to all or some of them and features peculiar to each individual enzyme of the group. Clues are presented for a tentative classification of ATPases and a simple system is suggested for the designation of interaction of ATPases with ions which is often used as the main feature of identification of individual ATPases.

Adenosine Triphosphatases↗
PubMed1981

Factors governing substrate-induced generation and extrusion of protons in the yeast Saccharomyces cerevisiae.

Experiments with respiration deficient (rho-), ADP/ATP transport deficient (op1) and double (op1 rho-) mutants, with glycolytic and tricarboxylic acid cycle substrates showed that the substrate-induced acidification of yeast suspensions is closely associated with glycolysis. The glucose/proton stoichiometry is 2.5 : 1 to 4 : 1 depending on glucose concentration. The kinetics of the process are complex, the acidification curve having a very fast initial component and two slower exponential components. The first component suggests an initial proton efflux from endogenous sources, triggered by exogenous substrates. The acidification process exhibits two Km values at about 1 and 15 mM D-glucose, indicating two distinct saturable pathways of proton extrusion. The total extent of acidification and thus the final pHout reaches a saturation value with increasing glucose concentration and suspension density. Both the total extent and the rate of acidification are subject to control by extracellular pH which reflects the tendency of the cells to build a fixed [H+]out/[H+]in ratio. When the control is lifted, both quantities are considerably increased. A crucial role in the substrate-induced acidification is thus played by active membrane processes and their control mechanisms.

Acid-Base Equilibrium↗
PubMed1981

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 nmol 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 K+ greater than Rb+ much greater than Li+ congruent to Cs+ congruent to Na+. 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.

Acid-Base Equilibrium↗
PubMed1981

Membrane potentials in yeast cells measured by direct and indirect methods.

The membrane potential, delta psi, of various yeasts estimated from the distribution of tetraphenylphosphonium cations ranged from -50 to -120 mV, depending on species, incubation conditions and technique of measurement. Values obtained directly with a microelectrode in Endomyces magnusii were consistently lower than those determined indirectly.

Ascomycota↗
PubMed1980

Gas chromatographic determination of extracellular metabolites produced by baker's yeast during glucose-induced acidification.

Gas chromatographic separation of metabolites extruded during the medium acidification by resting baker's yeast supplied with glucose is described. Silylation with bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane and a direct chromatography on Chromosorb G-AW-DMCS with 2.5% SE-52 (lactic, pyruvic, succinic, glyceric, fumaric, malic), polyols (glycerol, arabinitol) and sugars (glucose). The yeast was found to extrude glycerol, lactic, malic, and succinic acid.

Chromatography, Gas↗
PubMed1980

Extrusion of metabolites from baker's yeast during glucose-induced acidification.

Extrusion of metabolites (glycerol, lactic, malic, and succinic acid) during the medium acidification caused by resting baker's yeast supplied with 200 mM glucose was studied under aerobic and anaerobic conditions and in the absence and presence of 14 mM KCl. The maximum levels of glycerol and of the sum of acids (about 13 and 8 mM, respectively) were attained anaerobically; aerobiosis reduced the levels by 40-50% and the presence of K+ ions by another 10-20%. The time courses of glucose consumption and medium acidification were similar aerobically and anaerobically. The glucose consumption curves exhibited a short plateau about 2 min after glucose addition, caused probably by a rapid osmotic equilibration of glucose across the cell mambrane. Metabolite extrusion indicates that at high glucose concentrations the alcohol dehydrogenase reaction is supplemented by other reactions aiding in the maintenance of a balanced NAD+/NADH ratio in the cells.

Anaerobiosis↗